Table of Content

20 October 2015, Volume 48 Issue 20

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Overexpression of a Transcription Factor Gene SiNF-YA6 from Millet (Setaria italica) Enhanced the Resistance of Transgenic Plants to Nitrogen Starvation

FANG Guang-ning, HU Li-qin, WANG Er-hui, XUE Fei-yang, MA You-zhi, XU Zhao-shi, LI Lian-cheng, ZHOU Yong-bin, DIAO Xian-min, JIA Guan-qing, CHEN Ming, MIN Dong-hong

Scientia Agricultura Sinica. 2015, 48(20):  3989-3997.  doi:10.3864/j.issn.0578-1752.2015.20.001

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【Objective】Nuclear transcription factor Y (NF-Y) belongs to a transcription factor gene family widely existing in eukaryotic. NF-Y-like transcription factors are involved in the process of gene expression regulation of plant growth and development and stress responses. So far, it is never been reported that NF-Y-like transcription factors are involved in plant resistance to low nitrogen stress. In this study, a NF-Y-like transcription factor gene SiNF-YA6 was isolated from millet, and its characteristics and biological function were identified.【Method】A NF-Y-like transcription factor gene SiNF-YA6 highly up-regulated by low nitrogen stress was selected from the results of transcriptome sequencing of millet treated by low nitrogen. DNAMAN and MEGA6.0 software were used to make the phylogenetic tree of NF-YA6. The Real-time PCR was used to analyze expression patterns of SiNF-YA6 under different treatments. SiNF-YA6 was transformed into Arabidopsis protoplast to observe its subcellular localization. SiNF-YA6 was overexpressed in Arabidopsis to identify gene function.【Result】SiNF-YA6 is 714 bp in length and encoding 237 amino acids. SiNF-YA6 contains a CBF conservative domain with 62 amino acids. Phylogenetic tree analysis showed that NF-YA was fell into 6 classes, and SiNF-YA6 belongs to the fifth class. SiNF-YA6 was the highest homology with a rice NF-YA protein (Os03g07880) (77.4%). Expression pattern analysis showed that NF-YA6 was induced by low nitrogen stress, and its expression reach peak after treatment for 5 d that raised about 5 times compared with the expression level at the beginning of treatment. While transcription of SiNF-YA6 under high salt, drought, ABA treatment also showed a trend of increasing. SiNF-YA6 under SA and GA treatments was no obvious different. Tissue specific expression analysis showed that SiNF-YA6 mainly expressed in millet roots. Subcellular localization indicated that SiNF-YA6 was mainly located on the plasmalemma and nucleus in plant cell. Results of low nitrogen stress resistance test showed that the main root length and root surface areaof SiNF-YA6 transgenic Arabidopsis were significantly higher than in wild type Arabidopsis, and the fresh weight of transgenic plants was also significantly greater than the wild type plants (P<0.01) under low nitrogen stress (total nitrogen content of 1 mmol·L^-1), and the growthof SiNF-YA6 transgenic Arabidopsis showed no obvious difference with wild-type Arabidopsis under SA and GA treatments. Those results indicated that overexpression of SiNF-YA6 gene significantly enhanced transgenic plants resistance to low nitrogen stress. The downstream genes, including some nitrogen transporter genes (NRT2.1 and NRT2.4), were up-regulated in SiNF-YA6 transgenic Arabidopsis. However, SiNF-YA6 transgenic Arabidopsis showed no obvious difference with wild-type Arabidopsis under high salt and drought stresses.【Conclusion】Overexpression of SiNF-YA6 enhanced transgenic plants tolerance to low nitrogen stress by up-regulated expression of several nitrogen transporter genes, which will give new candidate for improvement of the nitrogen use efficiency in crops.

Salt Tolerance Analysis of TaNHX2 Over-Expression Transgenic Soybean

LIN Kang-xue, LIU Xiu-jie, SUN Shi, CHEN Li, HAN Tian-fu, HOU Wen-sheng

Scientia Agricultura Sinica. 2015, 48(20):  3998-4007.  doi:10.3864/j.issn.0578-1752.2015.20.002

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【Objective】 In order to obtain valuable salt-tolerance soybean germplasms, the productive potential of TaNHX2（wheat Na⁺/H⁺ transporter code gene）over-expression transgenic soybeans was studied through observing their salt induced phenotype, photosynthetic intensity and agronomic traits under salt stress.【Method】The T₄ generation of TaNHX2 over-expression transgenic soybeans, which the transgenic authenticity confirmed by Glufosinate-resistance assay, PCR and RT-PCR detections at V₃ stage were chosen as materials. The positive individuals were then treated with NaCl solution at V₄ stage, during the treatment phase, the salt induced phenotype of transgenic and wild-type plants was observed. Furthermore, the photosynthetic intensity of the soybeans at R₃ stage was measured. Finally, the plant height, node number on main stem, pod number per plant, seed number per plant and seed weight per plant of soybean plants which harvested at R₈ stage were measured. Two concentrations of NaCl solution (0 and 200 mmol·L^-1) were applied in Beipuchang research site, while three concentrations were applied (0, 150 and 200 mmol·L^-1) in Changping, Beijing. The observation of salt induced phenotype and measurement of photosynthetic intensity were only carried out at Beipuchang research site. 【Result】 The successful expression of foreign gene TaNHX2 was confirmed by molecular identification, its inheritance was stable. Transgenic soybean and wild-type plants had similar growth vigor, leaf size and photosynthetic intensity under normal growing condition. While both the transgenic soybean and wild-type plants became shorter under 200 mmol·L^-1 NaCl treatment, their leaves became smaller and their photosynthetic intensity decreased. When compared with wild-type, the transgenic soybean lines (C12, C21 and C19) had lower dwarf degree, larger leaf size and photosynthetic intensity, the photosynthetic rate (Pn) of lines C12 and C21 significantly larger than wild-type. In addition, the yield related agronomic trait value of transgenic soybean lines and wild-type were comparable under normal condition, while under 150 mmol·L^-1 and 200 mmol·L^-1 NaCl stress, the agronomic trait values of transgenic soybean lines were larger than wild-type. Under 150 mmol·L^-1 NaCl stress (Changping), seed weight per plant of lines C12, C21 and C19, pod number per plant of line C19 had a significant difference with wild-type. Under 200 mmol·L^-1 NaCl stress (Changping), pod number per plant, seed number per plant and seed weight per plant of lines C12, C21 and C19, plant height of lines C12 and C19 had a significant difference with wild-type. At Beipuchang research site, plant height of lines C12, C21 and C19, seed number per plant and seed weight per plant of line C19 were significantly larger than wild-type. 【Conclusion】Compared with wild-type, transgenic soybean lines (C12, C21 and C19) had lower salt damage degree, kept a higher photosynthesis and certain yield under salt stress, line C19 had an excellent yield potential both at two research sites, therefore, it is practical valuable for breeding and production.

