Table of Content

01 October 2014, Volume 47 Issue 19

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES

Development of a Wheat Variety Identification System Based on Fluorescently Labeled SSR Markers

ZHENG Yong-sheng, ZHANG Han, WANG Dong-jian, SUN Jia-mei, WANG Xue-mei, DUAN Li-li, LI Hua, WANG Wei, LI Ru-yu

Scientia Agricultura Sinica. 2014, 47(19):  3725-3735.  doi:10.3864/j.issn.0578-1752.2014.19.001

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【Objective】In this study, a wheat variety identification system based on fluorescently labeled SSR markers was developed to provide a high-throughput DNA profiling means for identification of Chinese wheat varieties.【Method】Information on SSR markers which have been mapped to the 21 pairs of wheat chromosomes was collected. Highly polymorphic markers among China released wheat varieties were screened by PCR amplification followed by denaturing polyacrylamide gel electrophoresis. Markers selected were labeled at the 5′ end of forward primer using one of the four fluorescent dyes: TAMRA, HEX, ROX and 6-FAM. DNA Analyzer was employed to detect the fluorescently labeled PCR-amplified fragments. Peak patterns, polymorphism and distribution on linkage maps of these markers were evaluated and makers with simple peak pattern, high polymorphism and even distribution were chosen. The sizes of the alleles at each marker locus were determined and reference variety corresponding to each allele was assigned. A wheat variety identification system based on these selected fluorescently labeled SSR markers was developed.【Result】Eight morphologically diversified China released wheat varieties were PCR amplified using 2 438 mapped SSR markers for screening polymorphic and stably amplifying primer pairs. Two hundred and sixty primer pairs met the criterion. After further screening, 130 out of 260 mapped SSR markers were chosen based on distribution on wheat linkage map, PCR amplification stability and polymorphism revealed in 48 wheat varieties released in China. These 130 markers were labeled with four fluorescent dyes and further screened using DNA Analyzer. Finally, 42 SSR markers with relatively even distribution on linkage map, simple peak patterns, high polymorphism and stable PCR amplification were chosen. Sizes of alleles at each marker locus were determined using DNA Analyzer. Each allele was named according to its DNA Analyzer determined size. Reference variety corresponding to each major allele was assigned to eliminate systemic error arising between batches of samples. To profile wheat varieties cost-efficiently, 42 markers was divided into seven groups according to the fluorescent type of the markers and range of allele sizes at each marker locus. As many as 9 markers can be analyzed in the same capillary tube. DNA profiling was conducted on 1 625 wheat cultivars using this system. A total of 434 alleles were detected. The number of alleles varied between 3 and 23, with an average of 10.3 per locus. The PIC values ranged from 0.291 to 0.829, with an average of 0.610. A DNA profile database of China released wheat varieties was constructed based on the DNA profiling information of 1 625 wheat cultivars.【Conclusion】 A wheat variety identification system based on fluorescently labeled 42 SSR markers was established, which will provide a high-throughput DNA profiling alternative for identification of wheat varieties.

Characterization of Two LEA Genes and Their Response to Abiotic Stresses in Wheat

LIU Lu-lu, CHEN Lei, ZHANG Chun-yan, SHI Rui-jie, REN Jiang-ping, MENG Fan-rong, YIN Jun, LI Yong-chun

Scientia Agricultura Sinica. 2014, 47(19):  3736-3745.  doi:10.3864/j.issn.0578-1752.2014.19.002

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【Objective】 Drought is one of the major factors significantly affecting the growth and yield of wheat crop. In this study, two LEA genes were characterized and their response to abiotic stresses, such as drought, salt, heat and cold, were investigated. The biological functions of these two LEA genes during the stress tolerance of wheat plant were also discussed, which would provide important information for the application of these two LEA genes in wheat molecular breeding for the improvement of stress tolerance. 【Method】RT-PCR technology was used in the cloning of LEA genes and proper bioinformatic methods were applied for characterizing the LEA genes and their deduced proteins. The gene expression patterns responding to various abiotic stresses were analyzed by using the real time qRT-PCR. 【Result】 Two wheat LEA genes TaLEA4 and TaLEA5, with complete coding sequences, were cloned and sequencing analysis showed that these two LEA genes encode 180 and 163 amino acids, with a predicted molecular weight of about 18.8 and 16.9, pI of 5.6 and 7.2, respectively. Genomic structuring showed that one intron with 100 bp in length was detected in both of the cloned genes. Amino acid sequence analysis indicated that both of the wheat LEA proteins were rich in polar amino acids, which accounted for about 71%, and so they showed high hydrophilic. Conserved domain analysis indicated that both of TaLEA4 and TaLEA5 included a typical LEA_4 (pfam: 02978) domain and belong to LEA_4 subfamily of LEA proteins. Protein structural analysis demonstrated that the region of helixes accounted for 96.7% and 96.3% of TaLEA4 and TaLEA5, respectively. Both of these LEA proteins can form a bow shape structure, and one binding site of ligand PEV (C₃₉H₇₈NO₈P) was found in TaLEA4, while two of that were detected in TaLEA5. The expression profiling revealed that both of the wheat LEA genes were induced by plant hormone ABA, and the expression level of TaLEA4 was significantly higher than that of TaLEA5. Under the abiotic stresses of drought, salt and heat, TaLEA4 was greatly up-regulated, while TaLEA5 was only induced by drought-stress and its expression level was significantly lower than that of TaLEA4. Both of these LEA genes did not response to cold-stress. Interestingly, the expression level of TaLEA4 was obviously higher in root tissues than that in leaf tissues under drought- and salt-stresses, while that was higher in leaves under heat-stress, which might be due to that the osmotic stresses were directly sensed by roots, but leaf tissues were more sensitive to heat-stress. 【Conclusion】 Both of the wheat LEA genes TaLEA4 and TaLEA5 belonged to the LEA_4 subclass LEA family, they showed high hydrophilic and were involved in the ABA dependent stress-responding gene network. It was deduced that TaLEA4 might play important roles during the processes of responding to the drought, salt and heat stresses, while the TaLEA5 was only involved in the drought-stress responding and its function might be weaker than that of TaLEA4.

Creating Low Grain Protein Content Barley by Suppressing B-hordein Synthesis Through RNA Interference

LI Jing-wen, ZHANG Zheng-ying, LING Li-jun, LI Shu-jie

Scientia Agricultura Sinica. 2014, 47(19):  3746-3756.  doi:10.3864/j.issn.0578-1752.2014.19.003

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【Objective】The objective of the study is to develop a transgenic barley germplasm with low protein content under high nitrogen dosage by RNA interference (RNAi) to specially down-regulate the expression of grain B-hordein genes.【Method】The conserved sequence of B-hordein gene (gi|18928) in barley was cloned based on recombinant PCR and confirmed by sequence analysis. The RNAi expression vector pBract207-zz-gp4, aiming at silencing barley grain B-hordein gene, was reconstructed by Gateway technology and transformed into barley cultivar Golden Promise by Agrobacterium-mediated method using immature embryo. Transgenic plants were obtained after resistance selection, differentiation and regeneration, and then the transgenic progeny was screened by PCR, Southern blotting, and semi-quantitative RT-PCR. Protein content were determined by near-infrared spectroscopy analyzer, SDS-PAGE analysis and nitrogen management.【Result】Two B-hordein fragments (Gp4 and Gp5 ) were identified in this study. Sequence analysis revealed that the length of the sequence was 349 bp. Cluster analysis showed that the highest degree of identity (92%) was between Gp4/Gp5 and other known B-hordein. And also the highest degree of identity (98%) was between conserved sequence and the mRNA of B-hordein. The hpRNA silencing fragment was designed on the basis of Gp4 in sense and antisense orientation with the sequence of the i18 and iv2 intron as spacer region between the repeats and driven by Ubi promoter. Twenty-three T₁ transgenic lines were obtained by Agrobacterium-mediated method and eleven transgenic plants of T₁ generation were confirmed by PCR of selectable marker gene and sense and antisense reaction of B-hordein. Southern blot analysis showed that RNAi constructs were successfully integrated into the plant genomes in T₂/T₃ generations. Further, six transgenic barley positive lines of T₃ progeny were selected by random and analyzed by semi-quantitative RT-PCR after flowering. The results showed that the development expression of B-hordein genes of six T₃ transgenic plants was significantly lower than that of non-transgenic plant, especially on the 20 DAF (Day after flowering), which was not only reduced by 28.19%-55.19% compared with the control, but also was the lowest one among the developing periods after flowering. Then protein content analysis of eight lines of T₁/T₃ selected by random demonstrated that the total protein content decreased significantly compared with the control. At the same time, SDS-PAGE revealed that the ration of B-hordein of different transgenic barley plants or lines decreased compared with that of non-transgenic plants, that of three lines of them reduced significantly and the degree of decreasing was 49.42% on average. In addition, RNAi-20, which had low protein content, was used to carry out nitrogen management analysis. The result revealed that the protein content of the line significantly decreased compared with that of control in high nitrogen dosage (HN₂ and HN₃) treatment, and which gradually lower than the control with the change of nitrogen application from HN₂ to HN₃. SDS-PAGE demonstrated that the B-hordein content of transgenic barleys decreased and electrophoretic band changed.【Conclusion】The RNAi technology can effectively inhibit the expression of B-hordein gene in barley grain, decrease the B-hordein content of RNAi lines, reduce the protein content under high nitrogen dosage, improve the quality of barleys, and create new superordinary germplasm resources of barley.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Investigation of Endosperm Cell Development of Different Rice Varieties

