Table of Content

01 May 2014, Volume 47 Issue 9

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Identification of Wheat Key Autophagy-Related Factor ATG18 and Profiling of Their Expression Under Biotic and Abiotic Stresses

SUN Hong, ZHANG Wei, WEI Xiao-Jing, WANG Hua-Zhong

Scientia Agricultura Sinica. 2014, 47(9):  1657-1669.  doi:10.3864/j.issn.0578-1752.2014.09.001

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【Objective】 One objective of this study is to identify wheat homologues of autophagy-related gene ATG18, to analyze the sequence characteristics and 3-D structures of their encoding proteins. The other objective is to profile their expression patterns under biotic and abiotic stresses and under phytohormone applications, thus laying a foundation for elucidating their biological functions. 【Method】 EST assembly and RT-PCR were carried out to clone ATG18 cDNA sequences from RNA pool generated from the powdery mildew (Pm) causal fungus Blumeria graminis f.sp. tritici (Bgt)-challenged wheat leaves. Bioinformatics tools were employed to analyze gene exon-intron models, to predict protein domains, conserved amino acids and 3-D structures and to analyze the phylogenetic relationships among plant orthologues. Real-time fluorescence quantitative PCR was used to investigate gene expression patterns in responses to Bgt infection, treatments of various phytohormones or exposure to conditions of high salinity, drought, low temperature and darkness or nitrogen deficiency. 【Result】 Four members (TaATG18a, 18b, 18c and 18d) of the wheat ATG18 family were identified in this study. They are of great similarity and each has an ORF of 1 158 bp encoding a protein of 385 amino acids. The deduced protein sequences of TaATG18s consist of two classic WD-40 domains, one phosphatidylinositol 3-phosphate (PI3P) binding motif and the conserved amino acids at the ATG2-binding site. The four TaATG18s all have two kinds of alternatively spliced mRNA isoforms, respectively encoding the complete functional protein form and the N-terminal truncated non-functional form missing the WD-40 domain and the conserved functional residues. TaATG18a was predicted to fold into a six-blade β-propeller-like structure similar to other WD-40 proteins. The PI3P binding motif is located at the strand 4 of blade 5 and the loop connecting blades 5 and 6, and the ATG2 binding site is located at the loop connecting blades 2 and 3. Expression of TaATG18s can be upregulated by biotic stress of Bgt infection, however, the detailed regulation patterns by Bgt infection differed between wheat-Bgt compatible and incompatible reactions. In the two incompatible reactions triggered by the broad-spectrum Pm resistance gene Pm21 and the isolate-specific Pm resistance gene Pm3f, two times of transcripts accumulation of TaATG18s were observed during 0-36 hours after inoculation of Bgt, which are closely related to the progression of Bgt infection. Although TaATG18s of the moderately susceptible line Yangmai 158 also showed a bimodal Bgt-induced expression profile, the first time of transcripts accumulation had a shorter duration and a lower peak than that observed in the incompatible reaction on Pm21-carrying Yangmai 158 isogenic line. Expression of TaATG18s of the highly susceptible line Chancellor fluctuated little in response to Bgt infection. Differences were also found between the resistant line and the susceptible line as to their TaATG18s expression responding to the same exogenously applied phytohormone salicylic acid (SA) or ethylene, which showed negative effects on the resistance line but positive effects on the susceptible line. Phytohormones regulated the expression of TaATG18s not only at the transcription level, but also at the process of mRNA splicing. Expression of TaATG18s can also be upregulated by abiotic stresses of high salinity, drought, low temperature and darkness or nitrogen deficiency. 【Conclusion】 Proteins encoded by TaATG18s identified here probably participate in the wheat autophagy process through their PI3P-mediated autophagic membrane localization and the formation of ATG18-ATG2 complex. TaATG18s and their involved autophagy are implicated in the wheat immune response to Bgt infection and in the wheat responses to various abiotic stresses. Different expression responses of TaATG18s to one kind of phytohormone SA or ethylene in the resistant and the susceptible lines may partially account for the different phenotype outcomes of Pm resistance or susceptibility.

Relationship Between Microspore Abortion of CMS Lines Associated with Nutrient Metabolism Disorder in Tapetal of Anther in Wheat (Triticum aestivum L.)

