Table of Content

10 March 2009, Volume 42 Issue 3

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Morphological Identification and Analysis of EMS Mutants from Hexaploid Wheat Cultivar Yanzhan 4110


Scientia Agricultura Sinica. 2009, 42(3):  755-764 .  doi:10.3864/j.issn.0578-1752.2009.03.001

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【Objective】 The mutant library is the basic material of functional genomics. In wheat, this kind of mutant library was rarely reported. In this study, two wheat mutant libraries with EMS were constructed for laying a foundation for wheat functional genomics research. 【Method】 EMS was used as chemical mutagen to build the mutant library with early mature spring wheat Yanzhan 4110. Biological characters and agronomic traits were investigated in M2 generation. Some of mutants identified from M2 plants will be validated through M3 generation. 【Result】 During the growth period after sowing the M2 seeds, all the phenotypic changes of plants were observed and recorded. Phenotype mutants such as seedling habits, leaf appearance, plant height, spike appearance and mature period were obtained. Some of them were related to important agronomic traits and biological characters, specifically, some mutants were rarely observed in natural variation, such as extremely dwarf, with plant height at about 10-15cm. The rate of M2 phenotype mutation was 6.6%. 【Conclusion】 Two libraries of Yanzhan 4110 induced with EMS are ideal mutant libraries and they will be efficiently used in wheat functional genomic research and wheat improvement.


Effect of Different Wx Protein Combinations on Wheat Starch Biosynthesis in Developing Endosperm


Scientia Agricultura Sinica. 2009, 42(3):  765-771 .  doi:10.3864/j.issn.0578-1752.2009.03.002

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【Objective】 This study was to elucidate the difference in starch physiochemical properties among wheat cultivars differing in Wx protein combinations through the comparison of starch accumulation and activities of starch biosynthetic enzymes. 【Method】 The accumulation of amylose and amylopectin in developing endosperm from 14 wheat cultivars with six types of Wx protein combinations were investigated, together with activities of four key enzymes involved in starch biosynthesis such as ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), granule bound starch synthase (GBSS), and starch branching enzyme (SBE). 【Result】 The number and type of Wx protein had a significant influence on activities of starch biosynthetic enzymes and the accumulation of amylose and amylopectin in developing endosperm. The activities of starch biosynthetic enzymes increased with the number of Wx protein, especially for GBSS, together with the amylose content in grain. The Wx-B1 protein deficiency induced the lowest GBSS activity and amylose content in grain, then was the Wx-D1 protein, and the effect of Wx-A1 protein deficiency was the least. The activities of four starch biosynthetic enzymes correlated to each other significantly, and also correlated to the number of Wx protein, amylose and amylopectin content in grain, which suggested the biosynthesis of amylose and amylopectin in developing endosperm were interacted and synchronous. 【Conclusion】 The Wx allele is one of the most determinant factors for amylose content for it can control the activity of GBSS through the number and type of Wx protein and affect other starch biosynthetic enzymes indirectly, then the synthesis of amylose and amylopectin, which result in different starch physiochemical properties and noodle quality finally.


Separation and Analysis of Wheat Leaf Proteome by Combination of 2D-LC and Nano LC-MS/MS


Scientia Agricultura Sinica. 2009, 42(3):  772-780 .  doi:10.3864/j.issn.0578-1752.2009.03.003

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【Objective】 Two-dimensional liquid chromatography (2D-LC) can be used as a complementary approach to protein separation with two-dimensional gel electrophoresis (2-DE). The purpose of the study is to establish a protocol to separate and identify wheat leaf proteome by combination of 2D-LC and Nano LC-MS/MS. 【Method】 The soluble protein extracted from wheat leaf were desalted by gel filter chromatography and separated by strong anion exchange chromatography (SAX) in the first dimension. The elution fractions were subjected to SDS-PAGE and the fractions without the most abundant protein (s) were further separated by reversed phase liquid chromatography (RPLC) in the second dimension. To test the effectiveness of the separation method, some of the 2D-LC fractions including lower intensity peaks were digested and analyzed by Nano LC-MS/MS. The MS/MS data were used to search against NCBInr and EST database using MASCOT search engine. Meanwhile, de novo sequencing was performed manually and the data were used to search against nrdb95 database using MS BLAST search engine. 【Result】 A total of 15 collections were obtained through the first dimensional separation and the most abundant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in plant leaf, could only be observed in the 15th collection. After the second dimensional separation, 1551 collections were collected with 1867 resolved protein peaks from the other 14 fractions without RuBisCO. Nine proteins were identified from six 2D-LC collections with lower intensity peaks selected randomly. 【Conclusion】 Based on the experiment setup and results, it is tentatively concluded that the combination of 2D-LC and Nano LC-MS/MS could be an effective method in future wheat leaf proteomics analysis.


Contributions of Genetic Improvement to Yields of Maize Hybrids During Different Eras in North China


Scientia Agricultura Sinica. 2009, 42(3):  781-789 .  doi:10.3864/j.issn.0578-1752.2009.03.004

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【Objective】 To make a survey of the advances of maize breeding efforts in north China and find the strategies to continuously improve the yields of maize hybrids in the future, the regression of maize hybrid agronomic traits on year of introduction was calculated. 【Method】 The regression of agronomic traits in yields of hybrids, which was based on the split-spot experimental design with 25 hybrids released during 1970s-2000s eras in north China as sub-plot and population density as main plot, was carried out in Xinjiang and Beijing in 2005 and 2006, respectively. 【Result】 By means of comprehensive analysis on response between yields and eras from 1980s to 2000s, it showed that the yield response of medium density was higher than that of high density in Xinjiang, and the response of medium density was also not significant compared with that of high density in Beijing. It indicated that higher yield response of high density had not been achieved between 1980s and 2000s. In addition, increasing improvement of 1000-kernel weight and rows per ear was the major positive factor that enhances yield of hybrids between 1980-2000 while improvements for decreasing barren rate and shelling percentage were the important negative factors. Through the pooled ANOVA, it showed that there were no significant improvements for traits such as ears per plant, stock lodging, barren tip rate which were closely associated with adaptability of high density between 1980s and 2000s. 【Conclusion】 Therefore, it could be concluded that higher density strategy and technology routine of maize breeding activity should be strengthened and improved although some yield response of high density has been achieved in the last few decades. As a result, the future hybrids in China can be developed towards the modern breeding goals such as early maturity, tolerance to high density, higher shelling rate, no bare tip.


Developmental and Gene Expression Analysis of a Fuzzless Mutant of Gossypium hirsutum


Scientia Agricultura Sinica. 2009, 42(3):  790-797 .  doi:10.3864/j.issn.0578-1752.2009.03.005

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【Objective】 This study aims to compare the transcript profiles between the cotton fuzzless mutant and its isogenic line in order to identify the genes which are involved in cotton fiber development. 【Method】 Scanning electron microscope (SEM) was used to observe the differential fiber developmental patterns between the fuzzless mutant GZnn and its isogenic line TM-1, and differential display PCR was employed to identify the differentially expressed genes. These resulting fragments were sequenced and analyzed by BLASTx. 【Result】 Using SEM to examine the cellular events of fiber cell development in the mutant GZnn, it was found that not only the process of fiber cell formation and elongation delayed, but also the fiber number reduced in the mutant in comparison with TM-1. By using differential display RT-PCR, 12 differentially expressed cDNA fragments were obtained, among which DS3 showed high degree of similarity to myb family transcription factors and was expressed exclusively in ovule before anthesis (-3-0 DPA) in the mutant, but not in that of TM-1. Therefore, DS3 might be a negative regulator during fiber initiation. 【Conclusion】A large number of genes were expressed specifically during cotton fiber initiation and development. Further investigations on the genes which play key roles during this process are of help to understand the molecular mechanisms underlying the differentiation and development of cotton fibers, and this will contribute to cotton genetic improvement.


Selection of Parents for Breeding of Sweetpotato Food Varieties of High Carotene Content


Scientia Agricultura Sinica. 2009, 42(3):  798-808 .  doi:10.3864/j.issn.0578-1752.2009.03.006

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【Objective】 Sweetpotato varieties with high carotene content have higher value of healthy care to human. This paper aims to improve the breeding efficiency of special varieties with high carotene content by using the method of combining molecular markers and agronomic traits to evaluate and screen the parents. 【Method】 Cluster analysis was carried out to identify and screen the excellent parents by utilizing phenotypic results of the agronomic and characteristic traits, RAPD and ISSR markers of 15 parents and their descendants by group crossing. 【Result】 A great variation in two important characteristics of the carotene content and the storage root yield was observed among different parent materials. A negative correlation was found between the factor of the carotene content and both factors of fresh roots and dried root production. The most significant positive correlation between the carotene content of parents and that of their descendants was calculated, with the coefficient value of 0.7932 **. The phenotypic correlation calculated based on the agronomic characteristics of all the trial materials was not agreed with the known genealogy, while that based on the molecular markers data showed better result. ISSR markers could amplify 13.8 bands per primer on average, in which the rate of polymorphic loci was 89.6%. RAPD markers amplified 9.4 bands per primer, in which the rate of polymorphic bands was 74.46%. 【Conclusion】 A large genetic variation of carotene content was found among the parents. For analyzing the genetic diversity of sweetpotato, the method of molecular marker was found to be better than morphological traits. ISSR marker was thought to be more efficient than RAPD markers. The results from the molecular markers and agronomic traits analysis can supply a valuable theory reference for the selection of parents for breeding of new varieties with high carotene content.


TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Calcium Distribution in Anther of Photo-sensitive Genic Male-Sterile Rice and Its Relationship with Pollen Fertility


Scientia Agricultura Sinica. 2009, 42(3):  809-815 .  doi:10.3864/j.issn.0578-1752.2009.03.007

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【Objective】 This study was conducted to investigate the relationship between Ca2+ distribution in anther and pollen abortion of photo-sensitive genic male-sterile rice during the anther development. 【Method】 Potassium antimontate was adopted to observe the distribution of Ca2+ in parenchyma, anther wall and pollen of photo-sensitive genic male-sterile rice line Nongken 58S and fertile line Nongken 58N during their anther development. 【Result】 The results showed that there were many differences between sterile and fertile anthers. In the fertile pollen, the calcium precipitates gradually accumulated on its surface, but hardly distributed in pollen cytoplasm; pollen wall could form completely. Whereas, in the sterile pollen, the distribution of calcium granules was obviously fewer on its surface compared with fertile pollen, but more abundant in cytoplasm; moreover, the pollen formed an abnormal cell wall. The tapetal cells of sterile anther began to degenerate ahead of time and the progress of degeneration was very slow. The sterile anther formed ubish bodies earlier than fertile anther, but had no calcium granules on its surface until late microspore stage. Furthermore, the calcium granules on the surface of ubish bodies were obviously less than in fertile anther. In general, there were much more calcium granules in anther wall of sterile anther than in fertile anther, except ubish bodies. During the development of anther, the calcium precipitates in parechymatous cells increased gradually, but at the same developmental stages, the sterile anther had more calcium granules than fertile anther. 【Conclusion】 It is concluded that the premature degradation of the tapetal cell and the abnormal function of ubish bodies lead to the hindrance in transport of Ca2+ to locule, and cause the deficiency of Ca2+ on the surface of pollen subsequently, as a result, the exines can’t form normally. In addition, Ca2+ assembles in pollen cytoplasm abundantly at late microspore stage, this is one of the most important factors which lead to pollen abortion.


Starch Particle Size Distribution in Developing Endosperm of Wheat Cultivars Lumai 21 and Jinan 17


Scientia Agricultura Sinica. 2009, 42(3):  816-823 .  doi:10.3864/j.issn.0578-1752.2009.03.008

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【Objective】 The present study was carried out to characterize the distribution and development of starch granules during grain filling in two wheat cultivars differing in gluten. 【Method】 Two wheat cultivars, Lumai 21 (weak gluten winter wheat) and Jinan 17 (strong gluten winter wheat) were used as materials to investigate the distribution of starch granule size with laser-diffraction particle-size analyzer during endosperm development at Tai’an of Shandong Province in 2004-2006 growing season.【Result】 At different days after anthesis (DAA), the percent number distribution of starch granules in wheat endosperm exhibited single peak curves with peaks at 0.5~1.1 μm. The starch granules initiated before 7 DAA and increased in size to maximum diameter of 20 μm at 7 DAA. These early synthesized granules (A-type) continued to enlarge to their maximum diameter of 30 μm at 10 DAA. By 14 DAA, a burst of synthesis created a new population of small starch granules (B-type), which reduced the number proportion of big starch granules. A third group (C-type) of small granules was initiated at 21 DAA. From 24 to 28 DAA, the percent number of ＜0.6 μm starch granules increased and those of ＞9.8 ?m starch granules decreased continuously, indicating that new granules were still synthesized, however, the magnitude of increase reduced. These small granules continued to enlarge in diameter by 35 DAA. The percent number of ＜0.6 μm starch granules and that of ＞0.6 ?m in Lumai 21 was lower and higher, respectively, than in Jinan 17 at 7-14 DAA. However, there were opposite changes at 17-28 DAA. 【Conclusion】 It is concluded that Lumai 21 has higher starch granule accumulation rate and stronger capacity of new starch granule synthesization than Jinan 17, respectively, at early and late grain filling stage. At maturity, the percentages of volume and surface area of B-type starch granules in Jinan 17 are higher than those in Lumai 21, but on the contrary in the percentages of A-type starch granules.


Studies on the Programmed Cell Death in Barley During Starchy Endosperm Development


Scientia Agricultura Sinica. 2009, 42(3):  824-832 .  doi:10.3864/j.issn.0578-1752.2009.03.009

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【Objective】 Barley starchy endosperm cell has become dead cell during endosperm development, the purpose of this study was to reveal that the death of starchy endosperm was a special form of programmed cell death (PCD). 【Method】 Structural morphology of nucleus was observed with transmission electron microscope and fluorescence microscope, the breakage of nuclear DNA was analyzed with agarose gel electrophoresis and TUNEL, the activity of antioxidant enzyme and grain filling ratio were also investigated. 【Result】 The nucleus of barley starchy endosperm showed nucleus deformation, chromatin condensation, nuclear envelope disruption, and nuclear residue formation from degraded nucleus, which were typical features of PCD, along with the development of the starchy endosperm in barley. Breakage of nuclear DNA was observed with agarose gel and TUNEL method. Nuclear residues, which were not completely lost, were observed among starch granules in starchy endosperm cells after staining with DAPI. Evan’s blue staining indicated that starchy endosperm cell death occurred unsynchronously among the starchy endosperm cells with initiation points randomly distributed in the endosperm tissue. Antioxidant enzymes showed high activities, and grain weight increased during degradation of nucleus and development of cytoplasm without nucleus. 【Conclusion】 Barley starchy endosperm cell remained alive, kept its normal metabolism, the synthesis and accumulation of starch and storage protein during degradation of nucleus and developemt of cytoplasm without nucleus. The starchy endosperm cell finally become death cell when it is completely filled with reserves. The development of starchy endosperm in barley is a special form of PCD.


Relationship Between Nitrogen Concentration in the Subtending Leaf of Cotton Boll and Fiber Quality Indices


Scientia Agricultura Sinica. 2009, 42(3):  833-842 .  doi:10.3864/j.issn.0578-1752.2009.03.010

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【Objective】 To investigate the relationship between nitrogen concentration in the subtending leaf of cotton boll and fiber quality indices, and to provide theoretical basis for monitoring nitrogen status in fiber quality formation of bolls with different anthesis dates. 【Method】 Field experiments were carried out in Nanjing (118o50′E, 32o02′N) and Xuzhou (117°11′E, 34°15′N), representing different ecological conditions in the middle lower reaches of Yangtze River Valley and the Yellow River Valley in China. Two cotton cultivars and three nitrogen application levels were applied. The three N levels were 0, 240 and 480 kgN?ha-1, representing low, medium and high nitrogen level, respectively, and the two cultivars were KC-1 and AC-33B with different fiber quality indices. Responses of LMA (leaf mass per unit leaf area) and nitrogen concentration (NM, nitrogen content per unit leaf mass; NA, nitrogen content per unit leaf area) in the subtending leaf of cotton boll to nitrogen application levels were studied, and the relationships between nitrogen concentration and fiber quality indices were firstly groped. 【Result】 (1) NA in the subtending leaf of cotton boll contained both the information of LMA and NM, possessed the special sensitive characteristic in response to nitrogen application levels and anthesis dates. It can reflect the increasing trends with anthesis date postponing and significant difference in nitrogen treatments. (2) With the average values of NA increasing, the changing trends of pivotal quality indices (length, strength, micronaire and uniformity) of cotton fiber according with the curve of parabola with peak value. (3) With anthesis date, postponing, the difference of NA between low-nitrogen and medium-nitrogen treatments enlarged gradually, on the contrary, the difference between high-nitrogen and medium-nitrogen treatments diminished gradually, fiber strength and micronaire also showed similar trends between corresponding nitrogen treatments. 【Conclusion】 NA in the subtending leaf of cotton boll is tightly related to fiber quality indices formation. As the corresponding relationships between NA and fiber indices are not affected by cotton varieties and ecological sites, it is proposed that NA in the subtending leaf of cotton boll could be used as a practical physiologic index for timely monitoring nitrogen status for fiber quality in the future.


Relationship Between Nitrogen Efficiency and Root Architecture of Maize Plants: Simulation and Application


Scientia Agricultura Sinica. 2009, 42(3):  843-853 .  doi:10.3864/j.issn.0578-1752.2009.03.011

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【Objective】 The relationship between root morphology and its spatial distribution in the soil and nitrogen acquisition efficiency was studied. The visualization of root architecture was attempted by using root functional-structural model. 【Method】 A field experiment was conducted with two maize inbred lines, N-efficient 478 and N-inefficient Wu312. The lengths of embryonic and different whorls of node roots harvested at different developmental stages were scanned, and used as parameters to simulate root architecture based on the modified root functional-structural model. 【Result】 478 had a longer root lengths of embryonic and node roots, better root spatial distribution in the soil than that of Wu312. The roots of 478 had larger growth rate and higher branching density than those of Wu312 in terms of simulation. The root length of the embryonic roots and the early initiated three whorls of nodal roots, which represented a small portion of total root length, declined rapidly after reaching their highest values before silking. The shoot-born roots from the higher nodes initiated predominantly, which was synchronized closely with shoot development and demand for nutrients, reached their maximal lengths at 93 d after sowing and then declined. 【Conclusion】 The differences in root length and its spatial distribution in the soil play an important role in nitrogen acquisition efficiency between maize genotypes. The root architectures of both maize inbred lines at different developmental stages could be visualized by using root functional-structural model.


Effects of Topping on Potassium Metabolism and Expression of Potassium Channels in Tobacco Plants


Scientia Agricultura Sinica. 2009, 42(3):  854-861 .  doi:10.3864/j.issn.0578-1752.2009.03.012

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【Objective】 In order to provide a theoretical basis for the potassium regulation metabolism, this paper analyzed the effects of topping on the expression of potassium channel genes in flue-cured tobacco. 【Method】 Real-time fluorescence quantitative PCR was used to detect the relative expression change of potassium channel genes, and endogenesis auxin (IAA) was detected by ELISA in tobacco through pot experiments. 【Result】 As compared with the control, the potassium accumulation in whole plant of decapitated tobacco decreased and there was more potassium distribution in leaves and roots. The expressions of NKT1 and NTORK1 genes in leaves were inhibited by topping, but not in roots. The expression of both NKT1 and NTORK1 in leaves was higher than that in roots within 24 h, except 14 d while it was reversed in the two tissues. The variations of expression of potassium channel genes in roots could control potassium accumulation and regulate K+ concentration in aerial parts. Further results showed that the level of endogenesis IAA, which declined in leaves and increased in roots by 140.7% at 14 d compared to the control plant, was attributed to the expression change of potassium channels. 【Conclusion】 The variations of expression of potassium channel genes are likely to be caused by the endogenesis hormone in tobacco after topping, and it has a direct impact on the potassium accumulation and distribution simultaneously.