LI Dong-liang, LI Xiao-gang, GU Yun-jie, WANG Zhong

Scientia Agricultura Sinica. 2014, 47(19):  3757-3768.  doi:10.3864/j.issn.0578-1752.2014.19.004

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【Objective】The objective of this research is to clarify the difference of rice endosperm of different varieties during the development process.【Method】Four rice varieties, Nipponbare, Yangdao 6, Wuyunuo16 and Yangfunuo 4 were used as the experimental materials, and their caryopsis development days precisely were precisely recorded and their caryopsis development was closely observed. Starch accumulation and physiological activities were observed by I₂-KI and TTC staining. Structure changes of rice starch endosperm cells were observed by applying spur resin embedding and semi-thin sectioning, and light microscopy. The ultrastructure and element analysis of full ripe rice caryopsis were observed under scanning electron microscope and EDS. 【Result】Four stages occurred in caryopses development of all the rice varieties tested: formation stage, milky stage, dough stage, and full maturity stage. Compared to development stages of the endosperm, formation stage runs in parallel to the coenocyte and cellurization stages, which are the initial stage of endosperm formation; milky stage corresponds to the differentiation stage of endosperm cells, while the last two stages, dough and full maturity stages, are equivalent to that of the endosperm maturity stage. The shape of two rice sub subspecies, japonica and indica, varies significantly. Of the four rice varieties tested, two indica varieties showed a relatively faster growth rate than two japonica cultivars; Wuyunuo16 and Yangfunuo 4 had almost identical growth rate of dry matter accumulation. Nuclei of starch endosperm cells degenerated with the extension of amyloplasts, while amyloplasts were still enlarging and growing. Amyloplasts in the endosperm cells were compound granules, and their development pattern possibly affected a certain quality of endosperm. Amyloplast in endosperm cells was spherical or elliptic in shape during early developmental stage, but turns to polyhydric during the late development stage. Two glutinous rice cultivars tended to have a loosely stacked endosperm and as a result, caused the appearance to be less transparent. Aleurone cell volume of two indica cultivars were bigger than japonica ones, mature aleurone cells were rich in minerals like P, K, Mg, and S. Cultivar Yangfunuo 4 had some single amyloplast detected in aleurone cells and, showed a higher level of O element content.【Conclusion】The development of starchy endosperm is autonomous and is independent of the existence of endosperm nuclei. The two non-glutinous rice cultivars have a more densely packed endosperm, The types of starch contents in cells from scutellum, pericarp and endosperm are relatively distinct in two glutinous rice cultivars, which stresses the disparate set of mechanism of starch synthesis between maternal tissue, endosperm and embryo.

TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY

Characteristics of Air Moisture and the Effects of High Air Moisture at Booting Stage on Grain Yield of Wheat in Jianghan Plain

WANG Xiao-yan, ZHAO Xiao-yu, CHEN Hui-fu, WANG Xiao-ling, XIONG Qin-xue

Scientia Agricultura Sinica. 2014, 47(19):  3769-3779.  doi:10.3864/j.issn.0578-1752.2014.19.005

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【Objective】 Jianghan Plain is one of the most important wheat producing areas in Hubei Province, and high relative air moisture is a major limitation on wheat grain yield. Under the conditions of global climate change, relative air moisture increased as well as air temperature. This research was carried out to study the effect of increasing relative air moisture on wheat grain yield, in order to provide a theoretical basis for promoting wheat production in Jianghan Plain. 【Method】The research was carried out based on the analysis of air moisture characteristics in Jianghan Plain from 1983-2013. During the experiment, air moisture was increased to 100% by artificial chamber for 10 days at booting stage of wheat, and the normal air moisture treatment was used as CK, samples were taken at 5 d, 10 d after high air moisture control, and 5 d, 15 d after recovery from all treatments. The effects of high air moisture on photosynthetic characteristics, SPAD value in flag leaf and top 3rd leaf, course of leaf senescence, root activity, plant height, and accumulation of dry matter, grain yield and yield components were studied. 【Result】Air moisture from 1983 to 2013 in Jianghan Plain fluctuated with a 2-5-year circle, and the average air moisture was over 80% during booting stage in most of the years. The air moisture at booting stage in Jianghan Plain in 2011-2012 and 2012-2013 was high, and averaged 77.4% and 79.1%, respectively. Increasing air moisture at booting stage decreased SPAD value in flag leaf and top 3rd leaf, and the difference was not significant at 5 d later, but significant at 10 d later. When air moisture returned to normal, there was a difference in leaf SPAD value between high air moisture treatment and CK, but still inferior to CK. The effects of high air moisture on photosynthetic rate in flag leaf was in agreement with SPAD value. High air moisture reduced photosynthetic rate in flag leaf and removing high air moisture treatment, the difference between high air moisture treatment and CK reduced. Increased air moisture to 100%, MDA content in flag leaf and top 3rd leaf increased, suggesting that high air moisture quickened senescence in leaf. Further analysis showed that root activity reduced as well. Removing high air moisture treatment, the difference of MDA in leaf and root activity between CK and high air moisture treatment reduced, but was still significant, which mean the effects of high air moisture on the above index was not reversal. With the increasing of relative air moisture, the amount of dry matter and grain yield decreased. Compared with CK, at maturity, dry matter of high air moisture treatment decreased by 5%, and grain yield decreased 10.0%, respectively. For further analysis, the decrease of grain yield was attributed to spike weight and grain amount per spike, decreased by 6.0% and 4.2%, respectively, at maturity compared to CK. 【Conclusion】Under the conditions of this experiment, high air moisture at booting stage decreased SPAD value and photosynthetic rate in flag leaf, quickened membrane peroxidating, reduced root activity, and induced to the decrease of biomass and grain yield decreased as well.

Remote Sensing Prediction of Winter Wheat Protein Content Based on Nitrogen Translocation and GRA-PLS Method

LI Zhen-hai, XU Xin-gang, JIN Xiu-liang, ZHANG Jing-cheng, SONG Xiao-yu, SONG Sen-nan, YANG Gui-jun, WANG Ji-hua

Scientia Agricultura Sinica. 2014, 47(19):  3780-3790.  doi:10.3864/j.issn.0578-1752.2014.19.006

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【Objective】Prediction of grain protein content (GPC) can provide effective decision-making supporting information for acquisition and processing of high quality wheat. The objective of the study is to demonstrate the feasibility of remote sensing monitoring of wheat grain protein content based on nitrogen translocation theory, and its expansibility between regional and annual level. 【Method】Field experiments of four winter wheat cultivars by four nitrogen applications in Beijing during 2012-2013 growing seasons were carried out for model building. Firstly, the two main sources of grain nitrogen accumulation and their relationships were analyzed based on nitrogen translocation theory and agronomy parameters modeling. The nitrogen remobilization from vegetative organs to grain was considered as the key point, while the nitrogen uptake from the root absorption during grain filling stage was simply calculated as the nitrogen remobilization from vegetative organs to grain multiplied by a factor. Mechanism of predicting GPC with leaf nitrogen content (LNC) at the flowering stage was clarified through integrating agronomy parameters modeling. Meanwhile, the temperature factor was considered. Secondly, twenty-four vegetative indices were selected according to the good relationship between vegetative indices and leaf nitrogen content, and remote sensing estimating of LNC was established by using grey relational method and partial least squares method (GRA-PLS). Therefore, a prediction model of GPC with remote sensing was established. 【Result】The results showed that the selected five vegetative indices according to grey relational grade were mND705, NDVIcanste, Readone, DCNI and NDCI. For the LNC estimating, the determination coefficient (R2) and corresponding to root mean square error (RMSE) of modeling and validation results were 0.859, 0.257% and 0.726, 0.063%, respectively. Estimation of LNC has good robustness by using GRA-PLS method. The R2 and RMSE of predicted and measured GPC of modeling and validation results were 0.726, 1.30% and 0.609, 1.19%, respectively. The results indicated that it was available to estimate GPC by integration model of nitrogen translocation theory and GRA-PLS method. 【Conclusion】The integration model with explanatory and expansibility could explain the theory of “why the LNC is used to predict GPC”, achieved prediction of grain protein content between regional and annual levels, and had a wide range of potential applications.