Analysis of the Coefficient of Parentage Among Major Jute Cultivars in China

ZHANG Jia-qiang, CHEN Chang-li, LUO Xia-hong, JIN Guan-rong

Scientia Agricultura Sinica. 2015, 48(20):  4008-4020.  doi:10.3864/j.issn.0578-1752.2015.20.003

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【Objective】 We explored the genetic diversity and parentage among major Jute cultivars since the founding of new China in 1949. 【Method】The coefficient of parentage (COP) and cluster analysis were used to clarify the genetic variabilities and relationships among Jute cultivars in six varieties arising since 1949. Twenty Jute cultivars of Corchorus olitorius L. and 40 individuals of Corchorus capsularis L. were selected as test materials, based on where the extension area was largest and pedigree information for each clearest.【Result】A total of 190 cross combinations were made among 20 cultivars of C. olitorius, with 31.6% of the combinations sharing parental relationships, with an average of 0.0964. These crosses indicated that there were not close relationships among 2/3 of cultivars. A total of 780 cross combinations of C. capsularis were made among 40 cultivars with 50.3% of the combinations sharing parental relationships, but their average COP value was 0.1179. The crosses indicated that there were close relationships among more than half of the tested cultivars. Looking at all crosses, the genetic similarity relationships of C. capsularis cultivars were higher than that of C. olitorius cultivars. Clustering analysis based on coefficients of parentage examined the genetic relationships among the jute cultivars and the characteristics of the cultivars recent evolution since 1949. Cluster analysis suggested that the C. olitorius cultivars could be divided into five groups at a OUSHI distance of 0.95. Two imported cultivars were separated into 2 groups and the remaining 18 cultivars were classified into 3 groups based on several predominant parents, such as Cuilü, Guangfengchangguo, Bachang 4, Maliyeshengchangguo. C. capsularis could be divided into 6 groups at the OUSHI distance 0.90. Two regional cultivars were separated into 2 groups and the remaining 38 cultivars were classified into 4 additional groups based on several predominant parents, such as D154, Qiongshan, Yueyuan 1, Lubinyuanguo, and C46. The coancestry coefficient analyses showed that the corner stone parents for C. olitorius were Cuilü, Guangfengchangguo, Kuanyechangguo, Bachang 4. These had the highest breeding values for C. olitorius. The corner stone parents for C. capsularis were D154, Lubinyuanguo, Yueyuan 1, JRC-212 and C46 played a substantial role among the registered C. capsularis cultivars. 【Conclusion】The majority of jute core parents were earlier developed cultivars, so it is important to enhance the utilization of important jute varieties and introduce novel jute germplasm resources. Breeders’ reliance on backbone parents, such as Guangfengchangguo, Kuanyechangguo, Yueyuan 1, D154, JRC-212, facilitates the loss of plant genetic diversity and certainly increases genetic uniformity in new varieties.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Discussion on the Theory and Methods for Determining the Target Yield in Rice Production

ZOU Ying-bin, XIA Bing, JIANG Peng, XIE Xiao-bing, HUANG Min

Scientia Agricultura Sinica. 2015, 48(20):  4021-4032.  doi:10.3864/j.issn.0578-1752.2015.20.004

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【Objective】The aim of this study was to discuss the theory and methods for determining the target yield in rice production. 【Methods】The discussion and analysis were based on the results of field experiments conducted in five locations (Chengmai of Hainan Province, Huaiji of Guangdong Province, Binyang of Guangxi Province, Changsha of Hunan Province, and Xingyi of Guizhou Province) in South China with different N application rates (zero N application; moderate N rate: 161—176 kg·hm^-2; high N rate: 225 kg·hm^-2) and varieties (hybrid varieties Liangyoupeijiu and Y-liangyou 2 and inbred varieties Huanghuazhan and Yixiangyouzhan ) in 2012 and 2013, and the reports of relevant literature in China and abroad.【Results】The field experiments showed that the yield performance of even the same rice variety exhibited significant or extremely significant differences among the five locations. Under N application conditions (moderate and high N rates), Xingyi had the highest average yield (Liangyoupeijiu: 13.20-13.54 t·hm^-2, Y-liangyou 1: 13.50-13.78 t·hm^-2, Huanghuazhan: 11.26-11.42 t·hm^-2, Yuxiangyouzhan: 11.32-11.45 t·hm^-2), followed by Changsha, Chengmai, Binyang, and Huaiji had the lowest average yield (Liangyoupeijiu: 6.66-6.71 t·hm^-2, Y-liangyou 1: 6.96-7.20 t·hm^-2, Huanghuazhan: 6.96-7.11 t·hm^-2, Yuxiangyouzhan: 7.35-6.86 t·hm^-2). Similarly, the highest average soil-based yield (yield of no N application treatment) was recorded in Xingyi (10.52 t·hm^-2), followed by that in Changsha, Chengmai and Binyang, and the lowest average soil-based yield was recorded in Huaiji (4.53 t·hm^-2). The rice yield under fertilized conditions (namely fertilized yield) (Y_F) depended extremely significantly on the soil-based yield (Y_S). The regression equations under moderate and high N rates were Y_F=0.814Y_S＋3.337 (R²=0.824) and Y_F﹦0.864Y_S＋3.094 (R²=0.839), respectively. The contributions of the soil-based yield (the percentage of the soil-based yield in the fertilized yield) ranged from 64.8% to 85.5% on the average of five locations and from 72.7% to 79.3% on the average of four varieties. The analysis of the data (n=315) collected from previous studies also indicated that there was a significant positive linear relationship between the soil-based yield and the fertilized yield (Y_F=1.031Y_S+2.421, R²=0.523), and the average contribution of the soil-based yield was 67.7%. In addition, the results showed that yield increased by fertilization was tightly negatively related with soil-based yield contribution; grain yield was significantly quadratically related to plant N uptake and N application rate.【Conclusions】Target yield should be varied from site to site. Soil-based yield comprehensively reflects the paddy soil fertility and the climate productivity, and therefore can be used as the basis to determine the target yield in rice production. The target yield for high yielding cultivation of rice can be determined by the regression equation based on the soil-based yield. Improving soil fertility is an important approach for achieving the target yield.

Remote Sensing Estimation of Winter Wheat Theoretical Yield on Regional Scale Using Partial Least Squares Regression Algorithm Based on HJ-1A/1B Images

TAN Chang-wei, LUO Ming, YANG Xin, MA Chang, YAN Xiang, ZHOU Jian, DU Ying, WANG Ya-nan

Scientia Agricultura Sinica. 2015, 48(20):  4033-4041.  doi:10.3864/j.issn.0578-1752.2015.20.005

