Scientia Agricultura Sinica

Excision of Selectable Markers Based on Inducible AlcR/alcA and Cre/loxP Systems

ZHAO Qing, GUO Yang-Dong, XIE Hua, MA Rong-Cai, YAO Lei

Scientia Agricultura Sinica. 2011, 44(17): 3491-3500. doi:10.3864/j.issn.0578-1752.2011.17.001

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【Objective】To eliminate the potential risk in safety raised by selectable markers, an ethanol inducible excision system of selectable markers was constructed, which can be used during plant growth and development. 【Method】 The selectable markers can be removed based on AlcR/alcA inducible system and Cre/loxP site-specific recombination system. In the presence of the exogenous inducer, the activation of the downstream Cre gene was enabled. Cre recombinase identified and catalyzed excision of the intervening sequence between two directly oriented loxP sites, including selectable marker gene, AlcR/alcA and Cre/loxP system. The gene of interest, gus, was constitutively expressed before and after induction. 【Result】 The Arabidopsis thaliana transgenic plants were induced by ethanol. The results revealed that ethanol tightly control the “on” and “off” of the expression of Cre gene. After induction, the transgenic plants could not continuously grow on selective medium, which indicate the selectable marker was removed efficiently. The molecule analysis revealed the DNA fragment between two directly oriented loxP sites has been excised. 【Conclusion】 The results demonstrate that the excision of selectable markers based on inducible AlcR/alcA and Cre/loxP systems is reliable, and has a bright future.

Genetic Analysis of Purple and Waxy Grain in Wheat

HUANG Bi-Guang

Scientia Agricultura Sinica. 2011, 44(17): 3501-3507. doi:10.3864/j.issn.0578-1752.2011.17.002

Abstract ( 945 )

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【Objective】Waxy-grain wheat and purple-grain wheat are special germplasms with special values in nutrition and quality. Based on the genetic law of waxy and purple grain in concrete materials, purple-and-waxy grain wheat was bred which combined the advantages of both wheats.【Method】Reciprocal cross between waxy-grain wheat C75 and purple-grain wheat 03-Chu-3 was made through the F2:3 generation, and then backcross was done between F1 and C75 through BC1F2 generation. The inheritance of grain color and waxiness were determined according to the phenotypic ratios of each generation, and a new wheat germplasm producing purple-and-waxy grains (named as purple-and-waxy wheat) was created from the filial generation. 【Result】 The purple-grain color, behaving as maternal effect, was controlled by 2 pairs of dominant and complementary genes，and the dosage effect was also found for the purple gene, the ratio of 9purple:7red in F2:3 and 1purple:3red in BC1F2 was observed. The waxiness was controlled by 3 independent pairs of duplicate genes with the non-waxy being dominant to the waxy, the ratio of 63non-waxy : 7waxy in F2:3 and 7non-waxy : 1waxy in BC1F2 were observed. The genes controlling purple grain color are genetically independent to those of waxiness. Five purple-and-waxy wheat lines with homologous waxy and purple genotype were identified in this experiment by selfing purification, and thus a new wheat germplasm, purple-and-waxy wheat, was obtained. 【Conclusion】 Purple grain, having maternal effect, was controlled by two pairs of dominant and complementary genes, waxiness, being recessiveness, was controlled by 3 independent pairs of duplicate genes, and purple-grain wheat, a new germplasm, was obtained.

QTL Mapping for Grain Yield Associated Traits Using Ye 478 Introgression Lines in Maize

ZHAO Pu, LIU Rui-Xiang, LI Cheng-Pu, XING Xiang-Ru, CAO Xiao-Liang, TAO Yong-Sheng, ZHANG Zu-Xin

Scientia Agricultura Sinica. 2011, 44(17): 3508-3519. doi:10.3864/j.issn.0578-1752.2011.17.003

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【Objective】 The purpose of this investigation was to identify QTL (Quantitative Trait Locus) for maize yield related traits and those introgression lines containing favorable alleles. 【Method】 Two maize inbred lines, QB80 and Qi319 were used as the donor parents, respectively, and the Ye478 as the recurrent parent, two introgression line populations consisting of 61 and 72 family lines were constructed by backcrossing combined with directional selection, respectively. The two introgression line populations were evaluated across 4 environments in 2 years. The QTLs for yield and related traits were detected by stepwise regression (RSTEP-LRT) using Windows QTL ICI Mapping software. 【Result】 A total of 49 QTLs for 9 traits were identified in the population with QB80 as the donor, and 42 QTLs for 9 traits were identified in the population with Qi319 as the donor under four environments. Of which 16 QTLs were detected across not less than two environments. In addition, some QTLs for same trait detected in different environments were located in the same chromosome regions, and those QTLs for diverse traits were also located in the same or adjacent chromosome region, forming several multiple QTL-rich regions. The less consistent QTL was detected in two populations, indicating that the two donors contain different sets of favorable alleles. And the yield associated traits of those lines containing favorable introgression segments or alleles were significantly improved, implicating those lines are available for improving of Ye478 by QTL pyramiding. 【Conclusion】 The genetic difference between QB80 and Ye 478 is more than that of Qi319 and Ye478 , therefore, more yield trait QTL can be detected. Those introgression lines containing favorable alleles can be used to improve Ye478 by designed QTL pyramiding. The detected QTL-rich regions have given a subset of important chromosome regions for fine mapping and cloning of genes for yield associated traits.