PLANT PROTECTION

Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Tropomyosin Gene in Laodelphax striatellus Fallén

XU Qiu-fang, CHEN Qing-qing, NI Hai-ping, LI Shuo, ZHANG Jin-feng, ZHOU Yi-jun

Scientia Agricultura Sinica. 2014, 47(19):  3791-3798.  doi:10.3864/j.issn.0578-1752.2014.19.007

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【Objective】 By screening Laodelphax striatellus Fallén (small brown planthopper, SBPH) cDNA library, it was found that the tropomyosin (Tm) could interact with RBSDV P10 protein. To provide the antibody of Tm protein for analyzing the Tm gene’s function in the process of RBSDV and SBPH interaction, the Tm was cloned and expressed in prokaryotic system. Tm protein was purified and the polyclonal antibody was obtained by immunizing the rabbit. 【Method】 The complete open reading frame (ORF) of Tm from SBPH was determined by comparing the sequence to the highly homologous Tm in other species. The total RNA was extracted and the Tm ORF was cloned by reverse transcription polymerase chain reaction (RT-PCR). Tm was then inserted into the pMD-18T for sequencing. The nucleic acid sequence and the amino acids encoded by this ORF were analyzed by DNAstar software. Tm ORF was inserted into the expression vector pET-32a (+) by EcoR V and BamH I digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3) and expressed under the induction with 0.4 mmol?L^-1 IPTG for 4 h. Tm fusion protein was detected by SDS-PAGE. The E. coli BL21 (DE3) expressing the Tm fusion protein was collected and crushed by ultrasonic. The supernatant and the precipitate were collected, respectively, and SDS-PAGE was used to analyze the expression of the fusion protein. The soluble fusion protein was purified by Ni-NTA Agarose, washed with 100 mmol?L^-1 imidazole and dialyzed with 0.01 mol?L^-1 PBST. The purified fusion protein was used to produce polyclonal antibody via immunizing rabbit. The titer of rabbit anti-Tm antiserum was evaluated by indirect ELISA. To estimate the specificity of this antibody, the purified fusion Tm protein and the total proteins from SBPH were separated by SDS-PAGE and were tested by Western blotting assays using the prepared antibody.【Result】The sequence analysis results showed that the length of complete SBPH Tm ORF was 852 bp. It was amplified by RT-PCR and the sequencing result showed that the length of Tm ORF was 852 bp, encoded 283 amino acids, and its theoretical molecular weight was 32.6 kD. The blast results indicated that the encoded protein was conserved among different species. Tm was inserted into the vector pET-32a (+) and expressed in E. coli BL21 (DE3). The SDS-PAGE result showed that the Tm fusion protein of approximately 55 kD was mainly expressed as a soluble protein and there was also a small amount in inclusion body. The soluble fusion protein was purified and the polyclonal antibody was obtained by immunizing the rabbits using the purified fusion protein. The ELISA assay showed that the rabbit anti-Tm antiserum had a good sensitivity with the titer higher than 1﹕409 600. Using the polyclonal antibody to detect the purified Tm fusion protein and the total SBPH protein, specific band (55 and 37 kD, respectively) could be observed, suggesting that this antibody has strong specificity. 【Conclusion】 The ORF of SBPH Tm was cloned firstly. The Tm fusion protein was expressed in E. coli BL21 (DE3), purified successfully and used for preparing the antibody. The rabbit anti-Tm antiserum with high titer was obtained.

Cloning and Spatio-Temporal Expression of the Thermoreceptor Gene Painless in Pea Aphids (Acyrthosiphon pisum)

WEI Jin-jin, CAO De-pan, YANG Ting, WANG Gui-rong

Scientia Agricultura Sinica. 2014, 47(19):  3799-3809.  doi:10.3864/j.issn.0578-1752.2014.19.008

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【Objective】 The objective of this study is to clone acandidate thermoreceptor gene Painless in the antenna of pea aphids, Acyrthosiphon pisum, and to illustrate the protein structure encoded by this gene, further to characterize the expression profiles of this gene across developmental stages and in different tissues. 【Method】 Based on the sequence of Painless from Drosophila melanogaster, full-length sequence of the ApisPainless from AphidBase was predicted by bioinformatic analysis, andthe gene was cloned with cDNA template obtained from pea aphid antennae and specific primers were designed by Primer premier 5.0 software using RT-PCR. After determining the consistency of cloned sequence with predicted one by DNAMAN software, the protein structure of ApisPainless was depicted through the online tool SMART (simple modular architecture research tool) according to the analysis of ankyrin repeats at N-terminus and transmembrane domains. Subsequently, the Clustal Omega software was introduced to conduct alignment and analysis of ApisPainless with DmelPainless isforms (A, B and C). The phylogenetic tree was constructed for the analysis of insect TRP (transient receptor potential) channel superfamily, especially for the subfamily TRPA, which includes 4 members of TRPA1, Painless (ApisPainless), Pyrexia and Water witch, by comparing the length of their protein sequences and ankyrin repeats at N-terminus. Quantitative real-time PCR was applied to characterize the relative expression levels of ApisPainless at different developmental stages (1st-4th instar nymphs and adults) and in different tissues (antennae, heads, legs, and thoraxes and abdomens). 【Result】 The ApisPainless was successfully predicted and cloned, with a complete CDS of 2 832 bp, encoding 943 amino acids. Protein structure analysis indicated the ApisPainless had 8 ankyrin repeats and 6 transmembrane domains. The ApisPainless was more similar to the DmelPainless isoform A with the similarity of 42.3%, and clustered to the insect Painless cluster based on the results of multiple sequence alignment and phyl ogenetic analysis, respectively. TRPA1 was composed of about 1 200 amino acids, making it the longest among the TRPA subfamily, followed by Water witch, Pyrexia and Painless, with 920-1 000 amino acids. In addition, there were 8 ankyrin repeats observed at N-terminus in Painless, while 9 in both Water witch and Pyrexia, and 15-16 in TRPA1. ApisPainless was expressed at the highest level in the 1st instar nymphs, even though it was universally expressed across all the stages. Tissue-specific expression analysis elucidated that ApisPainless was expressed in a whole body-expressing manner, but mostly expressed in antennae, followed by legs. 【Conclusion】ApisPainless obtained in this study was clustered to Painless, which is a member of insect TRPA subfamily. The ApisPainless is highly expressed in antennae and legs of pea aphids, which implies that it may be involved in noxious heat sensation, mechanosensation and gustatory detection as its homolog Painless in D. melanogaster.