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【Objective】 It is a key research and application field in agricultural remote sensing to estimate crop yield through remote sensing technology, and provide timely and reliable yield information for regional field production. Accurate estimation of crop yield has important significance to ensure national food security, to constitute the social development planning, to guide and regulate the macro adjustment of planting structure, and to improve the management skills of agricultural enterprises and farmers. In order to further improve the accuracy of estimating winter wheat yields by remote sensing, and display application effect of domestic imaging products in agricultural production, this study constructs theoretical yield estimation model based on the domestic remote sensing image through screening sensitive remote sensing variables of estimating theory yield of winter wheat, so as to achieve theory yield estimation of regional winter wheat by remote sensing and provide a reference to timely understand yield tendency of winter wheat at the different ecological regions. 【Method】 The research used 2010-4-26, 2011-4-28, 2012-4-28 and 2013-5-2 HJ-1A/1B images at winter wheat anthesis stage as remote sensing data and extracted 13 remote sensing variables. In Jiangsu Province, the 5 counties of Taixing, Jiangyan, Yizheng, Xinghua and Dafeng were selected as the experimental sampling area, and representative samples were selected samples in the experimental sampling area, and were measured indoor. A total of 335 measured samples of winter wheat theoretical yield were divided into modeling datasets and testing datasets according to the ratio of 3﹕2. Based on the minimum value of predictive residual error sum of square (PRESS), the number of required principal component model was determined. The yield estimation model was assessed through determination coefficient (R²), root mean square error (RMSE) and determination coefficient (R²). This research was undertaken to make a systematic analysis on the quantitative relationships of satellite remote sensing variables to winter wheat theoretical yield. Depending on the partial least squares regression (PLS), the multivariable remote sensing estimation models and the space level distribution maps of winter wheat theoretical yield were constructed and verified through modeling and testing datasets, and the estimation effect of the PLS model was compared to linear regression (LR) and principal components analysis (PCA) algorithm models, respectively. 【Result】 The results of this research indicated that the majority of remote sensing variables were significantly related to theoretical yield, and there were significant multiple relationships among the majority of remote sensing variables. For the theoretical yield model based on PLS, the number of the best principal components was 4. Structure intensive pigment index, Normalized difference vegetation index, Green normalized difference vegetation index and Plant senescence reflectance index were identified as the sensitive remote sensing variables for estimating winter wheat theoretical yield. Through testing the theoretical yield model based on PLS algorithm with modeling and testing datasets, for the theoretical yield model, the R² were 0.79 and 0.76, respectively, the RMSE were 720.45 kg·hm^-2 and 928.05 kg·hm^-2, respectively, the RE were 11.45% and 13.92%, respectively. The PLS models with selected sensitive variables performed better to estimate winter wheat theoretical yield. PLS algorithm models to estimate winter wheat theoretical yield obtained the higher accuracy by above 25% and above 27% than the LR algorithm models, by above 15% and above 16% than the PCA algorithm models, respectively. The results of applying the PLS model were correspondent with the actual distribution of winter wheat theoretical yield on regional scale and had strong application ability. 【Conclusion】It was concluded that PLS algorithm could provide an effective way to improve the accuracy of estimating winter wheat theoretical yield on regional scale based on aerospace remote sensing, and contribute to large-scale application and promotion of the research results.

The Identification of High-Stable Yield Maize Varieties Using the Henan-Hainan Ecological Difference in Three Growing Season

HUANG Lian-fu, DONG Peng-fei, LI Hong-ping, LI Chao-hai

Scientia Agricultura Sinica. 2015, 48(20):  4042-4055.  doi:10.3864/j.issn.0578-1752.2015.20.006

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【Objective】In order to speed up the screening identification of a high-stable yield and eurytopicity maize hybrid.【Method】This experiment was conducted for three production seasons, using 11 maize varieties (combinations) under 6 kinds of evaluation environments of two ecological regions in Zhengzhou of Henan Province (Henan) and Sanya of Hainan Province (Hainan) during 2011-2012. Through comparisons with the evaluation results of these corn varieties (combinations) in 2011 in nine cities of Henan Province, we analyzed the difference between the two ecological environment conditions to examine the correlation and stability of yield and main agronomic characteristics between them.【Result】These results indicated that the ecological environments were appreciably different between Henan and Hainan, compared with Henan, lighting hours of phases Ⅰ and Ⅱ of Hainan increased by 2.69% and 11.17%, respectively. The average temperature increase was -0.39% and 11.02%, while the rainfall decreased by 15.49% and 8.15% respectively of the maize growth period. The sources of energy in Henan gave priority to shortwave radiation, with phases Ⅰ and Ⅱ in Hainan mainly being longwave radiation. The growth period of phases Ⅰ and Ⅱ in Hainan was shortened by 5.7 days and 7.02 days respectively more than in Henan, yet the main maize agronomic characters in the two places had certain correlations. It could be effective to use the natural environmental conditions of Hainan to identify the growth period duration, plant height, ear diameter, bare tip length, ear rows, numbers of tassel branch, ear-leaf length, and basal third eustipes length of varieties, it is more effective to select in phase Ⅱ of Hainan except the kernel rate. It was appropriate to identify the ear position height, spike length, grain numbers per spike, grain numbers per ear, thousand seed weight, yield, the number of internodes, the ear leaf width, and the base of the third quarter stem diameter. It was appropriate to identify the bacterial wilt and corn rust in Hainan, and leaf spot diseases and maize rough dwarf disease in Henan, with the consistent identification performance for those diseases in both Henan and Hainan. The suitable density in Henan and the arid barren treatment in phase Ⅱ of Hainan were the best among the 6 kinds of evaluation environments, with a strong discriminability and a good representativeness. The normal management of phases Ⅰ and Ⅱ in Hainan came second, while the high density in Henan and the arid barren treatment in phase of Hainan were suitable for the elimination of poor stability varieties. The results showed that the yield differences between different varieties were large under various environmental conditions. It was impossible to exactly evaluate the comprehensive performance of varieties using just one kind of ecological environment.【Conclusion】Partial characteristics of maize varieties (combinations) could be validly selected in Hainan, however, it was more effective to appraise high-stable yield maize varieties (combinations) in all directions using multiple environmental conditions of three quarters of the two locations in one year of Henan and Hainan.

PLANT PROTECTION

Antifungal Activities of Penicillium minioluteum ZF1 and Its Metabolites to Fusarium graminearum

SU Qian-fu, JIA Jiao, MENG Ling-min, LI Hong, ZHANG Wei, JIN Qi-ming, CONG Bin

Scientia Agricultura Sinica. 2015, 48(20):  4056-4063.  doi:10.3864/j.issn.0578-1752.2015.20.007

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【Objective】 Penicillium minioluteum ZF1 was isolated from the soil and identified as having the effect of biological control in previous studies. The objective of this study is to analyze the inhibition effect of the isolate and its culture filtrate with chemical fungicide to Fusarium graminearum, clarify the value of ZF1, and to offer a novel method for controlling the stalk rot and ear rot which are caused by F. graminearum. 【Method】 F. graminearum was inoculated at the mixed PDA and the growth speed was calculated. The effect of secondary metabolites on control of the mycelial growth of F. graminearum was known when cellophane removed and F. graminearum was inoculated. Fludioxonil, ZF1 culture filtrate and the two mixtures were compared each other, and their EC₅₀ and EC₉₅ were measured. The leaves at 5-leaf stage and kernels at milk-ripe stage were inoculated with P. minioluteum, and the process was observed. 【Result】 P. minioluteum ZF1 could obviously inhibit the mycelial growth of F. graminearum. The inhibition ratio reached 81.33%. The area of inhibited mycelial growth of F. graminearum was 16.21 cm² compared with the control groups. The effect of secondary metabolite of P. minioluteum was inoculated on PDA plate removed cellophane which inhibited the mycelia growth of F. graminearum reached 55.46%. The filtrate of P. minioluteum which were diluted 10, 20, 50 and 100 folds inhibited the growth area of mycelia of F. graminearum were 81.04%, 64.46%, 22.67% and 1.12%, respectively. The mycelia of morphology were not changed when ZF1 culture filtrate was added in PDA. The mixture of fludioxonil and 10% culture filtrate of P. minioluteum had the better effect than each single. The EC50 and EC95 values of fludioxonil to F. graminearum were 0.0162 and 0.5287 μg·mL^-1, respectively. The inhibition ratios of 10% culture filtrate of P. minioluteum and 0.5 μg·mL^-1 fludioxonil were 86.45% and 95.13%, respectively. EC₅₀ and EC₉₅ values of the two mixtures were 0.0023 and 0.4011 μg·mL^-1. The effect of fludioxonil inhibition was lost after 4 days. The inhibition effect of the mixture of 0.5 μg·mL^-1 fludioxonil and 10% culture filtrate of P. minioluteum prolonged for 10 days compared with only 0.5 μg·mL^-1 fludioxonil. Slight mold appeared in injured maize kernels and seedling leaves when P. minioluteum was inoculated on them. 【Conclusion】 The inhibition effect of P. minioluteum ZF1 and its culture filtrate on F. graminearum was obvious. The mixture of fludioxonil and culture filtrate of P. minioluteum could prolong the inhibition time to the mycelial growth of F. graminearum. A novel method that P. minioluteum acts as a biocontrol fungus for controlling maize stalk rot and ear rot can be used in the future.