Discussion on Development of Crop Models

CAO Hong-Xin, ZHAO Suo-Lao, GE Dao-Kuo, LIU Yong-Xia, LIU Yan, SUN Jin-Ying, YUE Yan-Bin, ZHANG Zhi-You, CHEN Yu-Li

Scientia Agricultura Sinica. 2011, 44(17): 3520-3528. doi:10.3864/j.issn.0578-1752.2011.17.004

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In order to deepen the studies of crop models (CMs), promote its development, and make it to join into international development tide, the methods of systematic analysis, classification, comparison, and mining of epistemology, etc were adopted in this paper. The roles in CMs were set forth through definition and classification of CMs, and the relationships between CMs and digital plant (DP) were analyzed in this paper. The latest studies and application progresses of CMs in China and overseas were reviewed, the issues in their current studies and application were analyzed through comparison of differences in plant growth models among four research groups (DSSAT, School of de Wit, APSIM, and China) and in functional structural plant models (FSPMs) among three research groups (L-Studio, GREENLAB, and China) in the world, and some countermeasures and suggestions for their further development were proposed. At present, although the application of CMs and DP is little, it will have a wide development and application foreground with development of agriculture in China in future, especially, changes in proportion of town and countryside.

Effects of Nitrogen and PP333 Application on the Lignin Synthesis of Stem in Relation to Lodging Resistance of Wheat

CHEN Xiao-Guang, SHI Yu-Hua, WANG Cheng-Yu, YIN Yan-Ping, NING Tang-Yuan, SHI Chun-Yu, LI Yong, WANG Zhen-Lin

Scientia Agricultura Sinica. 2011, 44(17): 3529-3536. doi:10.3864/j.issn.0578-1752.2011.17.005

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【Objective】 This study was carried out to elucidate the effects of nitrogen and PP333 application on lignin content in culm of wheat, and discuss the physiological mechanism of nitrogen and PP333 in regulating the lodging resistance of wheat. 【Method】A field experiment was conducted with two nitrogen application rates and two PP333 levels to study the changes of snapping resistance, culm lodging resistance index (CLRI), lignin content and activities of phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and 4-coumarate CoA ligase (4CL) of wheat, and the effect of nitrogen and spraying PP333 on the lignin content, related enzyme activities in the lignin biosynthesis of wheat and their relationships with lodging resistance.【Result】 Compared with the low N (225 kg•hm-2) rate, the high N (300 kg•hm-2) rate reduced the PAL, TAL, CAD and 4CL activities and lignin content, reduced snapping resistance and CLRI of the same cultivar. The application of PP333 improved the PAL, TAL and CAD activities, increased lignin content, snapping resistance and CLRI, thus reduced lodging area and lodging degree. Correlation analysis showed that the lignin content of basal stem had a significant and positive correlation with PAL, CAL and CAD activity and CLRI, respectively, yet had no significant correlation with 4CL activity.【Conclusion】The high N(300 kg•hm-2) rate reduced the PAL, TAL,CAD enzyme activities and lodging resistance. Spraying PP333 improved the PAL, TAL, CAD enzyme activities and lignin content, and thus enhanced the lodging resistance.

Cloning and Bioinformatics Analysis of Chitin Synthase Gene ThChsC from Trichoderma harzianum

JI Ying, LI Mei, TIAN Yun-Long, LIU Wei-De, NIU Jing, JIANG Xi-Liang

Scientia Agricultura Sinica. 2011, 44(17): 3537-3546. doi:10.3864/j.issn.0578-1752.2011.17.006

Abstract ( 1015 )

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【Objective】 The objective of this study is to clone and make bioinformatics analysis of the sequence of chitin synthase gene ThChsC from Trichoderma harzianum. 【Method】 The full length of ThChsC was cloned by polymerase chain reaction (PCR) and genome walking methods, the gene sequence obtained and the putative amino acid sequence were analyzed by bioinformatics software, and the three-dimensional structure of ThChsC was predicted by homology modeling. 【Result】 ThChsC (GenBank accession number HQ419000) coding sequence spans 2 835 bp and is comprised of 4 extrons inserted by 3 introns. The ThChsC contains an open reading frame (ORF) of 2 688 bp capable of encoding a polypeptide of 895 amino acids and shares 80% and 81% identity to Gibberella moniliformis chsB (ACY08039.1) and Colletotrichum graminicola chs2 (AAL23718.1), respectively. Phylogenetic analysis indicates that ThChsC belongs to the type III chitin synthase subfamily. The conserved Chitin_synth_1, Chitin_synth_1N and Chitin_synth_2 domains and three transmembrane domains were found in the predicted ThChsC protein. A conserved DHD domain of glycosyl transferase was found in the three-dimensional structure of ThChsC predicted. 【Conclusion】The chitin synthase gene ThChsC was cloned for the first time from Trichoderma harzianum Th-33. The further study of ThChsC will have an important significance in exploring the mechanism of chitin synthesis of Trichoderma harzianum.