Parasitism Potential of Three Indigenous Trichogramma Species on the Eggs of Soybean Pod Borer (Leguminivora glycinivorella)

LIN Ying, SONG Li-wei, ZANG Lian-sheng, RUAN Chang-chun, SHI Shu-sen

Scientia Agricultura Sinica. 2014, 47(19):  3810-3816.  doi:10.3864/j.issn.0578-1752.2014.19.009

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【Objective】The soybean pod borer (SPB), Leguminivora glycinivorella Matsumura, is the most serious pest of soybean production in North China. The objective of this study is to determine the parasitism potential of three indigenous Trichogramma species (T. leucaniae, T. ostriniae and T. chilonis) on their natural host eggs of SPB.【Method】The experimental population life tables on SPB eggs were established for each Trichogramma species reared on the eggs of rice moth (Corcyra cephalonica), and the reproductive property parameters of the net reproduction rate (R₀), mean generation time (T), intrinsic rate of natural increase (rm), finite rate of increase (λ), number of host eggs parasitized per female, average longevity of female and emergence rate were compared and analyzed. 【Result】 Three Trichogramma species all had the peak of fecundity at the first day after emergence, the percentages of eggs laid in total number were 68.1%, 69.1% and 64.0% for T. chilonis, T. ostriniae and T. leucaniae, respectively. With longevity increasing, T. chilonis and T. ostriniae exhibited a significant decrease in fecundity. Ten percent individuals of T. leucaniae survived for 7 days without any nutrient offered. Per female of T. leucaniae averagely parasitized 10.5 eggs of SPB (22.5% of total parasitization) 3 days after emergence. The R₀, T, rm and λ of T. leucaniae, T. chilonis and T. ostriniae on SPB eggs were 24.75, 23.78 and 21.13; 9.46, 10.34 and 10.37 d; 0.3393, 0.3064 and 0.2941; 1.4040, 1.3585 and 1.3419, respectively. However, there were no significant differences in longevity of female wasps and emergence rate in SPB eggs among the three Trichogramma species. Generally, the reproductive property parameters (R₀, T, rm and λ) of T. leucaniae were more ideal than those of T. chilonis and T. ostriniae. 【Conclusion】 Based on the parameters of life table on their natural hosts of SPB, the present study indicated that T. leucaniae could be a suitable biological control agent for controlling SPB.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION

Changes in Yellow Paddy Soil Organic Carbon Fractions Under Long-Term Fertilization

ZHANG Li-min, XU Ming-gang, LOU Yi-lai, WANG Xiao-li, QIN Song, JIANG Tai-ming, LI Zhong-fang

Scientia Agricultura Sinica. 2014, 47(19):  3817-3825.  doi:10.3864/j.issn.0578-1752.2014.19.010

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【Objective】 Soil organic carbon represents an high heterogeneity. Because of different chemistry and existing forms, soil organic carbon fractions show different decomposibility and fertility functions. Therefore, investigating soil organic carbon fractions is important for better understanding the stabilization and fertility mechanims of soil organic carbon. This study was conducted in order to examine the effects of long-term fertilization on yellow paddy soil organic carbon fractions and to guide proper fertilization. 【Method】 An 18-year paddy field experiment was conducted to investigate the effects of long-term fertilization on soil organic carbon fractions and allocation, and also to discuss the soil organic carbon saturation phenomenon by quantifying the relationship between soil carbon levels with annual carbon inputs under different fertilizer treatments. The designed treatments were: no fertilizer control (CK), chemical fertilizer (NPK), organic manure (M), low application rate of manure combined with chemical fertilizer (0.5MNPK) and high application rate of manure combined with chemical fertilizer (MNPK). The average annual carbon inputs ranged from 0.87 (in CK treatment) to 6.02 t·hm^-2·a^-1 (in MNPK treatment). 【Result】 The results showed that compared with CK, soil total organic carbon content was significantly increased by 10% under the NPK treatment, and by 24%-46% under the manuring treatments (0.5MNPK/M/MNPK). Compared with CK, soil free coarse particulate carbon, chemically-protected silt carbon and biochemically-protected silt carbon contents were significantly increased under the NPK treatment, and soil free coarse particulate carbon, physically-protected carbon, chemically-protected silt and clay carbon and biochemically-protected silt and clay carbon contents were significantly increased under the manuring treatments (0.5MNPK/M/MNPK). The proportion of physically-protected carbon to total carbon was higher under the treatments of 0.5MNPK, M and MNPK than under the CK and NPK trteatments. Soil free coarse particulate carbon concentration showed a significant linear increase with mean annual carbon input. Soil chemically-protected and biochemically-protected carbon concentrations and soil total carbon content showed a significant “saturation curve” increase with mean annual carbon input. 【Conclusion】 The data from the experiment indicate that the combined manure and chemical fertilizer is the best option for increasing the paddy soil organic carbon content, and that the relatively passive soil organic carbon fractions (chemically-protected, biochemically- protected) and total organic carbon existed saturation phenomenon and saturation limit under current conditions.

Optimization of Nitrogen Fertilizer Recommendation Technology Based on Soil Test for Winter Wheat on Weibei Dryland

CAO Han-bing, WANG Zhao-hui, SHI Yuan-chao, DU Ming-ye, LEI Xiao-qing, ZHANG Wen-zhong, ZHANG Lu, PU Yue-jian

Scientia Agricultura Sinica. 2014, 47(19):  3826-3838.  doi:10.3864/j.issn.0578-1752.2014.19.011

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【Objective】Nitrogen (N) is the main nutrient factor limiting wheat yield increase on dryland. Unreasonable application of N fertilizer not only makes it difficult to increase wheat yield, but also leads to increased nitrate N (NO₃^--N) residual in soil, enhanced N losses, and reduced N use efficiency. Therefore, it is of great significance to optimize the method of N fertilizer recommendation and solve the problem of unreasonable N fertilizer application for the sustainable production of wheat on dryland.【Method】 Based on balancing N output from soil by crop production and aimed at stabilizing crop yield, improving soil fertility and regulating NO₃^--N residual, the present method for Recommendation of N fertilizer Based on Monitoring NO₃^--N in 1.0 m Soil (RNBM, N application rate = N required for the target crop yield + Fertilizer N losses + Safety threshold of NO₃^--N in 1 m soil at harvest or before sowing (55.0/110.0 kg·hm^-2) - N input from environment - N input with straw retention - N input with seed - Soil N mineralization during winter wheat growing season - NO₃^--N in 1m soil at harvest or sowing) was optimized to be as: N application rate = N required for the target crop yield + Safety threshold of NO₃^--N in 1 m soil at harvest or before sowing (55.0/110.0 kg·hm^-2) - NO₃^--N in 1 m soil at harvest or before sowing. Using the optimized RNBM, two-year field experiments on 30 plots were carried out in six counties on Weibei Arid Tableland in Shaanxi Province. 【Result】 As the result of chronically unreasonable application of N fertilizer due to the lack of normative N fertilizer recommendation methods, it was found that in this region, the NO₃^--N residual in 1.0 m soil at sowing varied largely among different sites from 34.2 kg·hm^-2 to 708.4 kg·hm^-2, with the average to be 165.2 kg·hm^-2 and 17 of 30 sites higher than 110.0 kg·hm^-2. Nitrogen application rates determined by the optimized RNBM ranged from 30.0 kg·hm^-2 to 247.3 kg·hm^-2, with the average of 128.4 kg·hm^-2, which was 25.2% lower compared to that (171.6 kg·hm^-2）of the farmer’s practice (FP). The averaged wheat grain yield by the optimized RNBM and FP was respectively 5 658 kg·hm^-2 and 5 489 kg·hm^-2, grain N concentration was 20.8 g·kg^-1 and 20.3 g·kg^-1, showing no significant difference. Compared to FP, the optimized RNBM significantly increased N use efficiency (NUE) and partial fertilizer productivity of N (PFPN) by 24.0% (from 46.3% to 57.3 %) and 130.1% (from 34.9 kg·kg^-1 to 80.3 kg·kg^-1), respectively. At harvest time, NO₃^--N residual in 1.0 m soil under FP was found averaged to be 70.6 kg·hm^-2 (17.4 kg·hm^-2 to 203.4 kg·hm^-2), and that under the optimized RNBM was only 51.4 kg·hm^-2 (15.6 kg·hm^-2 to 113.9 kg·hm^-2), a little bit lower than the expected target of 55.0 kg·hm^-2. During the rainfall intensive summer fallow, NO₃^--N leached from 0 to 100 cm soil layer under the optimized RNBM was observed to be reduced by 47.9% compared to the FP. 【Conclusion】Therefore, the optimized RNBM is able to be flexibly used to determine and effectively regulate the N fertilizer rate, stabilize wheat grain yield, increase NUE and PFPN, and minimize NO₃^--N residue and leaching in dryland soil.