The Antifungal Activities of Bacillus spp. and Its Relationship with Lipopeptide Antibiotics Produced by Bacillus spp.

XIANG Ya-ping, CHEN Zhi-yi, LUO Chu-ping, ZHOU Hua-fei, LIU Yong-feng

Scientia Agricultura Sinica. 2015, 48(20):  4064-4076.  doi:10.3864/j.issn.0578-1752.2015.20.008

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【Objective】 The objective of this study is to clear the inhibition effect of 55 Bacillus spp. strains to pathogenic fungi, Rhizoctonia cerealis, Fusarium graminearum and Fusarium oxysporum f. sp. niveum, and the qualitative and quantitative relationship withlipopeptide antibiotics produced by the Bacillus strains.【Method】Inhibition rates of the 55 Bacillus spp. strains and their crude lipopeptides extracts against three pathogenic fungi were tested, respectively, by dual culture on plate. Three pairs of primers designed based on the known lipopeptide genes were used to amplify the corresponding genes, srfA, ituA and fenA, from the genome of the 55 strains. The lipopeptides antibiotics were identified by high performance liquid chromatography (HPLC) and mass spectrum (MS) techniques.【Result】 The 55 strains of Bacillus spp. and their crude lipopeptides extracts had certain biological activities to the tested pathogenic fungi. The crude lipopeptides extracts of 16.5% strains had higher antifungal activities than the Bacillus strains in vivo, the antifungal activities against the pathogenic fungi of crude lipopeptides extracts of 55.8% strains were weaker than living cells of the strains in vivo, while the antifungal activities of 27.7% strains did not change basically. The results of PCR amplification showed that among the 55 Bacillu strains, the genomes of 89.1% Bacillus strains contained srfA, 87.3% strains contained ituA, only 56.4% strains contained fenA. Among the 55 Bacillus strains, 53 Bacillus strains were identified with surfactins in the crude lipopeptides extracts by the MS analysis, 48 strains were identified with iturins, only Bs916 (B. subtilis 916) was identified having fenfycins in the crude lipopeptides extracts. Those strains that were not identified having the three kinds of lipopeptides had weaker or no antifungal activity. There were 48 strains which were found to have iturins all identified with bacillomycin D, only 2 strains were identified with bacillomycin L, 12 strains were identified with bacillomycin F, iturin A or mycosubtilin. Among the 55 Bacillus strains, 53 strains were detected with suefactins by HPLC, 51 strains were detected with iturins, only Bs916 strain was detected with fengycins and 2 strains were not detected with the 3 kinds of lipopeptide antibiotics. According to the time and type of chromatographic peak, iturins were divided into 4 types, named as types 1, 2, 3, and 4. The antifungal activities intensity of 4 types of iturins were type 1＞type 2＞type 3＞type 4. There were two unknown lipopeptides (named as Unknowns: Unknown1, 2 ) which were speculated as new lipopeptides were detected with MS analysis in 3 strains JCC-1, and JCC-9 and JCC-18. 【Conclusion】 Lipopeptides antibiotics produced by Bacillus spp. were the main composition against plant pathogenic fungi and had diversity characteristics. The structure changes and secretion of iturins had certain relevance to antifungal activities. There were some unknown new resisted resources in lipopeptides compounds from Bacillus spp..

Identification and Expression Analysis of 1-Aminocyclopropane- 1-Carboxylate Oxidase Gene from Quinclorac-Resistant Barnyardgrass (Echinochloa crus-galli)

DONG Ming-chao, YANG Xia, ZHANG Zi-chang, LI Yong-feng, GUAN Rong-zhan

Scientia Agricultura Sinica. 2015, 48(20):  4077-4085.  doi:10.3864/j.issn.0578-1752.2015.20.009

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【Objective】The objective of this study is to clone barnyardgrass (Echinochloa crus-galli) 1-aminocyclopropane-1- carboxylate oxidase gene (EcACO), analyze its expression and test its enzyme activity, and to unravel the quinclorac-resistant mechanism of E. crus-galli to quinclorac.【Method】The partial sequence of EcACO obtained from E. crus-galli transcriptome pyrosequencing was used to design primers for cloning EcACO from quinclorac-resistant and susceptible E. crus-galli. EcACO was then cloned and sequenced. The nucleotide and putative amino acid sequence analysis were compared using DNAman and GenDoc softwares. The transcript levels of EcACO between resistant and susceptible biotype E. crus-galli were determined by real-time quantitative PCR (qRT-PCR) with β-actin gene as the reference. Finally, the open reading frame (ORF) sequences of EcACO from resistant and susceptible biotypes E. crus-galli were inserted into the expression vector pMAL-c5x, respectively. After the recombinant plasmids were transformed into Escherichia coli strain BL21, the fusion proteins were expressed by the induction with 0.4 mmol·L^-1 IPTG for 16 h at 18℃. The soluble proteins were purified with MBP column for the measurement of ethylene released from MBP::EcACO fusion protein. 【Result】EcACO was isolated from E. crus-galli with quinclorac-resistant and susceptible biotypes of E. crus-galli. The ORF of EcACO was 936 bp, encoding 311 amino acids, with pI 5.4 and Mw 35 kD. The deduced amino acid sequences shared high identity with other ACO sequences from Setaria italica (93%), Zea mays (92%) and Sorghum bicolor (91%). Compared with EcACO from the susceptible biotype, five site mutations of EcACO were found in the resistant biotype, of which three site mutations were located in the putative conserved domain. Furthermore, qRT-PCR results showed that there was no significant difference in expression level of EcACO between resistant and susceptible biotype. Using the prokaryotic expression system and the measurement of MBP::EcACO activity, the released amount of ethylene in the MBP::EcACO from susceptible biotype was 2.15 folds higher than that from resistant biotype.【Conclusion】EcACO was identified from quinclorac-resistant and susceptible E. crus-galli. Compared with the susceptible biotype, the EcACO from the resistant one had five amino acid mutations, of which three site mutations were in the conserved domain. This might probably contribute to the reduction of released amount of ethylene and result in quinclorac resistance of E. crus-galli.