PLANT PROTECTION

Molecular Cloning and Activity Analysis of a Rice Gene Promoter OsBTF3p


Scientia Agricultura Sinica. 2009, 42(3):  862-868 .  doi:10.3864/j.issn.0578-1752.2009.03.013

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【Objective】 To make a cloning and functional analysis of OsBTF3p, a rice gene promoter directing GUS expression. The functional analysis of OsBTF3p is helpful to understand the mechanisms of gene expression and to utilize in generation of transgenic plants. 【Method】 A 1378bp promoter of an inducible rice gene OsBTF3 upon infection by Xanthomoas oryzae pv. oryzae (Xoo) was amplified by PCR and named as OsBTF3p. OsBTF3p fused in frame with the gus reporter gene and the resulting construct (OsBTF3p::GUS) was transformed into the rice calli through Agrobacterium-mediated transformation. Thirty-nine transgenic plants of rice （T0 generation） were successfully obtained. The promoter activity, tissue specificity and pathogen inductivity were analyzed as well. 【Result】 The cloned DNA sequence of OsBTF3p is same as that present in the GenBank database. GUS activity can be detected in the OsBTF3p::GUS -transformed but non-transgenic calli (control). GUS expression was also observed in the OsBTF3p::GUS-transformed vascular tissues of leaves and roots. The transgene OsBTF3p::GUS in rice is up-regulated upon Xoo infection. 【Conclusion】 OsBTF3p might function effectively in promoting GUS expression in transgenic rice plants in tissue-specific and pathogen-inducible fashions.


Secreted Expression of the Combinant Defensin alfAFP (M. sativa) in Pichia pastoris and Its Antimicrobial Activity Against Rice Pathogens in Vitro


Scientia Agricultura Sinica. 2009, 42(3):  869-875 .  doi:10.3864/j.issn.0578-1752.2009.03.014

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【Objective】 Most plant defensins involve with defense and exhibit antimicrobial activity against various plant pathogens. Expression of the defensin AlfAFP from Medicago sativa in transgenic potato and tomato plants has been proved to possess high resistance to the pathogen. Study on antimicrobial activity of alfAFP against three main rice disease, blast, bacterial blight and sheath blight, will be significant for further research of disease resistance in transgenic rice. 【Method】 alfAFP was inserted into yeast expression vector pPIC9K, and then transferred into Pichia pastoris GS115 strain by electroporation, and alfAFP was expressed in yeast and utilized to assess antimicrobial activity in vitro. 【Result】 A molecular mass of expressed alfAFP was about 6.5 kD by 15% SDS-PAGE analysis, and the recombinant protein showed certain antimicrobial activity against Rhizoctonia solani, Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, especially for Rhizoctonia solani. 【Conclusion】 Defensin alfAFP was successfully expressed in Pichia pastoris, and antimicrobial activity was detected for rice tested pathogens in vitro, indicating its potential function in breeding rice for disease resistance.


Cloning and Expression of a Neutral Protein Gene BS2 from Bacillus subtilis B111 in Pichia pastoris


Scientia Agricultura Sinica. 2009, 42(3):  876-883 .  doi:10.3864/j.issn.0578-1752.2009.03.015

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【Objective】 In order to exploit novel disease-resistance gene and biological pesticide sources, antifungal protein secreted by Bacillus subtilis B111 was purified and identified, then its gene was cloned and expressed in Pichia pastoris. 【Method】Antifungal protein was purified by ultrafiltration, 90% ammonium sulfate precipitation and QAE Sephadex A50 ion exchange chromatogram column, then it was identified via CapLC-ESI-MS-MS. Its gene encoding antifungal protein was cloned. The recombinant expression vector pPIC9K-PT was constructed and transformed into P. astoris GS115. The transformants were identified by PCR analysis．The products of the transformants induced by methanol were identified by neutral protease and antifungal activity assay. 【Result】 An antifungal protein BS2 was purified and identified to be a neutral protease from B. subtilis. Antifungal gene BS2 with pro-sequence was cloned. The recombinant expression vector pPIC9K-PT was constructed and BS2 gene was expressed in P. pastoris. The recombinant BS2 protein exhibited a molecular mass of approximately 69 kD and presented neutral protease activity of 27 800 U?g-1 and antifungal activity. Its production reached up to as high as 29.77 mg?L-1. 【Conclusion】 In this study, an antifungal neutral protein BS2 was purified and identified, then its gene was cloned and successfully expressed in P. pastoris.


Preparation of Monoclonal Antibody Against PthA-NLS and Construction of the Relative ScFv Gene


Scientia Agricultura Sinica. 2009, 42(3):  884-890 .  doi:10.3864/j.issn.0578-1752.2009.03.016

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【Objective】 The present study aimed at preparation of monoclonal antibody against the recombinant PthA-NLS and isolaton of the relative ScFv (Single Chain Variable Fragment) genes, providing a possibility to better understand the pathogenesis mechanism via PthA, and to develop proper construct for future experimentation to obtain citrus plants resistant to citrus canker disease by transformation and plant antibody techniques. 【Method】 The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti-PthA-NLS McAb with Trizol reagent, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR (splicing by overlap extension), and then the ScFv gene was constructed. The recombinant ScFv gene was cloned into pGEM-T and pET32a(+) vector. The later plasmid was transferred into E. coli BL21(DE3) and the expression of the recombinant protein was induced. 【Result】 Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6 and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 KDa of recombinant fusion protein was obtained. 【Conclusion】 Three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS were obtained, and the relative ScFv gene was constructed and successfully expressed in E.coli. These results may play an important role in further understanding of the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.


Cloning and Prokaryotic Expression of cDNA Encoding General Odorant Binding ProteinⅠfrom Spodoptera litura


Scientia Agricultura Sinica. 2009, 42(3):  891-899 .  doi:10.3864/j.issn.0578-1752.2009.03.017

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【Objective】 The objective of this study aims at cloning, sequence analysis and prokaryotic expression of a new cDNA encoding the general odorant binding protein Ⅰ from Spodoptera litura (named as SlGOBPⅠ). 【Method】 The cDNA encoding SlGOBPⅠ was isolated from the antennae of S. litura (SlGOBPⅠ, GenBank Accession No. EU086372) by homology cloning and rapid amplification of cDNA ends (RACE), and then the open reading frame (ORF) of SlGOBPⅠ was cloned into prokaryotic cells to test its expression. 【Result】Results of sequencing and structural analysis showed that the ORF of SlGOBPⅠ was 495 bp, encoding 164 amino acid residues, with the predicted MW of 19.3 kD and pI of 5.54, respectively. It shared the typical structural features of odorant binding proteins from other insects, including the six conservative Cys lotus in the sequence. The deduced amino acid sequence showed a high identity to the reported sequences of GOBPⅠ from other lepidopteran insects, and showed that SlGOBPⅠ may belong to the family of GOBP. The SlGOBPⅠ was constructed into pET-32a vector and expressed in Escherichia coli BL21 (DE3) after induction with IPTG. The analysis of SDS polyacrylamide gel electrophoresis and Western blot indicated that the molecular weight of fusion protein was about 32.0 kD. The fusion protein was purified by one step Ni-NTA affinity chromatograph column and injected into New Zealand White rabbit to raise antiserum successfully. The analysis of ELISA showed the titer of antiserum was 1﹕5 000. Western blot analysis showed that the expressed SlGOBPⅠ protein was crossreactive with the anti-SlGOBPⅠ antiserum, which indicated the expressed fusion protein belonged to GOBPⅠ of the insect. 【Conclusion】The successful cloning and expression of the coding sequence of SlGOBPⅠhave laid a basis for further study on the structure and function of SlGOBPⅠ.


Influence of Prey Species on Growth, Development and Reproduction of Orius sauteri


Scientia Agricultura Sinica. 2009, 42(3):  900-905 .  doi:10.3864/j.issn.0578-1752.2009.03.018

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【Objective】 The objective of the study is try to find a new diet on rearing of Orius sauteri massively. 【Method】O. sauteri was reared by Tyrophagus putrescentiae, Tetranytrus cinnabarinus or Brevicoryne brassicae as preys at 17.5℃, 22.5℃, 27.5℃, respectively, and its life tables were made in laboratory. 【Result】 The results revealed that more than 70% of O. sauteri nymphs were developed to adults when they fed on T putrescenti, which was no significant difference compared to those fed on B.brassica, but was significantly higher than those fed on T. cinnabarinus (survival rates＜ 50%). At 17.5℃, O. sauteri reared with T. putrescentiae had no oviposition. The net reproduction rate (Ro) of O.sauteri reared with T. cinnabarinus and B. brassicae at 17.5℃ were 1.18 and 1.09, respectively. The Ro of O. sauteri reared with T. putrescentiae, T. cinnabarinus and B. brassicae were 12.86, 15.93 , 6.11 at 22.5℃ and 0.88, 3.02 , 21.70 at 27.5℃, respectively. At 27.5℃, the intrinsic rate of natural increase (rm) was negative as O.sauteri reared with T. putrescentiae. 【Conclusion】 These results indicate that T. putrescentiae can be used to rear O. sauteri nymphae massively.