Effect of Water and Nitrogen on Yield and Nitrogen Utilization of Winter Wheat and Summer Maize

Lü Li-hua, DONG Zhi-qiang, ZHANG Jing-ting, ZHANG Li-hua, LIANG Shuang-bo, JIA Xiu-ling, YAO Hai-po

Scientia Agricultura Sinica. 2014, 47(19):  3839-3849.  doi:10.3864/j.issn.0578-1752.2014.19.012

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【Objective】Water and fertilizer are the major limiting factors in crop production. At present, the utilization of water and nitrogen is not reasonable, not only giving rise to a waste of water resources, but also making a threat to the environment. In order to discuss a reasonable collocation of water and nitrogen in winter wheat and summer maize rotation system, grain yield and nitrogen uptake and utilization were analyzed based on a 5-year experiment carried out in North China Plain (NCP).【Method】In the winter wheat and summer maize rotation system, the split plot design was employed with main plot of water (limiting water and suitable water), sub-plot of nitrogen fertilizer ((0+0), (60+60), (120+120), (180+180), (240+240) and (300+300) kgN·hm^-2, respectively, for wheat and maize rotation system), and three replicates in each sup-plot. Limiting and suitable water was employed according to winter wheat and summer maize irrigation system in the piedmont plain of North China, and the irrigation treatments included two, limiting water treatment irrigated 1 water at jointing stage and suitable water treatment irrigated 2 water at jointing and blossom stages for winter wheat, while for summer maize which irrigation time was confirmed according to the different years precipitation, and 1 water before sowing, 2 water before sowing and 12 leaf, and 3 water before sowing, 12 leaf and blossom stages.【Result】When water supply was more than 609.5 mm for wheat and maize, limiting factors of effect of N fertilizer was not water, and the contribution of N fertilizer to yield was larger; while water supply was lower, the effect of N fertilizer was restrained, and the contribution of water to yield was larger. Water and N had an obvious coupling effect, the N application rate for getting a high yield were 134.8 kg·hm^-2 and 126.4 kg·hm^-2 for wheat, and 176.8 kg·hm^-2 and 127.2 kg·hm^-2 for maize under limiting and suitable water conditions, respectively. When N fertilizer rate was 300 kg·hm^-2 and 240 kg·hm^-2, respectively, under limiting and suitable water for the two seasons crop, shoot N was amounted to a higher value, but N fertilizer rate was more than 60 kg·hm^-2 for maize under two water conditions and for wheat under limiting water, and 120 kg·hm^-2 for wheat under suitable water conditions, straw residual N significantly increased. Partial productivity of N fertilizer (PPNF) decreased with the increase of nitrogen application rate, and with the years passing, PPNF decreased significantly, especially the treatments of nitrogen application rate at 60 kg·hm^-2 for wheat season, which PPNF decreased rapidly. The soil organic matter and total nitrogen content did not decrease when nitrogen application rate reached 240 kg·hm^-2 of limiting water and 120 kg·hm^-2 of suitable water treatments for winter wheat and summer maize rotation system.【Conclusion】The major factor for limiting fertilizer efficiency was water under the condition of limiting water, therefore the amount of nitrogen application should be reduced in dry years. For winter wheat and summer maize rotation system, the N application rates for getting maximum yields were 311.6 kg·hm^-2 and 253.6 kg·hm^-2 respectively, and which grain yields were 16 127.5 kg·hm^-2 and 17 272.9 kg·hm^-2, respectively, under limiting and suitable water conditions.

HORTICULTURE

Effects of Carbonized Straw on the Nitric Oxide Formation and Nitrate Metabolism in Apple Roots and Its Root Zone Soil

YAN Li-juan, YANG Hong-qiang, SU Qian, MEN Xiu-jin, ZHANG Wei-wei

Scientia Agricultura Sinica. 2014, 47(19):  3850-3856.  doi:10.3864/j.issn.0578-1752.2014.19.013

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【Objective】The soil nitrate is not only fruit tree’s nitrogen nutrition but also the latent environmental pollution factor. The carbonization straw, a product of incomplete combustion of crop straw, is applied into the soil to improve soil physical and chemical properties. This study focused on the changes of nitric oxide (NO) and nitrate metabolism related enzymes in the roots and the root zone soil of apple tree applied with carbonized corn straw. The purpose was to reveal the regulating effects of carbonized straw on the nitrate metabolism of apple roots and it’s root-zone soil, so as to provide a theoretical basis for the control of soil nitrate transformation and the improvement of orchard soil management.【Method】In the spring, the carbonized corn straw and soil was mixed according to the mass ratio of 0.5%-8.0% (w/w) and loaded to the clay pots, and then the 3-year-old ‘Fuji’ apple trees (rootstock for Malus hupehensis Rehd) in similar growth were transplanted to the pots. After 120-190 d of transplantation, the change of NO formation and the activity of nitrate reductase (NR) in roots and root zone soil was investigated, the activity of nitric oxide synthase (NOS) in roots and activity of hydroxylamine reductase (HyR) and the nitrification intensity in root zone soil were measured regularly.【Result】It was significantly different that the responses of nitric oxide formatiom and nitrate metabolic in roots and root zone soil to the applicationrate of carbonized corn straw to the soil. The rate of NO formation, the activity of NR and NOS in root increased significantly when carbonized straw applied into the soil at 1%-2% (w/w), and they all decreased significantly while the application rate reached 8%. The NR activity in root increased significantly when carbonized straw applied into the soil at 0.5%-2.0% (w/w) and dropped significantly while the application rate at 4.0%-8.0%. It was more significant after 120-170 d of treatment that the effects of carbonized straw on the nitric oxide formation and nitrate reduction in root. The rate of NO formation, the activity of NR and NiR in the soil of root zone all increased significantly when carbonized straw applied into the soil at 1%-2% (w/w). But the rate of NO formation decreased significantly when the carbonized straw applied over 1%; the activity of NR and NiR in root zone soil dropped significantly when the application rate over 2.0%. It was 120-155 d after treatment that the effects of carbonized straw on the nitric oxide formation and nitrate reduction in root were most significant. The application of carbonized straw promoted the nitrification in the soil of root zone. The nitrification strength of root zone soil reached maximum when the dose of carbonized straw applied at 2%, and soil HyR activity increased gradually with the application rate of carbonized straw increased from 0.5% to 8.0%. It was more significant after 120-170 d of treatment that the effects of carbonized straw on the nitrification of root zone soil.【Conclusion】The application of carbonized straw into soil influenced the NO formation in apple roots and the nitrate metabolism in root zone soil significantly. The reduction of nitrate to NO in apple roots and root zone soil is promoted by carbonization straw in the lower application rate of 0.5%-1.0%. The nitrification in the soil of root zone was promoted by carbonization straw in the higher application rate of 2.0%-4.0%. It is most significant that the nitrate reduction promoted by carbonization straw in the soil of root zone when the application rate was 0.5%. The rate of NO formation in roots was the highest when the application rate of carbonization straw was 1.0%. The nitrification strength of root zone soil was the highest when the application rate of carbonization straw was 1.0%.

Effects of Pseudomonas fluorescens Inoculation on Root Respiration and Seedling Growth of Cherry

ZHANG Peng, QIN Si-jun, ZHOU Wen-jie, Lü De-guo, MA Huai-yu

Scientia Agricultura Sinica. 2014, 47(19):  3857-3865.  doi:10.3864/j.issn.0578-1752.2014.19.014

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【Objective】 Rational utilization of soil microbial for improving soil environment is helpful to promote plant yield and quality. The adaptation of cherry root is bad, so it is sensitive to the change of soil environment. The objective of the experiment is to discuss the mechanisms of Cerasus sachalinensis (Fr.Schu) seedlings responding to soil sterilization and inoculation of Pseudomonas fluorescens, thus providing an academic and practical basis for regulating the rhizosphere soil.【Method】In order to find the influence of inoculation P. fluorescens on plant root function, C. sachalinensis(Fr.Schu) seedlings were used as materials to measure the correlative indexes of root respiration metabolism.【Result】Compared with soil sterilization, glycolysis, tricarboxylic acid cycle, and cytochrome oxidase pathways were also the dominant respiratory pathways from 5 d to 50 d after C. sachalanensis seedlings were subjected to CK and SS+Pseu treatments. On 5th d, there was no significant difference in total respiration rate of the roots between SS+Pseu treatment and CK, however, on 20 and 35th d, in SS+Pseu treatment, the total respiration rate of the roots was lower than that in the CK, while the total respiration rate of the roots in the SS+Pseu treatment was higher than that in CK on 50th d. On 5th d, the activities of PK, MDH, G-6-PDH, SDH were significantly increased in the SS+Pseu treatment, however, the activity of PFK was significantly decreased. On 20th d, the activity of PK was significantly increased in the SS+Pseu treatment, however, the activities of PFK and G-6-PDH were significantly decreased. On 35th d the, activity of PFK was increased by 127.0%, On 50th d the activity of G-6-PDH was increased by two times. Though, there were some differences between CK and SS+Pseu treatment, however, these differences were not significant. In SS+Pseu treatment the stem diameter and leaves number were significantly increased by 9.3% and 16.7%. Inoculation with P. fluorescens was helpful to plant growth.【Conclusion】Inoculation with P. fluorescens could influence plant growth and root function. In general, 20 d after the end of continuous inoculation (six times) with the bacterial liquid whose OD value was 0.4 could change the rate of respiration pathways, key enzyme activities via influence the function of roots, and then promoted plant growth.