Effect of Bemisia tabaci (Gennadius) B-Biotype on Photosynthetic Characteristics and Activity of Defense Enzyme in Different Varieties of Tomato

CHEN Yi-Lei, CUI Xu-Hong, CAI Chong, CAO Feng-Qin

Scientia Agricultura Sinica. 2011, 44(17): 3547-3556. doi:10.3864/j.issn.0578-1752.2011.17.007

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【Objective】The objective of this study is to evaluate the selection of Bemisia tabaci B-biotype to different tomato varieties in the sight of physiological metabolism. 【Method】 An experiment was carried out at the artificial climate condition. The changes of the number of B. tabaci B-biotype, the value of photosynthetic parameter (i.e., Fv/Fm, Fv/Fo, Gs, Pn, Ci) and the defense enzyme (POD, CAT, PPO, LOX) activity on 9 species of tomato plant were comparatively studied by selection testing, respectively.【Result】 After sorting the 9 species of tomato plant by the number of the B. tabaci B-biotype, the descending order was arranged to be Huangshengguo (HSG)＞Zheza 205 (ZA 205)＞Zheza 203 (ZA 203)＞Zheza 206 (ZA 206) = Zheza 809 (ZA 809) = Hezuo 903 (HZ 903) = Fenniang (FN)＞Zhefen 202 (ZF 202) = Hongshengnv (HSN). Significant changes of the value of the photosynthetic parameter and the activity of the defense enzyme were observed in the plants HSG and ZA 205, but not in the plants ZF 202 and HSN, respectively. It indicated that the plants HSG and ZA 205 were stressed by the B. tabaci B-biotype feeding but not the plants HSN and ZF 202. As a result, two kinds of positive and significant correlations, i.e., the value change of the photosynthetic parameter and the number of the B. tabaci B-biotype, and the value change of light inhibition parameter (i.e., Fv/Fm) and the activity change of defense enzyme, were observed after 9 hours of selection testing. 【Conclusion】 Selection diversity of B. tabaci B-biotype to different tomato varieties could be reflected by the value changes of photosynthetic parameter and the activity change of defense enzyme. The B. tabaci B-biotype exhibited the highest selection to the plants HSG and ZA 205 and lowest selection to the plants HSN and ZF 202. The number of B. tabaci B-biotype showed a positive significant correlation with the degree of stress of B. tabaci on tomato plant.

The Quality Analysis of Cultivated Land in China

CHEN Yin-Jun, XIAO Bi-Lin, FANG Lin-Na, MA Hong-Ling, YANG Rui-Zhen, YI Xiao-Yan, LI Qian-Qian

Scientia Agricultura Sinica. 2011, 44(17): 3557-3564. doi:doi: 10.3864/j.issn.0578-1752.2011.17.008

Abstract ( 741 )

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Quality of cultivated land can be termed as the summation of such factors as soil quality, environmental quality, management quality and economic quality of the cultivated land. Quality of cultivated land is generally low in China. The area of cultivated land with moderate and low quality accounts for above 2/3 of the total. However, there is no common understanding about how the quality of cultivated land will change among the scientists thought most believe that the quality is generally decreasing. Results from the farmland soil monitoring campaign show that soil fertility of cultivated land is gradually increasing in China as indicators of the fertility present. This confirms the argument that quality of cultivated land as a whole is getting better and better in China though some factors may still be a problem in some parts of China.

Impacts of Returning Organic Materials on Soil Labile Organic Carbon Fractions Redistribution of Mollisol in Northeast China

LIANG Yao, HAN Xiao-Zeng, SONG Chun, LI Hai-Bo

Scientia Agricultura Sinica. 2011, 44(17): 3565-3574. doi:10.3864/j.issn.0578-1752.2011.17.009

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【Objective】 The objective of this study was to clarify the effect of returning different organic materials on characteristics of soil labile organic carbon fractions, and discuss fertilization system for controls of soil fertility. 【Method】 A six-year field experiment was carried out in Hailun county to study the effects of returning different organic materials on changes of soil total organic carbon (TOC) and redistribution of labile organic carbon fractions, as well as a greenhouse experiment with different soils was established to analyze soil productivity. 【Result】 Compared with the initial soil sample, 6-years application of chemical fertilizers (NPK) alone resulted in significant decreases in TOC, microbial biomass carbon (MBC),water-soluble organic carbon (WSOC) and light fraction organic carbon (LFOC). However, in plots amended with chemical fertilizers and organic materials (NPK+OM), the SOC and labile organic carbon fractions increased markedly. Compared with other organic materials, application of maize straw could be more suitable for the accumulation of SOC and LFOC, which was 26% and 136% greater than that of NPK application, and application of pig manure favored buildup of MBC and WSOC, which was 52% and 85% greater than NPK application. Soil productivity reduced in NPK plot, however, increased in NPK+OM plots. The decrease order of soil productivity in different NPK+OM plots was NPK+ pig manure＞NPK+ cattle manure＞NPK+ wheat straw ≈NPK+ maize straw. 【Conclusion】There were significant differences in TOC, labile organic carbon fractions and soil productivity among treatments amended with various organic materials in black soil. Application of chemical fertilizer amended with maize straws could be prone to increase TOC and LFOC pool, as well as amended with pig manure was the most effective measure to promote MBC, WSOC and soil productivity.