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

Temporal and Spatial Variation of Soil Nitrogen Within Growth Stage of Winter Wheat

MA Ya-bin, SONG Xiao-yu, YANG Gui-jun, WANG Ren-hong, QUAN Bin, JING Xia, LIU Xiao

Scientia Agricultura Sinica. 2015, 48(20):  4086-4099.  doi:10.3864/j.issn.0578-1752.2015.20.010

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【Objective】 This study’s aim was to explore the temporal and spatial variation characteristics of ammonium nitrogen and nitrate nitrogen in a soil solution within the growth stage of winter wheat, and to analyze the influence on the change of their content under variable topdressing treatments, in order to provide references for the cultivation and management of field crops. 【Method】To take the 2013-2014 year winter wheat experiment at National Precision Agriculture Experimental Station as an example, two methods including conventional statistics and geostatistics were respectively used to explore the variation characteristics of the time series and spatial structure characteristics of the ammonium nitrogen and nitrate nitrogen, making the interpolation charts of ammonium nitrogen and nitrate nitrogen with ordinary kriging interpolation, and finally analyzing the spatial variation rules of nitrate nitrogen and ammonium nitrogen in a whole growth stage of winter wheat. 【Result】Through the conventional statistic analysis: From the upstage to the harvest stage of winter wheat, the ammonium nitrogen content in soil solution was not obviously influenced by topdressing and had a decreasing trend while the nitrate nitrogen content was influenced by topdressing, and the performance for that change was first a decrease then an increase and decrease; The change of ammonium nitrogen had no difference between different topdressing treatments in the up, jointing, flowering, and filling stages, and has a significant or great significant difference between the D treatment, S treatment, and others in the harvest stage; while that of nitrate nitrogen made no difference between different treatments in the up and flowering stages, and showed a great significant difference between the BH treatment and others in the jointing stage, and showed a great significant difference between the CK and T, D, and S treatments in the filling stage, and showed a significant or great significant difference between the D treatment and others in the harvest stage. Through the geostatistics analysis: The spatial distribution of ammonium nitrogen showed more even, continuous, and higher spatial autocorrelation in the up, jointing, and harvest stages, and showed larger differences and more serious influences by random factors in the flowering and filling stages while that of nitrate nitrogen showed a more even, continuous, and higher spatial autocorrelation in the jointing, filling, and harvest stages, and showed larger differences and a lower spatial autocorrelation in the up and flowering stages. Through analyzing the spatial interpolation charts of ammonium nitrogen and nitrate nitrogen, the content of nitrate nitrogen in the north of experimental area was lower than that in the south in the up and jointing stages, and showed high in the whole experimental area in the flowering and filling stages, and then became higher in the north of the experimental area than that in the south in the harvest stage, while that of ammonium nitrogen in the north of experimental area was lower than that in the south in the up stage, showed a rising trend gradually from the north to the south in the experimental area, and had a strip distribution. In the flowering and filling stages, its spatial distribution had a big difference, in the harvest stage the content of ammonium nitrogen in the north of the experimental area was obviously higher than that in the south. 【Conclusion】Variable topdressing has no influence on the change of ammonium nitrogen and increases the content of nitrate nitrogen in the flowering stage, and there exists something different in changing their content between different treatments within one growth stage.

Winter Wheat Irrigation Simulation in Arid Area Based on AquaCrop Model

TENG Xiao-wei, DONG Yan-sheng, SHEN Jia-xiao, MENG Lu-min, FENG Hai-kuan

Scientia Agricultura Sinica. 2015, 48(20):  4100-4110.  doi:10.3864/j.issn.0578-1752.2015.20.011

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【Objective】 It is important to take an irrigation measure in time according to the drought situation in resisting drought and improving water use efficiency. In view of practical field application, this study explores the applicability in arid area and irrigation simulation in drought year with AquaCrop model. It will provide a guideline in resisting drought and protecting the yield.【Method】 Field surveys and experiments were conducted at Rougu test area and Wugong test area, Shaanxi Province during the typical wheat growing seasons of 2012-2013 and 2013-2014. The model parameters were adjusted with the data of Rougu test area acquired in 2013-2014, and the model was validated with the data acquired in Rougu test area in 2012-2013 and Wugong test area in 2013-2014. The model parameters mainly include canopy growth and canopy senescence coefficient, maximum canopy cover, water productivity and reference harvest coefficient. According to the surveys of the factual irrigation situation in 2012-2013, four irrigation situations were developed to simulate their influence on biomass and yield. Consequently, the optimal irrigation strategy was obtained. Finally, the water use efficiency was calculated.【Result】The R² and RMSE between the simulated and the measured canopy cover were 0.464 and 8.0%, respectively. The R² and RMSE of simulated and measured biomass were 0.889 and 1.662 t·ha^-1, respectively. The RMSE of simulated yield and measured yield was 0.377 t·ha^-1. Under the two scenarios that the wheat was irrigated at the 77th day and the 172th day after planting, the largest biomass could be obtained. The highest yield approaching 6.451 t·ha^-1 could be obtained under the treatment that the wheat was irrigated on the 77th day, the 172th day and the 200th day after planting. The water use efficiencies of Rougu test area (from 2012 to 2013), Rougu test area (from 2013 to 2014) and Wugong test area (from 2013 to 2014) were 1.84, 1.69 and 1.82 kg·m^-3, respectively.【Conclusion】The AquaCrop model could well simulate the development of winter wheat in arid regions, and the different irrigation times and irrigation frequencies have an important effect on winter wheat biomass and yield. Water use efficiency in drought year was higher than in normal year when two more irrigation were added. The experimental results demonstrate that AquaCrop model has a good application prospect in making irrigation strategies in arid area.

HORTICULTURE·STORAGE·FRESH-KEEPING·PROCESSING

Study on Producing Area Classification of Newhall Navel Orange Based on the Near Infrared Spectroscopy

LIAO Qiu-hong, HE Shao-lan, XIE Rang-jin, QIAN Chun, HU De-yu, Lü Qiang1,YI Shi-lai, ZHENG Yong-qiang, DENG Lie

Scientia Agricultura Sinica. 2015, 48(20):  4111-4119.  doi:10.3864/j.issn.0578-1752.2015.20.012