Effects of Calcium on Mitochondrial Function in Malus baccata Borkh. Roots Under Changing Temperature

SU Hong, LI Li-jie, MA Huai-yu, Lü De-guo, SUN Jing

Scientia Agricultura Sinica. 2014, 47(19):  3866-3873.  doi:10.3864/j.issn.0578-1752.2014.19.015

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【Objective】 The responding mechanisms of mitochondrial function in Malus baccata Borkh. roots and the regulating role of calcium under changing temperature stress were studied, and the results would be a theoretical foundation for further study of the physiological mechanism of M. baccata Borkh. roots under such kind of temperature stress. 【Method】 The seedlings of M. baccata Borkh. were used as materials and treated with or without 2% CaCl₂ solution, to investigate the characteristics of mitochondrial function, such as mitochondrial membrane permeability transition (MPT), mitochondrial membrane potential (?ψm), super oxygen anion () generation rate, electron transfer pathway activity and contribution rate (cytochrome pathway, CP; alternative pathway, AP), CaM content, and CaN content under violently changing temperature (5℃→20℃→0℃). 【Result】 In control, MPT,  generation rate and MDA content in M. baccata Borkh. roots obviously increased under violently changing temperature, ?ψm decreased at the same time. Compared with the control, CaCl₂ treatment maintained the MPT at a relatively stable level during temperature changing. At the cooling stage, ?ψm significantly rose in CaCl₂ treatment, meanwhile ,the generation rate of  gradually dropped.CaCl₂ treatment kept MDA content at a relatively lower level. The activity and contribution rate of CP in control descended as temperature going up, and thenincreased with temperature going down, while AP activity kept in ascending under temperature treatment. Comparing with the control, CaCl₂ remarkably increased the activities of CP and AP at 20℃. Moreover, the varying trends of AP activity and contribution rate were opposite to that of CP. In control, CaM content consistently increased, while CaN content decreased firstly and then increased. The content of CaM in CaCl₂ treatment declined with temperature going up, and then increased as temperature going down, while CaN content steadily decreased.【Conclusion】Violently changing temperature inhibited the function of mitochondria in roots and increased electronic leak, finally exacerbated the membrane lipid peroxidation. The extent of membrane lipid peroxidation could be effectively alleviated by CaCl₂ treating through stabilizing MPT, adjusting CP and AP activities. At warming stage, CaCl₂ treatment alleviated temperature stress by the signal mode of decreasing CaM content and increasing CaN content, and at cooling stage, by the signal mode of increasing CaM and decreasing CaN content.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Structures and Functions Prediction and Expression Profiles of Calreticulin as Calcium Binding Chaperones in Chicken

WANG Li-li, LI Nan, CAO Chang-yu, GONG Du-qiang, YU Dong, WANG Wei, LI Jin-long

Scientia Agricultura Sinica. 2014, 47(19):  3874-3882.  doi:10.3864/j.issn.0578-1752.2014.19.016

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【Objective】 The aim of the current study is to reveal the evolutionary relationships, and investigate the protein structure and functions and the expression profiles of calreticulin (CRT) as a key Ca²⁺ binding molecular chaperone within the endoplasmic reticulum (ER) of chicken.【Method】The nucleotides and amino acids of CRT in 12 species of vertebrates recorded in Gene bank were analyzed for evolutionary relationships by Laser Gene, and the structures and functions of CRT protein in chicken were predicted by bioinformatics, and the expression profiles of CRT in 30 organizations of chicken was analyzed by real-time PCR.【Result】Results of homology analysis showed that compared with the other 11 species of nucleotide sequences of CRT gene in chicken, gallus gallus and oryctolagus cuniculus had the highest nucleotide sequence homology, which was 78.7%, in addition, gallus gallus and oncorhynchus mykiss had the lowest homology, which was 70.5%. In the homology of amino acid sequences, the relationship between gallus gallus and crotalus adamanteus cadam is the closest by 85.0%, and the furthest relationships with gallus gallus is oncorhynchus mykiss which was 69.0% in amino acid sequence, besides, the homology of gallus gallus with cricetulus griseus, macaca mulatta, homo sapiens, oryctolagus cuniculus, sus scrofa, bos taurus, and xenopus (silurana) tropicalisis relatively close to almost above 80.1%. The protein structure and function prediction revealed that the CRT of chicken was constitute with 404 amino acids, and had a relative molecular mass of 46.8802 kD and a theoretical isoelectric point of 4.41, moreover, the negative charge is 102 amino acid residues and the positive charge amino acid residues is 53. The molecular formula of the CRT was C₂₀₇₄H₃₁₀₇N₅₄₃O₆₈₄S₉. The CRT of chicken had 22 hydrophobic regions, which of the C end and the N end had a high hydrophobic, while the hydrophilic of C end is stronger than that of N, and the protein formed a secondary structure as α_1(7-17)-α_2(22-25)-β₁₍₂₆₎- β_2(38-41)-β_3(50-54)-β_4(69-70)-β_5(75-82)-β_6(92-99)-β_7(110-114)-β_8(129-133)-β_9(144-151)-β_10(171-178)-β_11(183-187)-β_12(314-322)-β_13(326-332)-α_3(337-348)-α_4(350-375)- α_5(377-379)-α_{6(395- 401)}. CRT belongs to the transmembrane proteins and secreted protein, has signal peptides. The enzyme classification of CRT was EC 3.2.1.55 or EC 3.4.24.68. Tissue expression assay indicated that the gene of CRT expressed widely in chicken tissues, in which the lleum, glandular stomach and duodenum were highly more than the kidney (control) by 20 times. 【Conclusion】The sequence of nucleotide and amino acid in chicken CRT is relatively conserved in 12 vertebrate species. CRT is a transmembrane secretion protein and acidic protein. It belongs to the alpha-N-Arabia-glucosidase or Tentoxilysin, which catalyzed the hydrolysis  of the end non-reducing α-L-arabinofuranosidase residues in the terminal of α-L-arabinoside, which effected the α-L- arabinofuranosidase, Arabic glycans containing (1,3) and/or (1,5) glucosidic bond, Arab xylan and arabinogalactan. CRT combined specifically with the carbohydrate molecules and Ca²⁺, which could monitor glycoprotein assembly and folding and Ca²⁺ regulation, and plays an important role in the digestive system.