Characteristics of Organic Carbon Mineralization of Maize Straw in Lime Concretion Black Soil

ZHANG Li-Juan, CHANG Jiang, JIANG Li-Na, XU Shan, ZHANG Li-Gan

Scientia Agricultura Sinica. 2011, 44(17): 3575-3583. doi:10.3864/j.issn.0578-1752.2011.17.010

Abstract ( 919 )

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【Objective】 In order to better understand the characteristics of organic carbon mineralization of maize stalk in soil, the influences of different temperatures and different maize straw additions on the mineralization and decomposition of maize stalk in lime concretion black soil were investigated under the controlled condition in the laboratory. 【Method】 Mineralization of maize stalk at different temperatures (10℃, 20℃, 30℃) and different levels of maize straw addition (0.3, 1.5 and 3.0 g straw at 50 g soil) were studied by aerobic incubation tests. 【Result】 Results showed that organic carbon mineralization of maize stalk in soil was significantly influenced by temperature. At the same maize straw addition level, the higher the mineralization temperature (from 10℃ to 30℃), the higher the amounts of the organic carbon accumulated in soil. The mean mineralization temperature coefficients (Q10) of soil organic carbon (SOC) were 1.229, 1.251, 1.572, 1.399 at lower temperatures (＜20℃), and 1.006, 1.249, 1.401, 1.374 at higher temperatures (＞20℃) for CK, Ⅰ, Ⅱand Ⅲ treatments, respectively. The Q10 values were higher at lower temperatures (＜20℃) than that at higher temperatures (＞20℃), which indicated that mineralization of SOC was more sensitive to temperature rise at lower temperature. The accumulation and daily mineralization of SOC were increased with the increasing of maize straw addition at the same incubation temperature. The dynamics of SOC mineralization could be well fit by the first-order kinetics equation , and the C0 value increased with the increasing of straw addition. However, the potentially mineralized carbon (C0) was not significantly affected by temperature. 【Conclusion】 The increase of temperature and maize straw addition may be effective strategies for enhancing mineralization of maize straw in soil, and accelerating accumulation of organic carbon in soil.

QTL Mapping of Resistance Genes to Powdery Mildew in Cucumber (Cucumis sativus L.)

ZHANG Sheng-Ping, LIU Miao-Miao, MIAO Han, ZHANG Su-Qin, YANG Yu-Hong, XIE Bing-Yan, GU Xing-Fang

Scientia Agricultura Sinica. 2011, 44(17): 3584-3593. doi:10.3864/j.issn.0578-1752.2011.17.011

Abstract ( 1335 )

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【Objective】 The cucumber inbred line K8 with high resistance to powdery mildew was used as materials to study.the inheritance of resistance gene to powdery mildew in K8 and to conduct QTL mapping for these genes so as to provide a theoretical basis for the resistance mechanism and molecular assistant selection (MAS) breeding. 【Method】 An artificial inoculation method was adopted to test the degree of resistance to Podosphaera xanthii (syn. Sphaerotheca fuliginea) for the F2:3 family lines derived from the cross of K8×K18 (susceptible lines). SSR analysis, combined with bulked segregation analysis (BSA), was done on the DNA of F2 and F2:3 population using 2 360 pairs of SSR primers. JoinMap 4.0 and MapInspect software were used to construct SSR linkages and to make sure the corresponding relations between these SSR linkages and cucumber chromosome. QTL analysis on powdery mildew resistance genes was conducted by MapQTL4.0 software. 【Result】 The inheritance of the resistance gene to powdery mildew in K8 fit to the inheritance law of quantitative trait. Four QTLs named pm5.1, pm5.2, pm5.3 and pm6.1 for the resistance gene to powdery mildew were detected in this study. pm5.1, pm5.2 and pm5.3 were detected repeatedly in two years and the location for pm5.1, pm5.2 and pm5.3 was consistent. The QTL of pm5.2 accounted for the highest phenotypic variation. Four NBS resistance genes were found in the region of pm5.2. The QTL of pm6.1 located on Chr.6 was a minor QTL. 【Conclusion】 The QTL of pm5.2 located on Chr.5 was the major QTL. This resistance gene may belong to NBS resistance gene. The results in this study will be of great benefit to fine mapping and gene cloning for the major QTL of powdery mildew resistance gene, also the results will lay a good foundation for cucumber MAS resistance breeding.

Studies on the Phenolic Compounds in Berries of Vitis vinefera cv. Thompson Seedless Lines

ZHANG Zong-Qin, SUN Feng, JIN Qiang, LIU Wen-Jie, TIAN Bei-Bei, WANG Jing-Jing, LUO Qiang-Wei, GAO Jiang-Sheng, WANG Yue-Jin

Scientia Agricultura Sinica. 2011, 44(17): 3594-3602. doi:10.3864/j.issn.0578-1752.2011.17.012

Abstract ( 983 )

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【Objective】 This research was aimed to evaluate the clonal lines of V. vinefera cv. Thompson Seedless for breeding of new table grape varieties of higher resveratrol concentration, or new raisin grape varieties of lower total phenolic compound (TPC) concentration and polyphenol oxidase (PPO) activity relatively. 【Method】 Using V. vinefera cv. Thompson Seedless and ten diverse lines of this variety as materials, fresh berry mass and soluble solid content (TSS), resveratrol，TPCs and PPO activity were evaluated. 【Result】 The results indicated that the berry mass of Big-berry Thompson Seedless and W3 were 1.3286-1.4682 times of that of the control (Thompson Seedless). Resveratrol contents of W2, W8, Long-bunch Thompson Seedless and W7 were 110.65%-128.41% of that of the control. TPC contents of each lines were significantly different, in which Long-berry Thompson Seedless, W8, W3, and Big-berry Thompson Seedless were with small amount TPCs. PPO activity of berry skin was 1.06-13.19 times of that of the whole berry.【Conclusion】Compared to the control, W3, Big-berry Thompson Seedless, Long-berry Thompson Seedless and W5 were superior for fresh table grape breeding based on berry mass, TSS, resveratrol content. TPCs of Big-berry Thompson Seedless, Long-berry Thompson Seedless and W3 were relatively lower than other lines, and PPO activity of W7, Long-bunch Thompson Seedless, W3, W8 were relatively low among the materials tested. These results could provide some indications for the prospective breeding of new lines of Thompson Seedless variety.