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【Objective】Newhall navel orange (Citrus sinensis L.) fruits from different producing areas in China, exhibit contrasting quality and market values, due to wide-spreading location of various ecologies. Developing a recognition method based on Near-Infrared (NIR) spectroscopy is very important to identify and distinguish fruits from different producing areas. 【Method】One representative orchard was selected from 17 main producing areas distributed throughout southern China, from which one 100 Newhall navel orange samples were collected. NIR spectra were collected with a SupNIR-1500 spectrograph from the surface of fruit equator and shoulder and the filtered juice for each sample, which were further preprocessed through principal component analysis (PCA) for reduced dimensions and noise. By studying artificial neural network (ANN), a classic three-layer ANN model was established with an input layer, a hidden layer of non-linear activation function and an output layer. By studying support vector machine (SVM) with the radial basis function (RBF) being the kernel function and the principal components of NIR spectra being the input, a one-to-one extended SVM model was established with 126 classifiers. Gene algorithm (GA) with excellent natural selection was used to identify the best Genetic character subset from the principal components as inputs of a SVM classifier, thus a GA-optimized SVM model was composed. These three models were used to classify the NIR spectra of filtered juice, thus the production areas of the oranges, the classification accuracies of which decided the best classifier. Furthermore, the accuracies of the best classifier were tested with the NIR spectra from fruit equator and shoulder surface being the inputs. As a comparison, the best NIR spectra could be identified. 【Result】 Producing area classification was implemented with the three-layer ANN classifier with NIR spectra of Newhall orange juice as the input, where the classifying accuracy reached up to 81.45% when there were 11 input neurons and 13 hidden neurons. The studied one-to-one extended SVM classifier with radial basis function being the core function, exhibited higher accuracy of 86.98% when the number of PC was 20, better than ANN classifier. For GA-SVM classifier took into account the interaction of individual inputs, where the PCA-processed results were optimized by GA. During the experiments, classification accuracy hit 89.72% when the population, generation, mating probability, and mutation probability were 200, 100, 0.7 and 0.01 respectively, surpassed ANN and SVM classifier. Subsequent research found the highest accuracy of GA-SVM classifier was 80% when taking the spectra from the fruit equator, and 69% from the shoulder, not good enough as that of orange juice.【Conclusion】Considering the accuracy, GA-SVM classifier was regarded with the most excellence among three investigated classifiers. Spectra of orange juice were selected as the best data to analyze origins traceability. Accuracy of spectra of fruit equator was inferior to juice but superior to the shoulder, thus had the potential for non-destructive origins classification.

Analysis of Aroma Components in Xihu Longjing Tea by Comprehensive Two-Dimensional Gas Chromatography- Time-of-Flight Mass Spectrometry

ZHU Yin, YANG Ting, SHI Jiang, YU Fang-lin, DAI Wei-dong, TAN Jun-feng, GUO Li, ZHANG Yue, PENG Qun-hua, Lü Hai-peng, LIN Zhi

Scientia Agricultura Sinica. 2015, 48(20):  4120-4146.  doi:10.3864/j.issn.0578-1752.2015.20.013

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Aroma is one of the most important factors affecting the quality of tea, and identifying the chemical composition of tea aroma will enrich the basic theory of aroma chemistry of tea and establish an important theoretic ground to improve and enhance the aroma quality of tea. 【Method】 Aroma components of Xihu Longjing tea were analyzed by using a comprehensive two- dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and gas chromatography-mass spectrometry (GC-MS), and the separating capacities of GC×GC-TOFMS and GC-MS were compared. Then, the qualitative and relative quantitative analyses of aroma components separated by GC×GC-TOFMS were carried out according to the standard mass spectra in available databases, retention time, structured chromatograms and peak areas. Furthermore, the characteristic aroma compositions of Xihu Longjing tea were discussed according to the odor characteristics of its compounds with relatively high contents (≥0.5%). 【Result】 GC×GC-TOFMS technique displayed the strong superiority on the separating capacity by comparing the total ion chromatograms and chromatographic peaks with GC-MS. A total of 522 common volatile components were identified with a good match, and then further classified into 20 types of compounds, such as enols, alkenes, amines, alkanes, aldehydes, olefin aldehydes, ethers, alcohols, esters, lactones, allyl esters, ketenes, ketones, phenols, organic acids, sulphur compounds, nitrogen heterocyclic compounds, oxygen heterocyclic compounds, aromatic hydrocarbons, and alkynes; and the largest numbers of aromatic hydrocarbons (77), large numbers of alkanes (50), alkenes (43), esters (43) and ketones (41), and the minimum numbers of alkynes (3) were included. Relative quantitative analysis showed that 50 volatile components, including 1-penten-3-ol, cis-hex-3-en-1-ol, linalool, α-terpineol, geraniol, butane, methylcyclopentane, 2,2,4,6,6-pentamethylheptane, dodecane, heneicosane, tetracosane, hentriacontane, acetaldehyde, pentanal, hexanal, furfural, heptanal, phenylacetaldehyde, nonanal, 1-ethoxypropane, 2-ethoxybutane, 1-ethoxybutane, 1,1-diethoxyethane, 1-ethoxypentane, pentanol, tert-butanol, benzyl alcohol, phenethyl alcohol, phytol, diisobutyl phthalate, dibutyl phthalate, ethyl 2-(5-methyl-5-vinyltetrahydrofuran-2-yl) propan-2-yl carbonate, (Z)-hex-3-en-1-yl hexanoate, methyl palmitate, acetoin, 2,4-di-tert-butylphenol, 3-methylbutanoic acid, nonanoic acid, hexadecanoic acid, linoleic acid, octadecanoic acid, dimethyl sulfoxide, benzothiazole, indole, caffeine, linalool oxide (pyranoid), 2,3-dihydrobenzofuran, ethylbenzene, phenylpropane, and 1-methylnaphthalene, were found to be relatively high contents in aroma components of Xihu Longjing tea, suggesting that they played an important role in effecting the aroma quality of Xihu Longjing tea. The analysis of characteristic aroma compositions indicated that enols, aldehydes, alcohols, esters and aromatic hydrocarbons with agreeable smell would be responsible for the outstanding aroma quality of Xihu Longjing tea, and the effects of alkanes, ethers, organic acids and sulphur compounds with no special smell or offensive odor and aroma compounds with low odor threshold values were also worth future research. 【Conclusion】 The successful application of GC×GC-TOFMS technology in tea aroma analysis has improved the number of identified compounds significantly, which could make up for the defects of GC-MS analysis to some extent and supply the advanced technical support for the future in-depth studies on the chemical composition of tea aroma and formation mechanism of tea aroma quality.

Optimization of the Process Conditions and Change of Volatile Flavor Components of Longan Pulp Fermented by Lactic Acid Bacteria

LIU Lei, WANG Hao, ZHANG Ming-wei, ZHANG Yan, ZHANG Rui-fen, TANG Xiao-jun, DENG Yuan-yuan

Scientia Agricultura Sinica. 2015, 48(20):  4147-4158.  doi:10.3864/j.issn.0578-1752.2015.20.014