Effect of Chlorimuron-ethyl on Biochemical Mechanism in Tolerant Sugar Beet


Scientia Agricultura Sinica. 2009, 42(3):  906-911 .  doi:10.3864/j.issn.0578-1752.2009.03.019

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【Objective】 Effect of chlorimuron-ethyl on biochemical mechanism in tolerant sugar beet was investigated to provide basic data on using the tolerant genotype to chlorimuron-ethyl properly. 【Method】 Tolerant sugar beet was used to analyze its biochemical mechanism under chlorimuron-ethyl stress with frame culture in field and water culture. 【Result】 Glutathione S-transferase (GST) activity in leave of tolerant sugar beet was remarkably increased as chlorimuron-ethyl was used at 0.5 ga.i.ha-1 and 1.5 ga.i.ha-1 field dose , at the same time glutathione (GSH) content increased by 50.0-490.1 μg g-1. GST activity of sensitive sugar beet decreased by 122.6 U?(min?mg)-1 compared with tolerant sugar beet and GSH content only increased by 7.4 μg g-1 as chlorimuron-ethyl was at 0.5 ga.i.?ha-1 field dose in sensitive sugar beet. The higher GST activity and GSH content could conjugate chlorimuron-ethyl which had been absorbed in tolerant sugar beet and made it lost activity. Acetolactate synthases (ALS) activity of the tolerant sugar beet increased by 62.5% and 70.6%, respectively, in seedling and leaf growth period, at the same time ALS activity of the sensitive variety decreased by 36.8% and 64.8%, respectively. 【Conclusion】 The rapid enhancement of GST,GSH and the target enzyme activity of ALS is the important reason for the tolerant sugar beet enduring to chlorimuron-ethyl.


SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Response of Element Distribution in Various Organs of Maize to Fertilizer Application


Scientia Agricultura Sinica. 2009, 42(3):  912-917 .  doi:10.3864/j.issn.0578-1752.2009.03.020

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【Objective】 The effect of fertilizer application on biomass dry weight and element distribution in various organs of maize (Zea mays L.) was investigated to disclose the internal mechanism of fertilizer effect on crop. 【Method】 Five fertilizer treatments were designed, i.e.no fertilizer (CK), nitrogen-phosphorus fertilizer (NP), and nitrogen-potassium fertilizer (NK), phosphorus-potassium (PK), and nitrogen-phosphorus-potassium fertilizer (NPK). A field experiment on the response of biomass dry weight and N, P, K, Cu, Zn, Fe, Mn element distribution in stem, leaf and seed of maize to different fertilizer application treatments was carried out. 【Result】 The results showed that dry weight, element content of various organs of maize were different. N content in various organs was in the order of leaf ＞seed＞stem; P content was in the order of seed＞leaf＞stem; K content was in the order of stem＞leaf＞seed; Fe content was in the order leaf ＞stem＞seed. Fertilizer application reduced the differences in N, P, K, and Fe content in various organs, and changed the ratio of between dry weight and the Cu, Zn contents in leaf, seed and stem. Except for a few treatments, the response trend of N, P, Fe elements to fertilizer application was similar to Cu and Zn. The response of element content in various organs of maize to different fertilizer combinations was also different. The response extent of the major elements of N, P and K to fertilizer application was larger than microelements Cu, Zn, Fe, and Mn. The response extent of stem and leaf was larger than seed. The response of elements to NPK fertilizer treatment was bigger than NP, NK, and PK. On the whole, response differences of element distribution to various fertilizer treatments were not remarkable. Besides, element distribution in various organs of maize was also influenced by the mutual effect of fertilizer varieties. 【Conclusion】 The response trend and extent of various element distributions to different fertilizer treatments had both similarities and differences. Adjusting fertilizer application could change the biomass dry weight and element distribution in various organs of maize, thus promoting the uptake and cycle of nutrients. This investigation has provided a useful information for high yield production.


Nitrate-N Leaching in 23-Year Winter Wheat Field Combined with Application of Nitrogen and Phosphorus


Scientia Agricultura Sinica. 2009, 42(3):  918-925 .  doi:10.3864/j.issn.0578-1752.2009.03.021

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【Objective】 To provide a reference for scientific fertilization in the dry-land，present study on the changing trend of NO3--N leaching and the relationship between NO3--N accumulation and fertilizer rates in the farmland ecosystem based on data of the long-term fertilizer experiment. 【Method】Soil samples were collected per 20 cm in 0-300 cm soil layer, after being extracted with 1 mol?L-1 KCl, the NO3--N concentration in soil was analyzed with AA3 automated flow injection analyzer. 【Result】 When N fertilizer was applied alone, the accumulation peaks of NO3--N occurred in 80-100 cm and below 300 cm soil layer. If the annual rate of N reached 180 kg hm-2, total NO3--N accumulation amount in 0-300 cm soil layer was 8 times the amount of annual N application. When P fertilizer was applied alone, it had no significant effect on NO3--N distribution. NO3--N accumulation amount in the soil enhanced significantly with increasing N application rates. When N and P fertilizer was applied in combination, the application of P fertilizer alleviated NO3--N leaching and the positive effect increased with increasing N application rates. When N rates were excessive, even combined with P fertilizer, NO3--N penetrated into deep soil layer and accumulation peaks were found in 100-120 cm and 240-260 cm soil layers. Quadratic polynomial regression equation could reflect the relationship between NO3--N accumulation and N, P fertilizer rates. 【Conclusion】 When N fertilizer was applied excessively for 23 years, NO3--N penetrated largely into deep soil layer and two accumulation peaks were observed. However, NO3--N leaching could be mitigated by combined application of nitrogen and phosphorus. Long-term fertilizers application at rates for maximum yield would result in a great amount of NO3--N accumulation in deep soil layer in dry-land wheat field.


Effects of Rice Straw Incorporation in situ and ex situ on Soil Organic C and Active Organic C in Agricultural Soils in Red Soil Hilly Region


Scientia Agricultura Sinica. 2009, 42(3):  926-933 .  doi:10.3864/j.issn.0578-1752.2009.03.022

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【Objective】 To investigate the effects of rice straw incorporation in situ and ex situ on soil organic carbon and active organic carbon in red soil hilly region. 【Method】 This study was conducted on slope upland and upland-paddy rotation in a long-term field experiment in red soil hilly region for 6 years, and three treatments were established, including CK (no fertilizer), NPK and straw + NP (S+NP). 【Result】 Soil organic carbon (SOC) in slope upland significantly increased by 14.6% and 36.2% in NPK and S +NP treatments compared to the initial value, respectively. Microbial biomass carbon (MBC) remained an increasing trend on the whole, and it significantly increased by 16.1% and 33.5% in NPK and S+NP treatments compared with CK. Dissolved organic carbon (DOC) was increasing yearly, and there was no significant difference among 3 treatments. SOC in paddy-upland rotation was almost steady in 6 years, but was lower than initial value in NPK and CK treatments. MBC remained the same trend as that in slope upland, and significantly increased by 14.2% in S+NP compared with CK, but declined in NPK. DOC had a decline trend in the late 2 years, and it was lower in NPK and S+NP treatments than that in CK. 【Conclusion】 Rice straw incorporation in situ and ex situ increased SOC and MBC compared with single application of chemical fertilizer, and the effect was more significant for straw incorporation ex situ in only few years. DOC had little response to fertilizer treatments, and the content was higher in slope upland with only 6 years cultivation than in paddy-upland rotation land with long-term cultivation.


Effect of Wheat-Residue Application on Physical and Chemical Characters and Enzymatic Activities in Soil


Scientia Agricultura Sinica. 2009, 42(3):  934-942 .  doi:10.3864/j.issn.0578-1752.2009.03.023

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【Objective】 The purpose of this study was to investigate the effects of wheat-residue application on soil physical and chemical properties and enzymatic activities in soil as well as their relations. 【Method】 A mid-season japonica rice cultivar Yangjing 9538 was grown in field. Three treatments of N-fertilizer application, farmers’ N-fertilizer practice (FFP), SSNM based on chlorophyll measurement (SPAD) readings, and no nitrogen application, and with or without wheat residue application (the straw was incorporated into soil) were conducted. Organic acid content, pH value, respiration rate, and enzymatic activity in soil were determined. 【Results】 The peak value of decomposition of wheat-residue was observed during the first month after rice transplanting. The contents of organic acids were increased significantly at mid-tillering and panicle initiation stages. Activities of urase and hydrogen peroxidase were enhanced initially and then decreased, and showed a peak value at 30 and 60 days after transplanting, and varied with development and growth stages. The activity of alkaline phosphatase in soil showed two peaks. The value of pH declined significantly, while the contents of organic acids were increased, under wheat-residue incorporation, and there was a negative correlation between them. The straw application increased the activities of urase, hydrogen peroxidase and alkaline phosphatase, organic matter content and respiration rate, while reduced ion contents of Na+, Mg2+ and Ca2+ in soil. Increase and decrease of the enzymatic activities and ion contents were associated with the decay degree of wheat-residue. Urase activity was higher under FFP than SSNM. There was no significant difference in other measurements of soil between FFP and SSNM. Activities of urase and alkaline phosphatase were significantly correlated with organic matter content and contents of total N, P and K. Hydrogen peroxidase activity was significantly correlated with the total N content, but not significantly correlated with the contents of total P and K in soil. 【Conclusion】 The results suggest that wheat-residue application could improve soil fertility through increase of organic matter content, enhancement of activities of enzymes, and reduction of contents of Na+, Mg2+, and Ca2+ in soil.