Development of EST-Derived SSR Markers and Their Application in Strawberry Genetic Diversity Analysis

DONG Qing-Hua, WANG Xi-Cheng, ZHAO Mi-Zhen, SONG Chang-Nian, GE An-Jing, WANG Jing

Scientia Agricultura Sinica. 2011, 44(17): 3603-3612. doi:10.3864/j.issn.0578-1752.2011.17.013

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【Objective】 The objective of this study was to analyze the SSR distribution in strawberry ESTs and to develop new EST-derived SSR markers, and application of EST-SSR markers in strawberry genetic diversity analysis was also validated. 【Method】 A total of 55 750 EST sequences of strawberry were obtained from NCBI. These sequences were screened by using MISA software to search for SSR motifs. Sixty pairs of primers were designed by the software Primer3.0 Plus. The PCR products of these primers were detected by PAGE and some of them were recovered for sequencing. 【Result】 A total of 1 490 SSRs were identified from 1 334 strawberry EST sequences. The dinucleotide, trinucleotide and hexanucleotide repeats were the dominant types with the frequency of 36.17%, 26.51% and 19.87%, respectively. Among the 60 EST-SSR primers, 42 amplified distinct bands and expected products, and 36 were polymorphic. Eleven polymorphic EST-SSR amplicons were validated by resequencing the PCR products. A dendrogram tree generated by the 11 validated polymorphic EST-SSR markers revealed reasonable genetic relationships among 20 strawberry cultivars. 【Conclusion】 It is an efficient and economic way to develop SSR markers from the strawberry EST sequences, and the EST-SSR markers can be used in strawberry genetic analysis.

Effect of Undernutrition and Compensation on the Growth of Small Intestinal Mucosa in Lambs

LIU Xiao-Gang, LI Da-Biao, HOU Xian-Zhi, KAO Gui-Lan, WANG Hai-Rong, YANG Jin-Li, ZHANG Chong-Zhi, XIA Wei

Scientia Agricultura Sinica. 2011, 44(17): 3613-3621. doi:10.3864/j.issn.0578-1752.2011.17.014

Abstract ( 757 )

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【Objective】 The effects of undernutrition (0-60 d) and compensation (60-150 d) on live weight (BW) and development of small intestinal mucosa in lambs were investigated. 【Method】 Eighty Wu Zhu Mu Qin lambs at average live weight (14.72 ± 1.10) kg, were divided into control group (CG), restricted group 1 (RG1), restricted group 2 (RG2) and restricted group 3 (RG3). Dietary nutrition level were (ME: 10.61 MJ•kg-1, CP: 150.8 g•kg-1), (ME: 10.49 MJ•kg-1, CP: 92.4 g•kg-1), (ME: 8.36 MJ•kg-1, CP: 150.1 g•kg-1) and (ME: 8.10 MJ•kg-1, CP: 62.7 g•kg-1) during undernutrition period for CG, RG1, RG2 and RG3, respectively. However, in nutritional compensation phase all groups were offered the same levels diet (ME: 9.77 MJ•kg-1, CP: 124.8 g•kg-1). Weight of lambs was measured every week. Four lambs of each group were slaughtered at 0 d (at the beginning of undernutrition period), 60 d (at the end of undernutrition period) and 150 d (the end of nutrition compensation period). The samples of duodenum, jejunum and mid-ileum were taken and fixed by using paraformaldehyde phosphate buffer. After HE staining, morphological structure of intestinal mucosa were observed under light microscope. 【Result】 The results showed that at the end of nutrition restriction period, the average live weight of RG1, RG2 (P＜0.05) and RG3 (P＜0.01) was significantly lower than that of CG. However, during the nutrition compensation period, the growth rate of RG3 was significantly higher than that of CG (P＜0.05). Development and growth of the small intestine mucosa were affected by nutrition level of diets. The energy restricted group (RG2) showed better compensatory potency than that of the protein restricted group (RG1). 【Conclusion】 The results indicated that after restriction by different levels of energy and protein all lambs showed compensatory effect in live weight and small intestinal mucosa growth. The development of small intestine mucosa was more prone to be influenced by dietary protein content other than energy concentration. After 60 d restriction by the nutrition level of ME: 8.36 MJ•kg-1, CP: 150.1 g•kg-1, the development of small intestinal mucosa of RG2 reached complete compensation at the end of nutrition compensation period, and nutrients absorption and growth rate also increased correspondingly.