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【Objective】The optical fermentation conditions of longan pulp by lactic acid bacteria were established, and the change of volatile flavor compounds of longan pulp before and after fermentation was clarified. This research can guide the development of functional longan beverage. 【Method】Lactic acid bacteria (Lactobacillus bulgavicus﹕Streptococus thermophilus=1﹕1) was domesticate in the medium containing 60%, 70%, 80% and 90% longan pulp and 10% skim milk. The acidity and pH of longan pulp were analyzed during the domestication. The conditions of fermented longan pulp by lactic acid bacteria were optimized through Box-Benhnken central composite experimental design based on the indicators of total acid. A regression model of four factors was established including the fermentation time, fermentation temperature, the content of skim milk powder and inoculation amount of lactic acid bacteria. Separation and analysis of the main volatile flavor compounds in longan pulp before and after fermentation were performed by headspace solid phase micro extraction and gas chromatography - mass spectrometry. 【Result】The lactic acid bacteria with good fermentation ability was obtained in longan pulp with high concentration. After 12 h of fermentation, the acidity of 90% longan pulp increased from 9.5ºT to 104.4ºT, and pH decreased from 7.12 to 4.44. The acidity of 90% longan pulp was significantly higher than that of 60% and 70% longan pulp (P<0.05), there was no significant difference of pH between longan pulp with four concentrations. The results showed that the optimum conditions are as follows: fermentation time 12h, fermentation temperature 45℃, 5% the content of skim milk powder and 3% inoculation amount of lactic acid bacteria. Under this condition, the acidity of fermented fruit pulp increased from 9.5ºT to 105.1ºT. The relative error to the value predicted by the model was 1.2%. It indicated that this model can be used in practical production. The volatile flavor compounds in longan pulp significantly changed after the fermentation. 53 volatile flavor compounds were detected in fermented longan pulp, which contained 15 terpenes, 6 alcohols, 18 esters, 7 ketones, 3 aldehyde, 2 acids and 2 others. 18 volatile flavor compounds were newly produced by fermentation. After the fermentation, the contents of alcohols, aldehydes, esters, ketones and acids increased, and the contents of terpenes decreased. The main volatile flavor compounds in fermented longan pulp were ethanol 25.68 μg·g^-1, 4-(1-methylethyl)- benzenemethanol 2.42 μg·g^-1, acetaldehyde 1.95 μg·g^-1, benzaldehyde 1.25 μg·g^-1, ethyl acetate 2.19 μg·g^-1, methyl benzoate 1.05 μg·g^-1, methyl salicylate 1.93 μg·g^-1, 3-hydroxy-2-butanone 1.085 μg·g^-1, (E)-β-ocimene 97.81 μg·g^-1, allo-ocimene 1.923 μg·g^-1, and acetic acid 1.84 μg·g^-1, respectively. Benzaldehyde and ethyl acetate were newly generated. The contents of ethanol, acetaldehyde, allo-ocimene and acetic acid increased after the fermentation. 【Conclusion】 Lactic acid bacteria fermentation significantly increased total acid and volatile flavor compounds of longan pulp. Longan pulp beverage fermented by lactic acid bacteria with a unique flavor can be developed by controlling fermentation conditions.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

The Optimization of Transfection Conditions for Transfecting Pluripotent Transcription Factors mRNA to GEF Cells

XIAO Tian-rong, ZHANG Lei, QIU Feng-long, LI Wei, ZUO Qi-sheng，LI Dong, LI Bi-chun

Scientia Agricultura Sinica. 2015, 48(20):  4159-4169.  doi:10.3864/j.issn.0578-1752.2015.20.015

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【Objective】To optimize the conditions for transfecting pluripotent transcription factors oct4、sox2、klf4 and c-myc mRNA to GEF cells, this study focused on transfection reagents and transfection methods. 【Method】GEF cells were separated from goat fetus leg muscle tissue via trypsin digestion method, and transcription factors then purified Using plasmid mini kits obtained from bacteriums. Then each downstream of the target genes in transcription carrier was extracted with a mono-restriction endonuclease enzyme. DNA purification kits were used to purify all the linear plasmids. Based on the linear plasmid, 5' end cap structure of mRNA was synthesised. TURBO DNase was used to eliminate the DNA template and the mMESSAGE mMACHINE® T7 Ultra Kit was use to add "tail" to the mRNA. Finally, MEGAclear™ Kit was used to purify the full mRNA. Five sheep derived recombinant plasmids: pCDNA3-oct4、pCDNA3-sox2、pCDNA3-klf4、pCDNA3-c-myc and pCDNA3-EGFP were linearized and then transcribed into mRNA in vitro respectively. GEF cells were transfected with different proportions of total mRNA (each of the five transcription factors mRNA was 0.2g). We used Lipofectamine 2000 by ratios of 1:0.5、1:1.0、1:1.5 and 1:2.0 to get the best transfection proportion. Using the best transfection proportions, transfection efficiencies were compared within the 2, 4, 6 generations of GEF cells. Immunofluorescence assay was performed to verify the transfection effect. 【Result】 The best transfection proportion was 1:1.0 (total mRNA/ Lipofectamine 2000), and the transfection efficiency of 2 generation was significantly higher than other generations of GEF cells under the best transfection proportion（P＜0.05）.This group of cells was good in both morphology and cell growth. Immunofluorescence assays showed that four pluripotent transcription factors mRNA were located in the nucleus of GEF cells, and obtained stable expression, while there was no expression of these four pluripotent transcription factors in the cytoplasm of GEF cells. After 24h of transfection by 4 pluripotent transcription factors, GEF cells turned slowly from fusiform to circular, and the cell activity was lower than the control group (normal GEF cells). 【Conclusion】 The best transfection condition for transfecting pluripotent transcription factors mRNA to GEF cells was at the 2 generation of GEF cells and under the 1:1.0 transfection proportion of total mRNA/ Lipofectamine 2000. This study provides proper parameters for transfecting cells or ES by mRNA , and also for obtaining iPS cells. We find indirect evidence for exogenous pluripotent mRNA in GEF cells and lay the foundation for further study of pluripotent transcription factors oct4、sox2、klf4 and c-myc.

The Geographical Distribution and Multivariate Analysis of Chinese Indigenous Sheep Breeds and Their Meat-Related Traits

WANG Hui-hua, ZHAO Fu-ping, ZHANG Li, WEI Cai-hong, DU Li-xin

Scientia Agricultura Sinica. 2015, 48(20):  4170-4177.  doi:10.3864/j.issn.0578-1752.2015.20.016

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【Objective】 China has among the world’s most abundant animal genetic resources; the number of mammal and poultry breeds account for 1/6 of global known taxa. Animal Genetic Resources in China was investigated and edited under the ministry of agriculture’s leadership and organization over 8 years. This book summarizes the geographical distribution and characteristics of China's indigenous and imported cultivars and species of imported livestock. We analyzed some production traits of Chinese indigenous sheep breeds using the section, sheep and goats of Animal Genetic Resources in China, We expected to determine cultivation material suitable for meat breeding. 【Method】 13 meat-related traits including data on body size, carcass and reproductive traits of indigenous sheep breeds were summarized from this resource. First, the indigenous breeds were divided into Mongolian, Kazakh and Tibetan groups and then their traits were analyzed using multiple comparison, principal component and hierarchical clustering methods within the statistical program R environment. 【Result】 Chinese indigenous sheep breeds are widely distributed according to our generated geographical distribution map. Four provinces (autonomous regions) of Xinjiang, Yunnan, Inner Mongolia and Shandong have relatively high level of diversity which respectively contain 13, 7, 5 and 5 breeds. Chinese indigenous sheep were mainly concentrated in the north, northwest and southwest plateau of China. The Kazakh group breeds mainly concentrate in the Xinjiang area, while the Tibetan group is on the Yunnan-Guizhou plateau. The Mongolian group is the largest and most widely distributed in the northern, central, and eastern parts of China. The multiple comparison analysis indicates that the productions performance of the Mongolia group is best, and Kazakh is worst. Three major lineages are distinguished via principal components 1 and 2, These results infer that Large-tailed Han, Guangling Large-tailed and Yecheng sheep belonged to the Mongolia group; Minxian Black Fur sheep belonged to the Tibetan. Chinese indigenous sheep were split into three categories by cluster analysis which we called low-yield & reproduction, high-yield & low-reproduction and high-yield & reproduction respectively. The high-yield & reproduction group held 7 breeds: Dulang, Large-tailed Han, Wadi, Taihang Fur, Yuxi Fat-tailed and Hu sheep. 【Conclusion】 Our analysis indicates that unclassified sheep breeds can be distinguished and inferences made as to which recognized group they belong based on 13 quantitative traits; moreover this method can be applied to the classification of newly discovered sheep breeds resources. We also found seven breeds from 42 Chinese indigenous sheep breeds were close to a specialized meat breed, and supporting to sheep bred or hybrid as the ideal material. This study not only recognizes Chinese indigenous sheep genetic resources, but also provides some references to help analyze other livestock types by the use of Animal Genetic Resources in China.