Dynamics of Soil Microbial Biomass on the Abandoned Cropland in Loess Hilly Area


Scientia Agricultura Sinica. 2009, 42(3):  943-950 .  doi:10.3864/j.issn.0578-1752.2009.03.024

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【Objective】 Soil microbial biomass is an important parameter representing material cycle and energy flow in soil ecosystem. Study on dynamics of soil microbial biomass in the abandoned cropland in loess hilly area so as to understand the change and effect evaluation during process of ecological restoration is of great importance. 【Method】 The research was carried out in Zhifanggou small watershed of typical erosive environment, the croplands abandoned in different times were selected as objects, interchangeablely spece-time method was adopted and slope croplands and the plots with Platycladus orientailis were used as contrasts. Several indexes were analyzed such as soil microbial biomass, respiration and metabolic quotient (qCO2) and involvement characteristics of physical and chemical properties. 【Result】 The research indicates that the content of microbial biomass and physical and chemical properties under erosive slope cropland was low. However, there was a significant improvement on physical and chemical properties after abandoning. Soil microbial biomass C increased dramatically one year abandoned later, rose sharply in the first 7 years of abandonment, then took on wavelike rising and reached maximum 50 years later. Soil microbial biomass N increased slowly at early stage and reached significant level after 40 years. Soil microbial biomass P dropped significantly at early abandoned stage, reached minimum 5-7 years later and then rose gradually, after 20-25 years, there was no significant difference compared with the slope cropland but reached maximum 50 years later. After abandoning 50 years, soil microbial biomass C, N and P increased by 166%, 146%, and 52%, respectively, compared with slope cropland, which were only 43.42%, 45.06%, and 51.47% of that in soil with Platycladus orientailis. Respiration increased sharply at early abandoned stage and tended to be stable, which did not differ significantly compared with Platycladus orientailis. Metabolic quotient (qCO2) increased drastically at early abandoned stage, then took on wavelike dropping and dropped to minimum which was also higher greatly than that of Platycladus orientailis. Correlation analysis showed that there was a significant relationship between soil microbial biomass C, N, P and soil nutrition. Soil microbial biomass C, N, P, organic C, total N, available N and available K presented a linear growth. 【Conclusion】 The results suggest that it is impossible to recovery soil fertility and soil microbial biomass by natural vegetation recovery under erosive slope cropland in the loess hilly area, but much longer time, compared with degradation of soil fertility after deforestation, is required to reach to the climax before vegetation destruction.


HORTICULTURE

Effect of Solid Matrix Priming for Watermelon Seed and Influence on Activity of Endo-β-Mannanase and DNA Replication


Scientia Agricultura Sinica. 2009, 42(3):  951-959 .  doi:10.3864/j.issn.0578-1752.2009.03.025

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【Objective】 With solid matrix priming (SMP) technique, germination barriers of triploid watermelon seeds were overcome and germination percentage was enhanced by 40%. 【Method】 The investigation of priming mechanism indicated that the water absorbing capacity was slower and not reached saturation of moisture content all the time and rate and the concentration of RNA accumulation increased during SMP process. Using single-seed gel-diffusion assay and electrophoresis method to measure the endo-β-mannanase activity in endosperm envelope of watermelon seeds during imbibition. 【Result】 The result showed that average activity of the endo-β-mannanase and the ratio of seeds with higher enzyme activity in primed seeds were higher than that in control seeds. Investigation of DNA replication levels in primed seeds with flow cytometric determination showed that the 6C:3C ratio in embryonic root tip cells was higher than the unprimed seeds at all stages of seed imbibition. Seed structure inspection with soft-x ray showed that the endosperm envelope of root tip area become thinner in SMP treated seeds. 【Conclusion】 These results indicate that during and after SMP, the influence of seed physiology, DNA replication and seed structure in triploid watermelon seeds have a certain correlation with overcoming germination barriers and enhancement of germination capacity.


Construction and Preliminary Analysis of the SSH Library of Chinese Wild Vitis pseudoretioulata Resistance to Downy Mildew


Scientia Agricultura Sinica. 2009, 42(3):  960-966 .  doi:10.3864/j.issn.0578-1752.2009.03.026

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【Objective】 Isolating disease-resistance ESTs against downy mildew from Chinese wild Vitis pseudoretioulata Baihe-35-1. 【Method】 To obtain a differentially expressed genes library, a forward-suppression subtractive hybridization library and a reverse-suppression subtractive hybridization library were constructed. The young grape leaves was inoculated with Plasmopara viticola as the treated materials, and the untreated leaves were used as control materials. ESTs from the library were sequenced. Three ESTs was analized with the Semi-Quantitative RT-PCR. 【Result】 The inserted segments were 150-900 bp in length. Altogether 85 forward SSH library ESTs and 29 reverse SSH library ESTs were obtained. BLASTn and BLASTx analysis revealed that 78 ESTs have high homology with known genes in GenBank, and 31 ESTs are unknown genes. 114 ESTs of them had been submitted to GenBank, and the accession number is FG106789-FG106902. Three genes were selected and proved that these ESTs from the library could express in the plant by reverse transcription semi-quantitative. 【Conclusion】 The differentially expressed genes are involved in signal transduction, energy metabolism, protein and nucleic acid metabolism, photosynthesis and transmembrane transport, and 23 ESTs of them are probably related to disease resistance. The next step is to obtain downy mildew resistance gene span sequence through rapid amplification of cDNA ends (RACE) technology. And carries out researches on the nucleus and the eukaryon expression confirmation gene for providing a basis for further study of the grape genes resistance to downy mildew.


Development of Sex Linked AFLP-Derived SCAR Markers in Carica papaya


Scientia Agricultura Sinica. 2009, 42(3):  967-973 .  doi:10.3864/j.issn.0578-1752.2009.03.027

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【Objective】 Papaya (Carica papaya L.), native to tropical America, is a widely distributed fruit crop throughout the tropical and warmer subtropical regions of the world. Papaya is a polygamous plant species with three sex forms: female, male, and hermaphrodite. Sex determination in the young seedling stage is very important for papaya plantation. 【Method】 By cloning sex-linked AFLP markers, 31 Carica papaya sex linked SCAR markers were developed and validated at different sex types of papaya plants and segregation population. 【Result】 Sixteen SCAR markers were found to be polymorphic between different sex types and were co-segregated with male and hermaphrodite sex, and therefore can be used to discriminate the female and hermaphrodite or male tree, while the other 15 SCAR markers were monomorphism yielding same size DNA fragments at three sex types. 【Conclusion】These AFLP-derived SCAR markers can be used as diagnostic tools for molecular discrimination of different papaya sex types at seedling stage and as probes to screen papaya BAC library to construct physical map around the papaya sex determination gene.


Analysis of Cold-Tolerance and Determination of Cold-Tolerance Evaluation Indicators in Chrysanthemum


Scientia Agricultura Sinica. 2009, 42(3):  974-981 .  doi:10.3864/j.issn.0578-1752.2009.03.028

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【Objective】 Low temperature is one of the most important factors affecting the growth of chrysanthemum. To evaluate the cold tolerance of chrysanthemum, physiological and anatomical characteristics in chrysanthemum were studied under low temperature, the relationship between different indexes of cold tolerance were revealed, and mathematical evaluation model on cold tolerance was established too, which will provide a basis for cold tolerant germplasm discovery, breeding of new cold-tolerant varieties and evaluation on cold tolerant of a large number of chrysanthemum varieties. 【Method】 Physiological parameters such as SOD activity, CAT activity, content of MDA, soluble sugar and soluble protein and anatomical structure of leaves of rhizomes were studied in six chrysanthemum cultivars. Several indexes closely related to cold tolerance were screened out via correlation test. The validity of those screened indexes were further verified by main component analysis, regression analysis, hierarchical cluster analysis and path analysis. 【Result】 Analysis of related indexes with cold tolerance showed that significant negative correlations (P＜0.01) were presented and changing rate of SOD activity at -14℃ to -17℃ , soluble sugar content (SS1) at -11℃ to -14℃, thickness of palisade tissue (L2), thickness of upper epidermis cells (L4), and ratio of palisade tissue against spongy tissue (L6), compactness of palisade tissue (L7) were observed, while a significant positive correlation (P＜0.01) between LT50 and looseness of palisade tissue (L8) was found. 【Conclusion】 Four indexes closely related to the cold tolerance were screened. Mathematical evaluation model for chrysanthemum cold tolerance, i.e., Y=16.370-0.029SS1-0.075L7-0.314L8-12.250L6 (R2=1.000), was established too, and the predicting accuracy of the model is higher than 94%.


STORAGE·FRESH-KEEPING·PROCESSING

Effect of Freeze-Thawing Cycles on the Quality Properties and Microstructure of Pork Muscle


Scientia Agricultura Sinica. 2009, 42(3):  982-988 .  doi:10.3864/j.issn.0578-1752.2009.03.029

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【Objective】 The effects of freeze-thawing cycles on pork quality properties and microstructure were investigated. The purpose of this study was to provide a scientific basis for meat preservation. 【Method】 The porcine longissimus dorsi (LD) were frozen for 7 d under -26℃, then thaw by lotic water (room temperature, 18℃). The effects of freeze-thawing cycles on thawing loss (TL), cooling loss (CL), cutting force (CF), microstructure, color (L*, a*, and b*-values), TBARS (thiobarbituric acid-reactive substances) of pork were studied. 【Result】 The results showed that freeze-thawing cycles significantly increased the TL, CL, TBARS, L*-values and b*-values, and decreased a*-values (P＜0.05). The scanning electron micrograph of pork muscle showed that freeze–thawing cycles increased the space between fiber, torn and confused muscle fiber bundles, and breakdown muscle endometrial. The pork CF subjected one freeze-thawing cycle was significantly higher than that of fresh pork, and was gradually lower after multiple freeze-thawing cycles. 【Conclusion】 The results demonstrated that freeze-thawing cycles seriously damaged microstructure of muscle and declined the quality properties of pork.


Determination of 2-Dodecylcyclobutanone in Irradiated Products


Scientia Agricultura Sinica. 2009, 42(3):  989-995 .  doi:10.3864/j.issn.0578-1752.2009.03.030

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【Objective】 A novel method was developed for quantitative determination of 2-dodecylcyclobutanone which is one of the specific markers of irradiated fat containing foods. 【Method】 The method includes extraction and separation by silica gel column and analysis by capillary column gas chromatography-mass spectrometry (GC-MS). And the correlative parameters of the concentration of eluting solvent, extent of deactivated silica, and collecting time for separation and chromatogram detection were optimized. 【Result】The limit of detection was 0.005 mg?L-1, the recoveries were 75.9%-103.9% according to adding concentration of 0.1-2.0 mg?L-1, and the relative standard deviations were all below 5.7%. 【Conclusion】It showed that the silica gel column separation method has lower cost, higher separative efficiency, better stability, lower detection limit, and larger analytic sample as compared to the EN1785 method.