Study on the Interspecies Crossbreeding Between Mithun (Bos frontalis) and Zebu (Bos indicus) Cattle

HE Zhan-Xing, ZHANG Ji-Cai, XU Kai-Xing, JIN Xian-Dong, WANG Zhe, WANG An-Kui, HUANG Mei-Fen, ZHAO Gang, YANG Guo-Rong, TU Xian-Yun, YUAN Xi-Ping, HUANG Bi-Zhi

Scientia Agricultura Sinica. 2011, 44(17): 3622-3633. doi:10.3864/j.issn.0578-1752.2011.17.015

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【Objective】 The potential of interspecies crossbreeding utilization and its foreground between Mithun (Bos frontalis) and Zebu (Bos indicus) cattle were studied. 【Method】 Firstly, after treated by CUE-MATETM (progesterone-releasing intravaginal device) and PG for synchronization estrous, Brahman cows were inseminated artificially with Mithun’s frozen semen. And gestation length and calving time of cows and calf sex ratio were investigated. Besides, the body weight of Mithun and Brahman crossbreed at birth, 6 months, 12 months and 18 months of age were measured and karyotype and reproductive performance of hybrids F1 (Mithun×Brahman) and so on were analyzed and investigated. 【Result】 The rates of synchronization estrous, pregnancy and calving of Brahman cow were 83.87%, 60.56% and 45.07%, respectively. The average gestation length was (290.83±5.26) d (n=24). The percentage of female calf in crossbred F1 (Mithun×Brahman) was 71.88%，higher than those of Mithun and Brahman (P＜0.05), and significantly higher than those of crossbred F1 (Mithun×Yellow cattle) and BMY cattle, respectively (P＜0.01). The average body weight at birth, 6 months, 12 months and 18 months of crossbred F1 (Mithun×Brahman)(n=29) was (26.69±4.84) kg, (153.08±37.58) kg, (219.34±31.31) kg and (302.06±28.92) kg, respectively, which were 68.18%, 37.12%, 45.07% and 40.13% higher than those of Mithun (P＜0.01), but 14.40% (P＜0.01), 5.23% (P＞0.05), 13.12% (P＜0.01) and 12.76% (P＜0.01) lower than those of Brahman. The average daily gain (ADG) of crossbred F1 (Mithun×Brahman) from birth to 18 months of age was (503.09±44.91) g and (502.89±53.54) g for male and female，which were 35.98% and 39.85% higher than those of male and female of Mithun (P＜0.01), but 16.49% and 6.63% lower than those of male (P＜0.01) and female (P＞0.05) of Brahman. The chromosome numbers of Mithun, Brahman and their crossbred F1 (Mithun×Brahman) were 2n=58, 2n=60 and 2n=59, respectively. The average age and body weight of crossbred F1 (Mithun×Brahman) at first pregnancy and their primiparous age were (16.00±2.83) months, (342.50±30.41) kg and (26.00±2.83) months, respectively, which gave one calf per year. The semen collection of 5 crossbred F1 (Mithun×Brahman) at 15-24 months of age was failed by electro-ejaculation treatment.【Conclusion】Interspecies crossbreeding of Mithun×Zebu performed better in rapid growth, wide adaptation and significant heterosis. And the crossbred female F1 was of better reproductive capacity, precocity and high reproductive efficiency. However, the fertility of male crossbred F1 will remain to be studied in future.

Effect of Exogenous Melatonin (MT) in Bovine Oocyte in vitro Maturation

GAO Chao, TIAN Xiu-Zhi, ZHANG Lu, XU Jing, WANG Feng, ZHUO Zhi-Yong, DAI Yun-Ping, LIU Guo-Shi

Scientia Agricultura Sinica. 2011, 44(17): 3634-3640. doi:10.3864/j.issn.0578-1752.2011.17.016

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【Objective】 To optimize the bovine embryo in vitro production system，the effect of exogenous melatonin (MT) on improvement of bovine oocyte in vitro maturation was studied. 【Method】 Immature oocytes were cultured in vitro inmaturation medium supplemented with different concentrations of melatonin, then IVF statistical analysis of embryo cleavage rate, blastocyst rate and blastocyst cell number was conducted. Hormone levels were detected by radioimmunoassay in follicular fluid and the maturation medium and the the oocyte viability and reactive oxygen (ROS) levels were determined.【Result】MT existed in bovine follicular fluid and similar with serum. In 10-11 mol•L-1 treatment, the progesterone (P) levels (2.01±0.33) ng•mL-1 (P＜0.05) of maturation medium was significantly higher than that of controls (0.87±0.10) ng•mL-1 (P＜0.05). Compared with the control group (67.32±7.30)%, cleavage rate in 10-9 mol•L-1MT treatment (77.81±7.06)% was significantly increased and the blastocyst rate and cell number of blastocyst (29.61±9.55)%, (90.30±28.74) were significantly higher than that of controls (19.64±9.22)%, (75.07±25.98) (P＜0.05). 10-9 mol•L-1 MT treatment improved the oocyte viability and decreased the ROS level (P＞0.05). 【Conclusion】 Melatonin exists in bovine follicular fluid and exogenous melatonin at a proper concentration can improve the in vitro maturation of bovine oocytes.