Cloning of Bos Grunniens HSP27 Gene and Its Expression in the Female Yak Reproductive Organs

HE Hong-hong, CUI Yan, PAN Yang-yang, FAN Jiang-feng, HU Wei, ZHANG Yi-fu, LIU Peng-gang，LI Qin, YU Si-jiu

Scientia Agricultura Sinica. 2015, 48(20):  4178-4187.  doi:10.3864/j.issn.0578-1752.2015.20.017

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【Objective】The objective of this study was carried out to study differences of the expression of Heat Shock Protein 27(HSP27) gene in the main reproductive organs of the female yak under normal physiological conditions by cloning the HSP27 gene and analyzing its biological characteristics. 【Method】 Samples from the tissues of ipsilateral Ovary, oviduct and uterus during follicular anaphase, luteal anaphase and early pregnancy phase were collected and cDNA were isolated from each of the collected tissues. RT-PCR was used to clone the HSP27 gene, and purified PCR products were cloned on pMD^TM18-T Vector to detect the sequence. The genetic characteristics of HSP27 gene were then analyzed utilizing bioinformation software to predict its product protein structure and potential functions. Next, RT-qPCR was employed to reveal the relative expression of HSP27 gene in the main reproductive organ during the reproductive cycle of the yak. Statistical analysis was performed using the software program SPSS (version 19.0, SPSS). 【Result】HSP27 gene sequence containing a complete coding sequence, with the coding region length of 450bp (GenBank accession No: KP716832), This length could encode 149 amino acids of which Leucine (10.7%) was the most, and Tryptophan (0.7%) the least abundant. We determined that the atom number, molecular formula, calculated molecular weight and theoretical isoelectric point of the encoded protein were 2366, C₇₄₇H₁₁₈₉N₂₀₅O₂₂₀S₅, 16.722 kD, and 5.33 respectively. The HSP27 encoded protein of HSP27 was predicted to be a type of soluble and non-transmembrane protein. Nucleotide sequence analysis revealed that the HSP27 gene nucleotide sequence of yak was similar to those of Bos taurus (99.8%), Bubalus bubalis (98.4%), Ovis aries (97.8%), Pantholops hodgsonii (97.6%), Orcinus orca (90.3%), Camelus ferus (89.7%), Sus scrofa (89.7%), Equus caballus (86.7%), Canis lupus (86.7%), Homo sapiens (85.5%) and Gorilla gorilla (85.3%). The similarity of resulting amino acid sequence of HSP27 gene between yak and bos taurus, bubalus bubalis, ovis aries pantholops hodgsonii, orcinus orca, camelus ferus sus scrofa, equus caballus, canis lupus, homo sapiens and gorilla gorilla were 100%、100%、100%、100%、96.3%、100%、96.8%、100%、100%、96.3% and 96.3%, respectively. The phylogenetic tree indicated that HSP27 gene of yak was close to bos taurus, bubalus bubalis, ovis aries and pantholops hodgsonii, but far from canis lupus, gorilla gorilla and homo sapiens. Moreover, the RT-qPCR indicated that HSP27 gene expressed universally in ovary, oviduct and uterus tissues during all reproductive periods, including follicular, luteal and pregnancy phases. The gene had its highest expression level in the follicle at follicle phase and its lowest expression in uterus in the luteal phase. In addition, the expression level of HSP27 gene in ovary was significantly higher than in the uterus during each of the different stages of the reproductive cycle. Its expression in ovaries, fallopian tubes and uterus changed dramatically because of pregnancy.【Conclusion】It is concluded from the present study that HSP27 gene in the yak is highly conservative in mammalian evolution according to the comparative nucleotide sequence analysis.. Analyzing HSP27 gene expression in central reproductive organs during the breeding cycle, it was found that HSP27 was related to follicular development and maturation and other important physiological processes related to reproduction and specifically may play an important biological role in pregnancy.

RESEARCH NOTES

Transient Silencing of HCP1 Gene in Wheat Mediated by Agrobacterium

LIU Peng, WANG Shuai-shuai, YANG Gao-shan, ZHANG Heng, HAN Sheng-fang, WANG Dong-mei

Scientia Agricultura Sinica. 2015, 48(20):  4188-4196.  doi:10.3864/j.issn.0578-1752.2015.20.018

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【Objective】Through transient silencing of the wheat HCP1 gene mediated by Agrobacterium, a simple, rapid, high efficient and reliable transgenic technique in wheat to identify the gene functions can be developed. The role of HCP1 gene in wheat in scavenging H₂O₂ that induced by inoculating Puccinia triticina was investigated using the transgenic plants overexpressing this target gene. The purpose of this study was to provide a theoretical basis for further understanding the signal transduction mechanism initiated by Puccinia triticina in wheat. 【Method】The Agrobacterium cells carrying the peGFP vector were cocultured into the wheat leaves, the explants for wheat transformation. The intensity and distribution of the fluorescence signal in leaves mediated by Agrobacterium strains of EHA105, LBA4404 and C58C1 were detected and the expressions of exogenous gene in leaves were examined. The transient silencing efficiency of HCP1 mediated by RNAi in the transgenic plants was detected by semi-quantitative PCR and real-time quantitative PCR as well as the Rohringer staining analysis. RT-PCR and qRT-PCR analyses were also performed to determine the expression of HCP1 after 12 h inoculation in strain C58C1, and after 72-96 h coculture in strains EHA105 and LBA4404. The changes of H₂O₂ stained by DAB were observed and the function of HCP1 was investigated in the transgenic leaves with transient silencing of the HCP1 gene inoculated race 260, an incompatible P. triticina strain.【Result】The GFP fluorescence signals were strongly detected at 48 h after inoculation of Agrobacterium strains with the explants. Three Agrobacterium strains all efficiently mediated the HCP1-RNAi transformation, with the strongest silencing efficiency by strain C58C1, followed by strains EHA105 and LBA4404. Rohringer staining suggested that strain EHA105 had a high efficiency to mediate the RNAi effects of HCP1, indicating that this strain had a high toxicity to cause the defensive reaction in cells. By contrast, strain C58C1 had higher silencing efficiency and less toxicity than strain EHA105, indicating that C58C1 could be used as the Agrobacterium strain to mediate wheat transformation. DAB staining revealed that H₂O₂ accumulated largely in wheat leaves with the transient silencing HCP1, showing comparable rate with the inoculation progress of P. triticina race 260. Thus, the accumulation of H₂O₂ was shown more amount in the transgenic leaves than the control group at times of 24 h and 48 h after inoculation of the leaf rust race.【Conclusion】Strain C58C1 inoculated with the binary plasmids for 72 h could transform wheat leaves with high efficiency. Transgenic analysis confirmed that HCP1 gene is involved in scavenging H₂O₂ when wheat inoculated with P. triticina strain.