Structural Stability and Degradation Mechanisms of Anthocyanins


Scientia Agricultura Sinica. 2009, 42(3):  996-1008 .  doi:10.3864/j.issn.0578-1752.2009.03.031

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Anthocyanins in plants are water-soluble pigments exhibiting bioactive functions, which can be used in food, pharmacy and cosmetics, but they are not stable. Main factors having impacting anthocyanins, degradation mechanisms and stabilizing methods of anthocyanins are reviewed in this article, it is of significance to enhance investigations on the anthocyanins degradation and stability.


Advance in Research on ε-Polylysine Biosynthesis and Its Degradation and Application


Scientia Agricultura Sinica. 2009, 42(3):  1009-1015 .  doi:10.3864/j.issn.0578-1752.2009.03.032

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ε-polylysine (ε-PL) is a home-poly-amino acid characterized by the peptide bond between the carboxyl and ε-amino groups of L-lysine, which is stable at high temperature and under both acidic and alkaline conditions. Due to this antimicrobial activity, ε-PL is now industrially produced as a natural antiseptic agont by a fermentation process. ε-PL producers commonly possess membrane-bound ε-PL-degrading aminopeptidase, which might play a role in self-protection. It is unknow about the biosynthetic mechanism of ε-PL. This review focuses on the physicochemical properties of ε-polylysine, antimicrobial activities of ε-polylysine, isolation and screening of producing strains of ε-polylysine, biosynthesis and molecular genetics of ε-polylysine, fermentation process optimization of ε-polylysine, purification and application of ε-polylysine.


ANIMAL SCIENCE·RESOURCE INSECT

Effect of Feed Intake Level on Progesterone Secretion, Embryonic Survival and Related Gene Expression During Early Pregnancy in Gilts


Scientia Agricultura Sinica. 2009, 42(3):  1016-1023 .  doi:10.3864/j.issn.0578-1752.2009.03.033

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【Objective】 Sixty-three Landrace×Yorkshire crossbred gilts were allotted to three treatment groups of high (H, 2×maintenace), medium (M, 1.2×maintenace), and low (L, 0.6×maintenace) after mating to study the effects of feed intake level on progesterone secretion, embryonic survival and mRNA expression of fibroblast growth factor receptor 2 (FGFR2), progesterone receptor (PGR) gene in embryo and retinol binding protein 4 (RBP4), folate-binding protein (FBP), uteroferrin (UF) gene in embryo and endometria. 【Method】 Gilts were slaughtered on day 12, 25 and 35 of pregnancy, and serum, uterine flushings, embryos and endometria were collected. ELISA was adopted to determine the progesterone concentration in serum and uterine flushings, and RT-PCR was adopted to determine the genes expression in embryos and endometria. 【Result】 Progesterone concentrations in serum were lower in H than M and L (P＜0.01) on day 12, 25, 35 of pregnancy. In uterine flushings progesterone concentrations was lower in H than M and L (P＜0.05, P＜0.01) on day 12 of pregnancy, and no difference was observed on day 25 of pregnancy. Total viable embryos were greater in M than H group (P＜0.05) on day 12 of pregnancy. No difference was observed between H and L group (P＞0.05). Medium feed intake level significantly increased the relative mRNA expression of FGFR2 and RBP4 in embryo and RBP4, FBP, UF in endometria (P＜0.05) on day 12, 25 and 35 of pregnancy. 【Conclusion】 Medium feed intake level had greater total viable embryos during day 1-35 of pregnancy in gilts. High feed intake level and restricted feeding induced higher or lower secretion of progesterone and decreased expression of FGFR2, RBP4 in embryo and RBP4, FBP, UF in endometrial, resulting in detrimental effects on the development of early embryo.


Effects of Different Energy Levels on Nutrient Utilization and Serum Biochemical Parameters of Early-Weaned Calves


Scientia Agricultura Sinica. 2009, 42(3):  1024-1029 .  doi:10.3864/j.issn.0578-1752.2009.03.034

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【Objective】 The current study was performed to investigate the effects of different energy levels on nutrition’s digestion metabolism and serum biochemical parameters of early weaned calves. 【Method】 Twelve neonatal Holstein calves were selected and assigned randomly to receive 1 of 3 treatments that contained 18.51, 19.66 and 20.80 MJ?kg-1 gross energy, respectively (n=4 per treatment). Digestion and metabolism trials were carried out at 5 phases (12-20, 22-30, 32-40, 42-50, 52-60 d). At the same time, blood metabolites were determined at 21, 31, 41, 51 and 61 days of age prior to morning feeding. 【Result】The results indicated that either DM, OM, and GE apparent digestibility or N and P retention tended to increase as age advanced. The average value of DM、OM apparent digestibility and Ca retention in high-energy treatment were significantly lower than that of other groups (P＜0.05). There were no significant differences in the concentration of serum glucose, triglyceride and urea N among treatments, whereas a significant effect of age was found on the level of triglyceride (P＜0.05). 【Conclusion】 To sum up, the medium-energy milk replacer seems to be more benefical for GE digestibility and N utilization, whereas low-energy milk replacer appears to be more rewarding for Ca and P utilization.


cDNA Cloning of lactoferin and Its Characteristics in the Tianzhu White Yak


Scientia Agricultura Sinica. 2009, 42(3):  1030-1038 .  doi:10.3864/j.issn.0578-1752.2009.03.035

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【Objective】 Lactoferrin play a very important role in antibiosis, antivirus, antitumor and immunomodulation for mammal. The coding region of LF gene of Tian Zhu white yak was cloned, and it’s molecular characteristics were analyzed in the study. 【Method】 Using mammary tissue of Tian Zhu white yak as material, the cDNA sequence containing coding region of LF gene was cloned by RT-PCR. The cDNA of LF gene of Tian Zhu white yak obtained by cloning was compared to corresponding sequence of cow. Sequence alignment and evolutionary tree were analyzed on Tian Zhu white yak and other species. The structure and properties of LF gene of yak were predicted. 【Result】 The 2 344 bp length cDNA of LF gene of Tian Zhu white yak was cloned for the first time, and the length of coding region of LF gene is 2 124 bp, coding 708 amino acids. There are 15 mutations in the cDNA between yak and cow, 12 mutations being in coding region of LF gene, causing 4 amino acid mutations in LF protein. There is high homeology of LF protein among different species. Amino acid similarities of LF protein compared yak to cow, human, mouse, goat, sheep, pig, dog, horse, camel, chimpanzee and chicken are 99.4%, 69.5%, 63.5%, 92.4%, 91.5%, 72.9%, 69.1%, 72.6%, 75.3%, 69.5% and 50.9%, respectively. The evolutionary tree of all species complies with laws of evolution. Homogenous 3D model of LF obtained by predict shows that LF protein forms two similar and symmetrical leaves, namely N-leaf and C-leaf, connected by an Alpha helix impressionable to prolease, and LF protein presents “two-folium-ginkgo-shaped” structure. 【Conclusion】 The coding region of LF gene in Tian Zhu white yak was cloned for the first time, and its molecular characteristics were shown. This work has laid an important foundation for researches of genetic engineering and protein function of LF protein of yak.


Effects of Seeds of Different Oil Plants on Fatty Acid in Ruminal Fluid, Blood Plasma and Milk CLA of Lactating Cows


Scientia Agricultura Sinica. 2009, 42(3):  1039-1046 .  doi:10.3864/j.issn.0578-1752.2009.03.036

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【Objective】 Three multiparous Chinese Holstein lactating cows with permanent rumen cannula were used to study the effects of adding different kinds of seeds of oil plants on fatty acid in ruminal fluid, blood plasma and milk CLA of lactating cows.【Method】 Experimental design used a 3×3 Latin square design (sunflower seed group, flax seed group and rape seed group).【Result】The proportion of CLA, t11-C18﹕1, PUFA and LCFA in milk fat in sunflowerseed group was significantly improved than that in flaxseed group and rapeseed group when adding different kinds of oil plants seed (sunflower seed, flax seed and rape seed) in the diets (P＜0.05). The proportion of t11-C18:1 in blood plasma in sunflower seed group was significantly improved than that in flax seed group and rape seed group (P＜0.05). There was an insignificant difference among the averages in three mensuration for all kinds of fatty acids in ruminal fluid after intaking the diets supplied different plant oil (P＞0.05), but the proportion of the saturation fatty acids gradually increased, and the proportion of unsaturated fatty acids gradually decreased, the proportion of CLA in ruminal fluid was always low in different times. The coefficient of determination of t11-C18﹕1 in ruminal fluid and blood plasma for CLA in milk fat from cows were 0.841and 0.766, respectively.【Conclusion】The sunflower seed is the best in improving the milk CLA and the composition of fatty acid in lactating cows.