Development of a Colloidal Gold Immunochromatographic Technique for Simultaneous Detection of Quinolones and Gentamicin in Milk

LI Xiang-mei, WANG Zhan-hui, XIAO Xi-long, WANG Zhao-peng, WEN Kai, WU Xiao-ping, XIA Xi, WU Jin-xiao, JIANG Hai-yang

Scientia Agricultura Sinica. 2014, 47(19):  3883-3889.  doi:10.3864/j.issn.0578-1752.2014.19.017

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【Objective】Quinolones and gentamicin are highly effective and broad-spectrum antibacterial drugs. They have significant antibiotic effects on gram-negative and gram-positive bacteria, and are widely used in agriculture in China. Because these two types of drug residues in foods of animal origin may cause harm to human health, therefore, in order to protect the consumers’ health, it is necessary to develop a detection method for simultaneous monitoring these two types of drugs residue level in food. A colloidal gold immunochromatographic method was developed for the simultaneous detection of 13 quinolones and gentamicin residues in milk.【Method】In this study, based on the quinolones and gentamicin monoclonal antibodies, the colloidal gold particles were prepared by sodium citrate reduction method, and mixed labeled with same ratio of these two types of monoclonal antibodies. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was investigated. Meanwhile, the coating condition of these two types of antigens was selected. A colloidal gold rapid test strip was developed to simultaneously detect 13 quinolones and gentamicin residue in milk on these bases, and the test strip using the principle of direct competition.【Result】The results showed that the method can simultaneously detect 13 quinolones and gentamicin. These 13 quinolones include enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, enoxacin, oxolinic acid, marbofloxacin, fleroxacin, orbifloxacin, danofloxacin and lomefloxacin. The test strip has no cross-reaction to other quinolones such as sarafloxacin, difloxacin, sparfloxacin and pazufloxacin, etc. At the same time, it has no cross-reaction to other aminoglycosides such as streptomycin, neomycin and kanamycin, etc. The limit of detection was estimated to be 20 ng·mL^-1 in milk for both the 13 quinolones and gentamicin, since the detection test line on the strip test completely disappeared at this concentration. The detection limit for milk sample of these two types of drugs fully meets the detection limit requirements of China. Samples were detected directly without treatment, and the entire testing process was completed within 5 min.【Conclusion】A parallel analysis of quinolones and gentamicin in 60 blind raw milk samples conducted by HPLC-MS/MS showed comparable results to those obtained from the strip test. All positive samples were detected while false positive and false negative phenomenon did not appear with this screening method. The results demonstrated that the developed method is suitable for the onsite determination of quinolones and gentamicin residues in a large number of samples. Since this method provides only qualitative and semiquantitative results, the determined positive samples should be further confirmed by more sensitive methods such as HPLC-MS/MS.

Effect of Salbutamol on the Pupation, Emergence, Life Span, Antioxidative Capacity and DNA Damage in Drosophila melanogaster

SUN Bing, LIN Xin-da, ZHANG Chun-hong

Scientia Agricultura Sinica. 2014, 47(19):  3890-3897.  doi:10.3864/j.issn.0578-1752.2014.19.018

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【Objective】The objective of this study is to explore the effect of salbutamol on the growth, development, life span, anti-oxidative capacity and DNA damage in Drosophila melanogaster, to further provide the basis for improvement of the more effective detection method.【Method】The 1st-instar larvae were fed with salbutamol and the pupate and emergence of flies were recorded, the pupation rate and the eclosion rate as well as pupation and eclosion time were counted. One day after eclosed, adult flies were fed up with normal medium and salbutamol medium, in which the concentrations of salbutamol were 0.48, 2.4,12 and 24 μg·mL^-1. Fifty flies for each medium(1﹕1 for male﹕female) and the experiment was repeated for 4 times. The number of lived flies was recorded every day. The medium was replaced every three days until all of the flies died. The 50 % lethal time, average longevity and average maximum time were recorded and calculated, respectively. The 3-day old adult flies which feed up with salbutamol or minimum medium were collected. Tissue homogenate of 30 adult flies (1﹕1 for male﹕female) were then prepared and centrifuged at 2 500 r/min and within 10 minutes. The enzyme activity kit were used to detect the enzymatic activities of SOD and CAT as well as the content of MDA. The 3-day old adult flies were used to detect the DNA damage of Drosophila mid-gut by the single cell gel electrophoresis (SCGE). The DNA damage of Drosophila cells were observed under a fluorescence microscope and photos were taken, then the images were analyzed by comet analysis software Cometscore^TM.【Result】Statistical analysis showed that the pupation rate and the eclosion rate decreased with the increasing concentration of Salbutamol, but there was no significant difference (P＞0.05). Statistical analysis also showed that the time of pupation and eclosion among the salbutamol treatments had no significant difference (P＞0.05). The enzymatic activities of SOD and CAT of the 3-day old adult flies were assayed, and the content of MDA of the 3-days old adult flies was also measured. The results showed that the content of MDA was increased significantly (P＜0.05) and the enzymatic activity of CAT was reduced significantly (P＜0.05) when the concentration of the salbutamol was increased. The antioxidant system was destroyed and the enzymatic activity of SOD increased significantly (P＜0.05). As the action of antioxidant system make the life-span of fly become longer. DNA damage detection experiments suggested that salbutamol caused the DNA damage of the fly cells. The SCGE experiment showed significant differences in the tail length (TL), olive tail moment (OTM), and tail DNA percentage (%DNA in tail) between flies grown in salbutamol treated medium and control.【Conclusion】Salbutamol has no influence on the pupation and eclosion of flies. It plays an important role in antioxidant system. Salbutamol restrains the enzymatic activity of CAT but increased enzymatic activity of SOD. High level of salbutamol can increase the life-span of flies significantly. It causes the DNA damage of the fly cells but not arouse the apoptosis.

RESEARCH NOTES

The Development and Identification of New Peanut Germplasm Materials with High Oleic Acid and Medium Pod

LI Li, HE Mei-jing, CUI Shun-li, HOU Ming-yu, CHEN Huan-ying, YANG Xin-lei, WANG Peng-chao, LIU Li-feng, MU Guo-jun

Scientia Agricultura Sinica. 2014, 47(19):  3898-3906.  doi:10.3864/j.issn.0578-1752.2014.19.019

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【Objective】 Germplasm resources is the base for crop breeding. The development of new high oleate peanutmaterials with various phenotypic characteristics is great significant for high oleic peanut breeding program. 【Method】Conventional variety Baisha 1016 and high oleic peanut line CTWE were used and their mutation types were determined by amplification and sequencing results of partial ORF of ahFAD2A and ahFAD2B. The oleic trait genotypes of two parents were confirmed by allele-specific PCR (AS-PCR). Oleic acid and linoleic acid content of each generation plants were detected by near infrared reflectance spectroscopy (NIRS) combined with gas chromatographic(GC) analysis. Double selection pressures of yield and oleic acid were used in 241 F_2:3 family lines and F₄ seeds, and the identification of phenotypic and AS-PCR were conducted on new selected materials.【Result】The amplification and sequencing results of partial ORF of ahFAD2A and ahFAD2B showed that the mutated sites of CTWE were the same as that of mutant F435 with a base substitution in (G448A) for ahFAD2A, and a single base pair insertion (442insA) for ahFAD2B while Baisha 1016 maintained wild type state on the same both sites. The AS-PCR analysis demonstrated that the parental lines differed in both genotypes and oleate content for ahFAD2A and ahFAD2B. The genotype of CTWE was o1_lo1_lol₂ol₂, and Baisha 1016 was OL₁OL₁OL₂OL₂, which was consistent with the sequencing results. Double selection pressures of yield and oleic acid were used in 241 F_2:3 family lines and 20 high oleate lines were selected, which the pods weight per plant was 30% higher than that of control of Jihua 2. Thirteen low-oleic family lines were eliminated by NIRS single particle detection on randomly picked 5 kernels of 20 high yield family lines respectively. All plants containing at least 1 kernels of high oleic acid type were detected, and four different genotypes in 7 lines were confirmed by AS-PCR. Four homozygous high oleate germplasm materials with various phenotypic characteristics and the yield 30% higher than that of control Jihua 2 were ultimately selected through field investigation and indoor test and ascertained by gas chromatography. Their 100-seed weights were between 54.00-75.29 g and belonged to medium-pod type. O/L ratio ranged from 28.66-41.19. The plant types of 11-3 and 13-2 were creeping type 3. They were both slight pod beak and slight pod reticulation. But the pod trait of 11-3 was bee waist shape with slight pod constriction, that of 13-2 was common shape with none pod constriction. The plant type of 15-2 was upright type and pod trait is common shape, none pod constriction, none pod beak and medium pod reticulation. The plant type of 16-1 was creeping type 2, pod trait was common shape, none pod constriction, slight pod beak and slight pod reticulation. 【Conclusion】 Four new medium-pod high oleic germplasm materials with different phenotypic characteristics were selected by AS-PCR assisted selection.