Cloning and Functional Expression of a Multi-Functional Cellulase Gene egx from Mollusca, Ampullaria crossean in vitro

HUANG Miao-Rong, LIU De-Wu, WU Zhen-Fang

Scientia Agricultura Sinica. 2011, 44(17): 3641-3648. doi:10.3864/j.issn.0578-1752.2011.17.017

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【Objective】 The objective of the study is to clone the multi-functional cellulase gene from Mollusca, Ampullaria crossean, and analyze its expression in vitro. 【Method】 The cDNA fragment was amplified by RT-PCR from the stomach of Mollusca, Ampullaria crossean, and then cloned into pMD18-T vector. Following sequencing, the gene was subcloned into the expression vector pET-32a (+) and pcDNA3.1 (+) using EcoRⅠand NotⅠrestriction sites, and the enzymatic activity was determined by DNS. 【Result】 Sequence analysis showed that the 1 326 bp amplicon consists of 1 185 bp coding sequence and part of flanking sequence. The DNA sequence and the putative amino acid sequence shared 99% and 100% identity with the reported sequence, respectively. The purified product from E.coli BL-21 (DE3) showed hydrolytic activities to various substrates including carboxylmethyl cellulose sodium salt (CMC-Na), 2-hydroxyethyl-cellulose, hydroxyethyl-cellulose, sigmacell and xylan with specific activities of 24.78, 15.67, 18.42, 600.91 and 175.43 U•mg-1, respectiviely, while the recombination protein expressed in PK15 showed hydrolytic activities of 0.84, 0.78, 1.01, 14.62 and 4.23 U•mL-1, respectively. 【Conclusion】 The multi-functional cellulase from Mollusca, Ampullaria crossean, was cloned, functional expressed in pro- and eukaryotic cells, and this could provide a foundation for further research and application of the multi-functional cellulase gene from Mollusca, Ampullaria crossean.

Advanced Research on Honeybee Proteome

LI Jian-Ke, FENG Mao, ZHENG Ai-Juan

Scientia Agricultura Sinica. 2011, 44(17): 3649-3657. doi:10.3864/j.issn.0578-1752.2011.17.018

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Honeybee plays an important role for the nature and the human. It is crucial for agriculture as a facilitator of pollination and indispensable to maintain the biological diversity of the ecological system. Also, bee products are widely used for their nutrition and health care functions to the mankind. The proteomic researches on honeybee have been ushered into a new stage since the completion of the honeybee genome sequencing project. And the combination of two-dimensional electrophoresis and mass spectrometry is the most popular method in honeybee proteome investigations. This paper reviewed the advances in honeybee proteomics concerning the development of honeybee egg, larva, pupa, hypopharyngeal gland and caste differentiation, the comparison between nurses and foragers, the analysis of some tissues and organs such as honeybee head, thorax, hemolymph, venom gland, queen spermathecal gland, male accessory gland and sperm, the studies on royal jelly, pollen, honeybee venom and so on, hoping to provide some clues for future studies.

Analysis of Fatty Acids Composition in the Liver of Mice with Non-alcoholic Fatty Liver Disease

WANG Xin, CAO Yu-Zhen, GUO Gui-Fang, LI Rui, ZHANG Xiu-Ying

Scientia Agricultura Sinica. 2011, 44(17): 3658-3665. doi:10.3864/j.issn.0578-1752.2011.17.019

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【Objective】 The aim of this study was to determine the liver fatty acid composition of an experimental mouse model of NAFLD induced by high-fat feed and CCl4 and to assess the association between the change of liver fatty acids content and NAFLD. 【Method】C57BL/6J mice were given high-fat feed for six consecutive weeks to develop experimental NAFLD. Meanwhile, these mice were given subcutaneous injections of a 40% CCl4-vegetable oil mixture twice per week. Fatty acids in mice liver were extracted with diethyl and derived by BF3/methanol, then analyzed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring. Qualitative analysis of fatty acids in mice liver was carried out according to the retrieval mass spectrometry database and the retention time of fatty acid standard. Quantitative analysis was determined by external standard method. 【Result】Pathological examination showed that NAFLD of C57BL/6J mice was successfully induced. Compared with the control group, high-fat feed and CCl4 led to significant increases in C14:0, C16:0, C18:0 and C20:3 (P＜0.01), and decreases in C15:0, C18:1, C18:2 and C18:3 (n-3) (P＜0.01) in the mouse liver. The treatment also led to an increase in saturated fatty acids (SFA) and decreases in other types fatty acids. An increase in the ratio of product/precursor n-6 (C20:4/C18:2) and n-3 [(C20:5+C22:6)/C18:3 (n-3)] and a decrease in the ratio of n-6/n-3 [C20:4/(C20:5+C22:6)] were also observed.【Conclusion】An increase of SFA, a decrease of unsaturated fatty acids (UFA), and metabolic disturbance of n-6 polyunsaturated fatty acids (PUFA) and n-3 PUFA in the liver of mice with NAFLD induced by high-fat feed and CCl4 were not only the results of NAFLD, but also the stimulating factors for the development of NAFLD.

Advances in Research of Short-Term Forecasting Methods of Agricultural Product Price

XU Shi-Wei, LI Zhe-Min, LI Gan-Qiong, DONG Xiao-Xia

Scientia Agricultura Sinica. 2011, 44(17): 3666-3675. doi:10.3864/j.issn.0578-1752.2011.17.020

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This paper reviewed the progress in the theories, methods and their application of short-term forecasting of agricultural product prices at home and abroad. Advantages and disadvantages of the existing research methods were comparatively analyzed and the future research directions were prospected. A conclusion drawn from the research was that the quantitative forecasting method could be divided into econometric forecasting, statistics forecasting, intelligent analysis and combination model as the wide application of mathematical statistics, econometrics, fuzzy mathematics and artificial neural network. In the future, the development of short-term forecasting methods of agricultural product prices will show these characteristics: quantitative analysis will be dominant and large-scale complex of intelligent hybrid model will be increased. Moreover, quantile regression model will be introduced as a new application of short-term forecasting of agricultural product prices.