Purification and Enzyme Property Comparison of Two Genetic Variants of Yak Lactate Dehydrogenase-1


Scientia Agricultura Sinica. 2009, 42(3):  1047-1052 .  doi:10.3864/j.issn.0578-1752.2009.03.037

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【Objective】 The aim of the present study was to investigate at the level of the glycolytic enzyme lactate dehydrogenase (LDH) the molecular mechanism by which yak (Bos grunniens) adapts to low oxygen environment. 【Method】 Yak LDH isozymes were assayed by polyacrylamide gel electrophoresis. Total LDH activities in heart, liver and skeletal muscle of yak, yellow cattle and buffalo were analyzed by spectrophotometry method. Dye affinity chromatography and DEAE-Sephadex ion-exchange chromatography were employed to purify lactate dehydrogenase-1 (LDH1, which is composed of four H subunits) genetic variants from yak cardiac muscle in order to compare the enzyme properties. 【Result】 Electrophoresis revealed two genetic variants of yak LDH1. According to the difference of electrophoresis mobility, the fast-band LDH1 was named as LDH1-F and the slow-band as LDH1-S. The relative activity of the purified LDH1-F and LDH1-S were 21.4 U?mg-1 and 17.8 U?mg-1 protein, respectively, and both of them exhibited only one band on SDS-PAGE or native PAGE. Kinetic analysis showed that Michaelis constants (Km) value for NADH existed no difference between the two variants, but significantly higher than that of bovine LDH1. The Km value for pyruvate of LDH1-F was lower than that of LDH1-S. Yaks carrying LDH1-F and LDH1-S variants showed similar total LDH activities and LDH isozyme profiles in heart, liver and skeletal muscles. Total enzymatic activity assay showed that the activities of LDH in heart, liver and skeletal muscles of yaks were significantly lower than those of yellow cattle or buffalo. 【Conclusion】 Results from this experiment indicate that the two genetic variants of yak LDH1 have differences in their Km value, and the Km(NADH) value is higher than that of bovine LDH1. The total LDH activities in heart, liver and skeletal muscles of yaks were significantly lower than that of cattle. These changes in yak LDH1 are attributed to the adaptation to hypoxic environment.


Effects of Propanediol Supplementation on Lactation Performance and Blood Metabolites in Early Lactation Holstein Dairy Cows


Scientia Agricultura Sinica. 2009, 42(3):  1053-1060 .  doi:10.3864/j.issn.0578-1752.2009.03.038

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【Objective】 The objective of this study was to observe effects of propanediol supplementation on feed intake, lactation performance, changes in body weight, energy balance, concentrations of blood metabolites and urine ketones in early lactation Holstein dairy cows. 【Method】 Thirty-two multiparous Holstein dairy cows, blocked by lactation number, corrected milk yield in last lactation, and expected calving date, were allocated into four treatments in a randomized block experiment. Graded levels (0, 150, 300, and 450 ml?d-1) of 1,2-propanediol were supplemented in the four treatments. 【Result】 The results showed that average feed intake, milk yield, milk protein percentage, milk lactose percentage, milk dry matter percentage and feed efficiencies were not affected significantly by propanediol supplementation (P＞0.05). Milk fat percentage was significantly depressed by the 450 ml?d-1 treatment compared to the control (P＜0.05). Cows receiving 300 and 450 ml?d-1 propanediol had a higher plasma glucose and serum insulin, lower plasma NEFA and β-hydroxybutyrate than control (P＜0.05). 300 and 450 ml?d-1 propanediol supplementation decreased the contents of ketones in excreted urine compared to 150 ml?d-1 and control (P＜0.05). The energy balance was improved in cows receiving the 300 and 450 ml?d-1 propanediol treatments as reflected energy balance calculations, and a lower body weight loss compared to cows in control (P＜0.05). 【Conclusion】 Results of the present study indicated that propanediol supplementation is beneficial to early lactating cows in improving their energy balance status, the optimum dose is about 300 ml per cow per day.


VETERINARY SCIENCE

Streptococcus Isolated from Pig with High Fever Syndrome and Its Pathogenesis in Mice


Scientia Agricultura Sinica. 2009, 42(3):  1061-1068 .  doi:10.3864/j.issn.0578-1752.2009.03.039

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【Objective】 The objective of this experiment is to study on serotype, distribution of virulence gene and pathology of 11 strains of Streptococcus, isolated from heart blood, liver, lymph node, kidney, lung and other tissues of swine from different areas in Hubei province, China. 【Method】 ATB aotu-biochemical reaction test system, PCR typing, PCR test of virulence factor genes and animal test assays were used. 【Result】 PCR typing assay indicated that 11 strains were Streptococcus suis including 1 serotype 7 strains, 2 serotype 9 strains, 8 other serotype strains, no serotype 1 strain or serotype 2 strain. Result of the genotypes of their mrp, epf, sly, orf2, fbps, gdh and mouse virulence test showed that the genotypes of high virulence streptococcus were epf+orf2+ gdh+ fpbs+ strains without mrp+ sly+. Pathobiology showed that acute septicaemia, extensive blooding, lung fever, nephritis, congestion-dropsy in spleen and lymphatic abscess in all over the body, espciouly in the acute death. 【Conclusion】 Serotype 7 and serotype 9 existed in Hubei Province, streptococcus suis has some pathogenicity to ardent fever of swine.


Synthesis of Foot-and-Mouth Disease Virus Empty Capsids in Insect Cells Through Acid-Resistant Modification


Scientia Agricultura Sinica. 2009, 42(3):  1069-1077 .  doi:10.3864/j.issn.0578-1752.2009.03.040

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【Objective】 Foot-and-mouth disease virus (FMDV), a non-enveloped picornavirus, is sensitive to acidic conditions. At pH below 7 the icosahedral virus capsid dissociates into 12 pentamers. It is difficult to synthesize FMDV capsids in insect cells for the influence of low pH of insect cell medium. This study aimed at assembling empty capsid-like particles of AsiaⅠFMDV in insect cells by site-specific mutation of related amino acid residues to improve pH-stability of empty capsids. 【Method】 Two amino acid residues were site-specifically mutated to get modified P12A gene named mP12A (H140L and H143L in VP3). Both P12A and mP12A were then inserted into a transfer vector pFastBacTM Dual under the control of PH promoter, respectively. The 3C gene was placed under the control of P10 promoter. Two recombinant baculoviruses were generated by transfecting recombinant bacmids into Sf9 cells. High Five? cells were infected with two recombinant baculoviruses after multiplication in Sf9 cells. 【Result】 The target proteins were successfully expressed in insect cells by Western blotting, and that expressed capsid proteins could be processed by expressed viral 3C proteinase. The capsid proteins were mainly expressed near the inner membrane of insect cells as revealed by immunofluorescent assay, and that small amounts were released into culture medium as showed by sandwich ELISA. Furthermore, empty capsid-like particles were observed in lystate of High Five? cells infected with recombinant baculoviruses with mutated P12A under electron microscope. Whereas, many small-sized particles were observed in lystate of High Five? cells infected with recombinant baculovirus without mutation in P12A. The observed empty capsid-like particles were about 25-30 nm in diameter.【Conclusion】 This is the first report on the observations of empty capsid-like particles of FMDV in insect cells by microscopy. The potential utility of these recombinant, non-infectious, FMDV empty capsids for diagnostic and vaccine purposes is apparent.


Inhibition of Hepatic Drug-Metabolizing Enzymes in Mice During Streptococcus suis Type 2 Infection


Scientia Agricultura Sinica. 2009, 42(3):  1078-1083 .  doi:10.3864/j.issn.0578-1752.2009.03.041

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【Objective】 The effect of Streptococcus suis infection on both phase I (oxidative) and phase II (conjugative) microsomal enzyme activities was investigated in a well-characterized mice infection model. 【Method】 One hundred and twenty ICR mouse were used and divided randomly into infection groups (HA9801 group and SS2-H group) and negative control group. Each infected mouse was inoculated intraperioneally with one ml inoculum containing 1/2LD50 of each strain. One control group of mice was injected with one ml of sterile water. Body weight were weighed every day and the levels of liver microsomal protein, activities of cytP450, cytb5, NCCD, ERND, AND, AH and GSH-S-T were determined on 3, 5, 10, 15 days, respectively. 【Result】 Intraperitoneal injection of Streptococcus suis resulted in a time-dependent modulation of hepatic drug- metabolizing enzymes activities in mice. The activities of some drug-metabolizing enzymes of infected groups were significantly lowered compared with the negative control group during different infectious days, especially on 10 day post-infection, the lowest levels of cytP450, cytb5, NCCD, AH and ERND were achieved. While between the two infected groups, there was no obvious difference in the levels of all the enzymes tested except the levels of cytP450 and cyt b5 at the early stage. 【Conclusion】 The present study is the first to show that the infection by S. suis can decrease the activities of some hepatic drug-metabolizing enzymes in mice. If such a response also occurs in humans, this has the potential to produce serious complications with drug and endogenous substrate metabolism in patients with an infectious disease. The results also suggest that drugs with narrow therapeutic indices are dangerous during therapeutic schedule and should be administered with caution during infectious diseases caused by S. suis or other bacteria and viruses.


Effects of Monochromatic Light on Structure of Small Intestinal Mucosa in Broilers


Scientia Agricultura Sinica. 2009, 42(3):  1084-1090 .  doi:10.3864/j.issn.0578-1752.2009.03.042

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【Objective】 This study was conducted to investigate the effect of various monochromatic lights on small intestine mucosal morphology and structure of chicken broilers, and to provide morphological data about the mechanism of monochromatic light action on development of broilers. 【Method】 A total of 120 day-old Arbor Acre male broilers were exposed to red (660 nm, R), green (560 nm, G), blue (480 nm, B), and white (400-700 nm, W) light from light emitting diode (LED) lamps for 7 d, respectively. All light sources were equalized under 15 lx light intensity for 23 h daily. There were six replication pens for each light treatment and 5 birds per pen. Then at the 0 and 7 day old, 1 chick from each pen was randomly sampled and killed. The proliferating cell nuclear antigen (PCNA) positive cells expression in jejunal gland epithelium of broilers, and intestinal goblet cells numbers, villous height, mucosa thickness, as well as the ratio of height villous to intestinal crypt (V/C) of broiler duodenum, jejunum and ileum were examined by histochemistry staining and immunohistochemistry methods. 【Result】Compared with other light groups, GL group increased obviously the related detection index at 7 day old. Compared with RL, GL increased broilers jejunal gland epithelium cells proliferation (115.5%, P＜0.05); GL increased the number of goblet cells of the duodenum, jejunum, and ileum of broilers by 71.8%, 35.8% and 27.2%, respectively (P＜0.05); increased the villus height of the duodenum, jejunum, and ileum of broilers by 207.9, 93.9 and 63.9μm, respectively (P＜0.05); increased the mucosal thickness of the duodenum, jejunum, and ileum of broilers by 262.2, 184.6 and 185.6μm, respectively (P＜0.05); as well as the ratio of V/C of the duodenum, jejunum, and ileum of broilers by 18.9%, 54.5% and 77.8%, respectively (P＜0.05). 【Conclusion】 The small intestine mucosal structure would be improved to an extent in chicken broilers when they were illuminated with green light at early growth stage (0-7 d) of broiler reared under 15 lx light intensity, accordingly improving the absorption function of small intestine, and accelerating broilers growth.