Response of Root Architecture, Nutrients Uptake and Shoot Growth of Malus hupehensis Seedling to the Shape of Root Zone

FAN Wei-guo, YANG Hong-qiang

Scientia Agricultura Sinica. 2014, 47(19):  3907-3913.  doi:10.3864/j.issn.0578-1752.2014.19.020

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【Objective】Root system architecture is an important morphological characteristic of root system and an important factor affecting plant nutrient absorption. The root morphology, architecture and root nutrient uptakes can be influenced by cultivation containers with specific shapes. The Malus hupehensis (Pamp) Rehd. is a good apple stock and sensitive to the environment of root zone. The project aimed to investigate the effect of different root zone shapes on shoot growth, parameters of root system architecture and nutrient uptakes of roots in M. hupehensis, for providing a basis for analyzing and creating ideal root architecture and improving the nutrient absorption and plant growth of apple stock. 【Method】 Different clay pots were used to set three root zones with different shapes, such as the ‘root zone of deep-narrow’ was set by the clay pot with the sizes of pot-diameter less than the pot height, the ‘root zone of shallow-wide’ was set by the clay pot with the sizes of pot diameter more than the pot height, the ‘root zone of equal height and diameter’ was set by the clay pot with the sizes of pot-diameter equal to pot height. In the early spring, the three different clay-pots were filled by similar nutrient soil with the same volume, respectively, and then the seedlings of M. hupehensis (Pamp) Rehd. with similar growth and roots were transplanted into the pots, respectively. The root morphology root architecture, shoot growth, root activity and root nutrient absorption rate were investigated after eight months of transplanting. 【Result】 The shoot growth, root architecture and nutrient absorption characteristics of the seedlings were significantly different after 8 months of growth in the three root zones with different shapes. When seedlings grown in the ‘root zone of deep-narrow’, the root top ratio was the biggest, the first and the second lateral roots were the shortest, the number of the first lateral root was more; the root activity and the potassium-uptake rates of the roots were the lowest, and the root uptake rates of phosphorus, calcium and zinc were lower and the iron-uptake ability of the roots was higher. When seedling grown in the ‘root zone of shallow-wide’, the shoots were thicker, longer and more leaves, and shoots grew the fastest, and the root/top ratio was smaller; the first and the second lateral roots were the thickest and longest, the number of the second lateral roots and fine roots was more; the potassium-uptake rate was the highest, the uptake rate of phosphorus and zinc were higher, the calcium-uptake rate of the roots were lower, and the iron-uptake ability of the roots was the lowest. When seedling grown in the ‘root zone of equal height and diameter’, the shoots were thinner, shorter and less leaves, and shoots grew the slowest, the root/top ratio was smaller; taproot was the thinnest, the number of the first lateral root and fine roots was less; the root activity was the highest, the uptake rates of calcium and zinc of the roots were higher, and the uptake rates of phosphorus, potassium and iron were lower. 【Conclusion】 There was a significant effect of root zone shape on the root morphology, root architecture, nutrient uptake, root/top ratio and shoot growth. The ‘root zone of deep-narrow’ decreased the length and diameter of lateral roots, increased the number of first lateral roots and the root/top ratio and improved the iron-uptake. The ‘root zone of shallow-wide’ increased the length and diameter of lateral roots, made fine roots richer, improved the uptake of phosphorus and potassium and promotes shoot growth. The ‘root zone of equal height and diameter’ decreased the diameter of taproot and the numbers of first lateral root and fine roots, promoted root activity and calcium-uptake.

Analysis on the Content of Major Heavy Metals in Top Ten Teas from Zhejiang Province

ZHANG Jian-yang, ZHANG Ying-bin, JIN Shou-zhen, MA Gui-cen, CHEN Li-yan, LIU Xin

Scientia Agricultura Sinica. 2014, 47(19):  3914-3921.  doi:10.3864/j.issn.0578-1752.2014.19.021

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【Objective】The rare earth (counted by total amount of oxides ∑ReOs), arsenic (As), chromium (Cr), cadmium (Cd), lead (Pb) content of top ten teas (West Lake Longjing included) in Zhejiang Province were investigated. The regularities of distribution of major heavy metals in top ten teas were analysed which will provide a scientific basis for safety evaluation and geographic origin in the famous teas. 【Method】A total of 78 samples were collected from 11 counties in 6 prefecture-level cities in Zhejiang province, which belongs to 11 species, i.e. West Lake longjing, Huiming tea, Jingshan tea, Kaihua longding, Anji white tea, Qiandao yuye, Dafo longjing, Yuexiang longjing, Wuyang chunyu, Lujian tea, and Songyang yinhou. The ∑ReOs, As, Cr, Cd and Pb contents were determined with the nitric acid-microwave digestion treatment method, and the results were analyzed by principal component analysis and cluster analysis.【Result】The contents of ∑ReOs, As, Cr, Cd and Pb in top ten teas were all in the allowed range of relevant standards, and significant differences were found in the content among top ten teas. The ∑ReOs content ranged from 0.305 mg·kg^-1 to 1.049 mg·kg^-1, where the highest content was in LJT, YXLJ had the lowest content. The average As content ranged from 0.053 mg·kg^-1 in SYYH to 0.121 mg·kg^-1 in WLLJ. The average Cr content ranged from 0.553 mg·kg^-1 in KHLD to 2.709 mg·kg^-1 in WLLJ. The average Cd content ranged from 0.033 mg·kg^-1 in WYCY to 0.099 mg·kg^-1 in KHLD. The average Pb content ranged from 0.510 mg·kg^-1 in KHLD to 2.763 mg·kg^-1 in DFLJ. Four principal components were obtained from the measured elements by principal component analysis, whose variance contribution rate was 88.159% in the total. Most kinds of teas could find their clusters by cluster analysis, and QDYY were all in cluster 7, while AJWT, HMT and JST were distributed in 4 different clusters.【Conclusion】The quality control is good in the planting, harvesting, processing and storage of top ten teas in Zhejiang province. There were significant differences in ∑ReOs, As, Cr, Cd and Pb among different species of famous teas.

Expression of Silkworm Vitellogenin Receptor in Vitro

LUO Juan, CHEN En-xiang, LIU Hong-ling, PENG Zhi-xin, YANG Cong-wen, SHEN Guan-wang, ZHANG Hai-yan, XING Run-miao, LIN Ying, XIA Qing-you

Scientia Agricultura Sinica. 2014, 47(19):  3922-3928.  doi:10.3864/j.issn.0578-1752.2014.19.022

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【Objective】 The objective of this study is to clone the silkworm BmVgR, to analyze its expression characteristics in insect cells and to investigate the expression patterns of the normal and mutational VgRs, thus providing a basis for its function identification. 【Method】 Specific primer sequences were designed according to BmVgR coding sequence, the amplified fragment was connected to the pET28a, and then transformed into E. coil BL21 (DE3), the fusion protein was obtained by IPTG induction. Ni-NTA affinity chromatography was used to purify the BmVgR protein peptide. Then the anti-BmVgR rabbit polyclonal antibody was obtained by BmVgR protein peptide as an antigen. The LBD1+EGF1 (C11D and C11V) domain of the normal and mutational VgRs was connected to the cell expression vector, the target protein was expressed with cell transfection. Immunohistochemistry was used to analyze the expression characteristics of the normal and mutational VgRs in insect cells. Western blot was used to detect the expression levels of the normal and mutational VgRs in the cell culture medium.【Result】 Prokaryotic expression gained a size of about 22 kD fusion protein, which was expressed as inclusion bodies. BmVgR protein peptide was expressed and purified, the anti-BmVgR rabbit polyclonal antibody was obtained, its titer was higher than 1﹕512 000. Purity was more than 95%. The endogenous genes of BmVgR and BmVg did not express in Sf9 and Spli221. The normal and mutational BmVgR-SP+LBD1+EGF1 was successfully expressed in the Sf9 cell (the BmVgR of the vit mutant, which was mutated in the 3rd Class B region of the EGF1 domain, coding 50 amino acid residuces). Immunohistochemistry showed that vit mutant and normal BmVgR could express normally in Sf9 cytoplasm. BmVgR rabbit polyclonal antibody and Myc-tagged antibody simultaneously detected the target protein, which indicated that the VgR antibody was better. As the SP + LBD1 + EGF1 peptide existed signal peptide, the expressed protein was secreted into the cell culture medium. Western blot indicated that the presence of amino acid deletion of the mutant vit did not affect the expression of the normal protein peptide, and the expression levels of the normal and mutational VgRs were not significantly different.【Conclusion】 The deletion of EGF1 domain didn’t affect the normal expression of SP+LBD1+EGF1 peptide protein, which may lead to the phenotype of the vit.