Comparison of Induction and Regeneration of Embryogenic Callus Initiated from Immature Embryos and Seedling-Derived Young Leaf Segments of Maize

CHEN Jing, DONG Hao, MA Hai-Zhen, SUN Quan-Xi, LIU Jiang, QI Bao-Xiu, LI Xin-Zheng, DONG Shu-Ting

Scientia Agricultura Sinica. 2011, 44(17): 3676-3682. doi:10.3864/j.issn.0578-1752.2011.17.021

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【Objective】 Using immature embryos and seedling-derived leaf segments of maize inbred lines that are commonly used in breeding as explants, the difference and correlation between these two explants in terms of their callus induction and regeneration were studied. The aim of this study is to provide a protocol for the establishment of an efficient and stable regeneration system in maize using leaf segments as an alternative explant. 【Method】 Six inbred lines of maize (Zea mays L.) that routinely used in breeding were tested in this study. The initiation, maintenance and differentiation of callus from their immature embryos and seedling-derived leaf segments were compared. The effect of 2,4-D on primary callus induction of different explants from all these six lines was studied. The callus induction and differentiation frequency were recorded, and the callus morphology was also observed. 【Result】 Different explants required different concentrations of 2,4-D to induce callus formation. Generally, the leaf segment was more difficult to induce and also required higher concentration of 2,4-D compared with immature embryos. The callus induction and differentiation rate of these two explants of all six genotypes were very different although that of the same genotype were similar. 【Conclusion】 Three genotypes, Qi 319, Mo17 and Luyuan 92, were selected after screening through six lines for their performance in higher callus induction rate and excellent morphology.

23S-5S rDNA Sequence Cloning and Analysis of Candidatus Liberibacter africanus and americanus of Citrus Huanglongbing

LIAO Hui-Hong, LI Yang-Rui, YANG Li-Tao, XU Ning

Scientia Agricultura Sinica. 2011, 44(17): 3683-3693. doi:10.3864/j.issn.0578-1752.2011.17.022

Abstract ( 700 )

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【Objective】 The objective of this study is to clone 23S-5S rDNA sequences of Candidatus Liberibacter africanus and americanus and compare to the corresponding region sequences of Candidatus Liberibacter asiaticus to clarify the ribosomal RNA operons relationships of HLB three species.【Method】The HLB DNA samples from africanus and americanus were amplified by the primers designed according to the 23S-5S rRNA gene conserved sequences of asiaticus. The PCR products were cloned and sequenced. The newly obtained sequences were further identified and analyzed.【Result】Sequences of 3 057 bp in length were obtained from africanus including 23S rRNA gene, cell wall hydrolase pseudogene and 5S rRNA gene. Sequences of 3 033 bp in length were obtained from americanus including 23S rRNA gene, glpK gene and 5S rRNA gene. The gene order is 16S rRNA, tRNAIle, tRNAAla, 23S rRNA, cell wall hydrolase pseudogene, 5S rRNA and tRNAMet for asiaticus. 16S rRNA, tRNAIle, tRNAAla, 23S rRNA, cell wall hydrolase pseudogene, 5S rRNA for africanus. 16S rRNA, tRNAIle, tRNAAla, 23S rRNA, glpK gene, 5s rRNA for americanus. 【Conclusion】The operon arrangement was special for three HLB species. The cell wall hydrolase gene coding in the reverse strand was found between 23S rRNA and 5S rRNA gene in three HLB species. It is a pseudogene for asiaticus and africanus while it is glpK gene for americanus.

Taxonomic Status Assays of Theileria equi Based on Hsp70 Gene

LUO Jin, LIU Guang-Yuan, TIAN Zhan-Cheng, XIE Jun-Ren, ZHANG Ping, SHEN Hui, DANG Gen-Sheng

Scientia Agricultura Sinica. 2011, 44(17): 3694-3700. doi:10.3864/j.issn.0578-1752.2011.17.023

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【Objective】 The objective of the study is to determine the taxonomic status of Theileria equi (T. equi). 【Method】 According to the Hsp70 gene sequence (AB248743.1) of Babesia equi (B.equi) in GenBank, the primers TeHsp70F and TeHsp70R were designed. And a fragment with the length of 1 920 bp was obtained by PCR. The gene sequence was compared with other 23 amino acids sequences. 【Result】 The fragment encods 639 amino acids, 208 hydrophobic amino acids and 167 polar amino acid. Identity analysis showed that T. equi has a closest relationship with B.equi (BAF02625.1), and has a little relationship with T. parva (XP764717.1) and T. annulata (AAA30130.1), while T. equi has a distant relationship with B. caballi (BAF02619.1), which infects equine. 【Conclusion】 It was a correct taxonomic status for B. equi to Theileria species. And the taxonomic status was error before, for T. equi to Babesia species. In this paper, a proof was provided for taxonomic status of Theileria equi.

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