Isolation and Comparison of Low-Molecular-Weight Metal Binding Protein in Testis and Liver of Rabbits Treated with Cadmium


Scientia Agricultura Sinica. 2009, 42(3):  1091-1099 .  doi:10.3864/j.issn.0578-1752.2009.03.043

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【Objective】 To isolate and compare the small molecular weight proteins in liver and testes of rabbits challenged by cadmium to recognize the mechanism of sensitivity of testes against cadmium. 【Method】 Ten male Chinchilla rabbits at the age of 6-month-old were injected subcutaneously by cadmium at different doses, nine days later, the animals were killed, livers and testes of the rabbits were removed and iced at ―70℃. Livers and testes were minced and homogenized and ultracentrifugated, the supernatant of livers and testes were collected, respectively, and a part of supernatant were passed a column with Sephadex G-75 and the column was calculated by marker. The wash solution from column were fractioned, proteins in each tube were measured by ultraviolet scanning, and cadmium and zinc in tubes were measured by atomic absorption spectrophotometry. 【Result】 There was a peak of protein in liver supernatant induced by cadmium measured at 254nm. The molecular weight of the protein within the peak was 6.0-10.0 kD, and heat-stable, cysteine was rich, could not be extracted by alcohol, no aromatic amino acid in the protein. The ratio of Cd/Zn was 4.7:2. The components of this protein was much similar as metallothionein. However, there was no peak of testis supernatant measured at 254nm, and no similar protein in testis supernatant of animals dosed by cadmium. 【Conclusion】 The reason of male rabbit testes are sensitive to cadmium challenge is that tastes can not produce matallothionein which can against heavy metals from damage.


RESEARCH NOTES

Resistance Monitoring of the Striped Stem Borer, Chilo suppressalis, (Lepidoptera: Pyralidae) in Jiangsu and Zhejiang Provinces in 2007


Scientia Agricultura Sinica. 2009, 42(3):  1100-1107 .  doi:10.3864/j.issn.0578-1752.2009.03.044

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【Objective】 This study monitored the resistance to nine insecticides in different populations of the striped stem borer, Chilo suppressalis (Walker), collected from Zhejiang and Jiangsu provinces in 2007, sensitivity of Donghai population to 20 insecticides was evaluated as well. The results have provided useful information for the resistance management strategies of the striped stem borer. 【Method】 Topical application method was used in the experiment. 【Result】 The results showed that Rui’an, Cangnan and Gaochun populations developed extremely high levels of resistance to monosultap (164.8 to 248.1 fold), and Lianyungang and Donghai populations had moderate levels of resistance (18.1 and 25.1 fold respectively). The resistance to triazophos varied greatly in five populations, from having extremely high levels of resistance in Rui’an, Cangnan and Gaochun populations (204.8 to 1395.2 fold), to being susceptible in Lianyungang and Donghai populations (1.8 and 3.1 fold respectively). Moderate resistance levels to fipronil were monitored in Rui’an and Cangnan populations (12.3 and 26.8 fold respectively), while other three populations tested were susceptible to fipronil (1.3 to 2.9 fold). A low level of resistance to abamectin was detected in Cangnan population (7.6 fold), and other four populations tested remained susceptible to abamectin (0.9 to 2.0 fold). Rui’an and Cangnan populations had moderate or high levels of resistance to cyhalothrin, fenvalerate, beta-cypermethrin, beta-cyfluthrin and deltamethrin (22.1-115.6 fold), while Lianyungang, Donghai and Gaochun populations had low or moderate levels of resisitance to cyhalothrin, fenvalerate, beta-cypermethrin, beta-cyfluthrin and deltamethrin (31.2-5.3 fold). The sensitivity to 20 insecticides was evaluated in Donghai population of Jiangsu province. The toxicity sequence of 20 insecticides to the population was as follows: abamectin, emamectin benzoate＞ivermectin, beta-cyfluthrin＞butene-fipronil, fipronil, fenvalerate, cyhalothrin, deltamethrin, beta-cypermethrin, tebufenozide≥phoxim, quinaphos, furan-tebufenozide, triazophos, pyridaphenthion＞malathion, chlorpyrifos, silafluofen＞monosultap. 【Conclusion】 The resistances to insecticides varied significantly with populations of the striped stem borer, so the resistance management strategies should be proposed according to the local situation.


Changes in Soil Respiration and Microbial Functional Diversity of Paddy Soil Under Different Fertilizer Amendments


Scientia Agricultura Sinica. 2009, 42(3):  1108-1115 .  doi:10.3864/j.issn.0578-1752.2009.03.045

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【Objective】 Microbiological aspects of soil quality under different fertilizer amendments were investigated to clarify the effect of over-use fertilizer, especially, chemical fertilizer on the agro-ecosystem sustainability. 【Method】 Soil microbial biomass, respiration intensity and functional diversity in Hapli-Stagnic Anthrosols were investigated by an incubation experiment with different fertilizer applications. 【Result】 During the incubation, the content of microbial biomass carbon was different under different fertilization: chemical fertilizer incorporated with 2% straw or 2% manure＞CK (no fertilization) and chemical fertilizer incorporated with 0.5% straw or 0.5% manure＞chemical input alone; Case in microbial biomass nitrogen was similar: chemical incorporated with 2% straw＞CK, chemical incorporated with 2% manure or 0.5% straw＞chemical alone. Respiration intensity and metabolic quotient declined with different fertilizer rates along the incubation. Soil respiration under chemical incorporated with 2% straw or 2% manure amendments were higher during the incubation and the patterns of metabolic quotient was: chemical plus 2% straw or manure and maximum level of chemical input alone＞chemical plus 0.5% straw or 0.5% manure and minimum chemical input＞CK and normal level chemical input. BIOLOG analysis indicated that over-use of fertilizer, especially chemical fertilizer decreased the potential of carbon utilization and functional diversity of soil microorganism. 【Conclusion】 As sensitive indicators, soil microbial biomass, respiration intensity and functional diversity corresponded to the changes in soil quality. Over-use of chemical fertilizer attenuated biological aspects of soil quality and interrupted the sustainability of agro-ecosystem.


Construction of Tall Fescue Chloroplast Expression Vector and Transient Expression in Tall Fescue Chloroplasts by Detecting GFP


Scientia Agricultura Sinica. 2009, 42(3):  1116-1122 .  doi:10.3864/j.issn.0578-1752.2009.03.046

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【Objective】 The study was made to detect transient expression of the tall fescue chloroplast expression vector in chloroplasts of tall fescue and provide a strong foundation for genetic engineering drought-resistant tall fescue through chloroplast transformation in the future. 【Method】 Firstly, the homologous fragment 16S/trnI-trnA/23S was amplified for site-specific integration from tall fescue chloroplast genome by PCR. Secondly, the yeast trehalose synthase gene tps1 was placed under the 16S rRNA promoter (Prrn) from rice and 3’ regulatory region of psbA gene from tobacco to construct tps1 cassette, which is ligated with phosphinothricin acethltransferase gene bar cassette and fused gene nptII-gfp cassette consisting of neomycin phosphotransferase gene nptII and green fluorescent protein gene gfp. Finally, the three expression cassettes were inserted into the homologous fragment to obtain tall fescue chloroplast stable expression vector gTKGB. Then vector gTKGB was introduced into young leaves of tall fescue through particle bombardment. Expression of GFP was analyzed under confocal laser scanning microscope. 【Result】 Gene accession numbers of 16S/trnI-trnA/23S cloned from tall fescue were DQ490947, DQ490948, DQ490949 and DQ490950. Chloroplast expression vector gTKGB was introduced into young leaves of tall fescue, and strong green fluorescence was observed in the chloroplasts of bombarded leaves under confocal laser scanning microscope. 【Conclusion】 Tall fescue chloroplast stable expression vector gTKGB can be used in tall fescue chloroplast transformation.


Balb/c Mouse Model of Neosporosis and the Development Process with Infection of Neospora caninum


Scientia Agricultura Sinica. 2009, 42(3):  1123-1128 .  doi:10.3864/j.issn.0578-1752.2009.03.047

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【Objective】 The main purpose of the current study was to construct a Balb/c mouse model of N. caninum infection in both acute phase and subacute phase, and to study the development process of N. caninum in mouse. 【Method】 In this research, Balb/c mice were divided into two groups, each group was inoculated with 8×106 tachyzotie /mice and 5×105 tachyzotie /mice, respectively. Mice were euthanatized at different time points. Tissues of heart, liver, lung, brain and muscle were used for pathological, immuohistochemical and microbiological examination. 【Result】 Both of the two groups were characteristed with vacuolar degeneration of the hepatocyte, hemorrhage in the lung and other visceras, nonsuppurative inflammation and necrosis in the brain. Cysts could be found in the brain 16 d after inoculation, and no cyst was observed in other tissues. The result of PCR revealed that N. caninum specific gene Nc-5 could be detected in lung and heart 12 h after inoculation, 8 d in brain. Nc-5 gene still could be detected in liver, lung and brain 25 d post infection. A tissue predilection to liver, lung and brain was found according to the results of histopathological examination. And male mouse showed more susceptivity to N. caninum than female did. 【Conclusion】 Acute and subacute N. caninum infection models were successfully built up in BALB/c mice. The spread of tachyzoites among the tissues was traced in the mouse model, and the dynamic process of N. caninum infection in BALB/c mice was discovered.