ZHANG Peng-Fei, SONG Yu-Long, ZHANG Gai-Sheng, ZHAO Xin-Liang, BA Qing-Song, LIU Hong-Zhan, ZHU Wan-Wan, LI Zhi-Kuan, WANG Jun-Wei, NIU Na

Scientia Agricultura Sinica. 2014, 47(9):  1670-1680.  doi:10.3864/j.issn.0578-1752.2014.09.002

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【Objective】 The aim of the experiment is to explore the relationship between tapetum degradation and nutrient metabolism in anther and pollen abortion in CMS of wheat. 【Method】 Wheat S-1376A CMS [(S)-1376 (A) ] and its maintainer line [(A)-1376 (B) ] were used as test materials. The development condition of the nucleus of pollens at tricellular was surveyed through dyed by DAPI, and stained by KI-I2 to observe the starch accumulated in pollens at tricellular. Semi-thin sectioning was employed to observe and compare tapetum development process, polysaccharides, lipids and proteins accumulation in the sterile line and maintainer line. The cell Sene Entry was utilized to collect images and to calculate the area of the microspores and the tapetum cells in the section. IPP 6.0 was adopted to analyze the mean optical density and IOD at each stage in 1376 and (S)-1376. 【Result】 The data demonstrated that in (S)-1376, compared with 1376, the tapetum cells in advance degradated at the tetrad stage, and the pollen nucleus development was retarded, the majority of which merely developed the late uninucleate. Pollens were dyed yellow by KI-I2 at tricellular in (S)-1376, which certified there was no starch present in the pollens result in them thorough abortion. Area of the microspores and the tapetum cells in the section was extremely huge at the tetrad in (S)-1376. In (S)-1376, compared with 1376, a great number of starches and proteins accumulated in microspores at the tetrad stage, but from late uninucleate stage this situation undergone dramatic difference that the starches and proteins significantly decreased; whereas the lipids were significantly poorer in each period. In (S)-1376, polysaccharides and proteins were significantly higher than 1376 in the tapetum in tetrad, however, polysaccharides were less in early uninucleate and proteins were low in late uninucleate, respectively. Notably, lipids were lower in each period in the tapetum in (S)-1376. The contents of polysaccharides and proteins were greater at tetrad stage in (S)-1376, while at the late uninucleate the polysaccharides, lipids and proteins were lower in (S)-1376 than that in 1376 ones. At bicellular and tricellular of (S)-1376, the substances of the cytoplasm were no longer increased, meanwhile starches and lipids stopped growing, too, the big vacuoles of the vegetative cell were still present and the pollens became malformation at last. 【Conclusion】 Tapetum of (S)-1376 degraded more earlier than (A)-1376 that caused nutrient metabolism abnormal, leading to the supplying of the polysaccharides, lipids and proteins were aberrant from the late development stage of uninucleate to binucleate, and then the nuclear of pollens fission was abnormal, ultimately, the pollens of (S)-1376 were aborted directly.

Genetic and QTL Analysis of Root Traits at Seedling Stage in Soybean [Glycine max (L.) Merr.]

LIANG Hui-Zhen-1, YU Yong-Liang-1, YANG Hong-Qi-1, ZHANG Hai-Yang-1, DONG Wei-1, CUI Wei-Wen-1, GONG Peng-Tao-2, FANG Xuan-Jun-3

Scientia Agricultura Sinica. 2014, 47(9):  1681-1691.  doi:10.3864/j.issn.0578-1752.2014.09.003

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【Objective】 Roots provide nutrition and affect crop yields. Root traits are believed to be complex and controlled by many genes, which show stability in different environments and present higher heritability. Study of such traits by QTL mapping of roots at seedling stage is needed for effective heredity breeding in soybean. The map spanned 2 047.6 cM across 27 linkage groups that contained 232 markers. 【Method】 With the methods of segregation analysis on the major-polygene mixed inheritance and composite interval mapping, a soybean SSR genetic linkage map constructed by a total of 447 recombinant inbred lines (RILs) derived from a cross between Jindou 23 (cultivar, female parent) and Huibuzhiheidou (semi-wild; male parent, ZDD2315) were used to analyze and identify the QTLs of root traits during seedling stage in soybean. Thirty seeds from each of the RILs and their parents were covered with pasteurized paper, and cultivated in clean water at 20-28℃ on 27 May, 2013, and the experiment was finished on 28 June, 2013. A complete random design with three replications was used. Root traits were measured on 8 June and 8 July at V2 stage.【Result】This grouping was supported by significant positive correlations among MRL, LRN, RW, RV and SW. There were highly significant positive correlations between HL and HW, and HL and SW. Root traits showed mutual influence and restriction, laying a foundation for selection of phenotypic traits. The inheritance analysis showed that the length of main root was controlled by three pairs of equivalent major genes; the number of lateral roots was controlled by two pairs of major genes; the weight and volume of root were controlled by four pairs of major genes; the length of hypocotyl was controlled by four pairs of additive major genes; the weight of hypocotyl was controlled by four pairs additive-additive × additive epistatic major genes. The polygene effect wasn’t found in all above root traits. The weights of leaf and stems were controlled by additive genes, but didn’t find major gene effects. Twenty-four QTLs associated with length of main root, number of lateral root, volume of root, weight of leaf and stems, length of hypocotyl and weight of hypocotyl were mapped on A1, A2, B1, B2, C2, D1b, F_1, G, H_1, H_2, I, K_2, L, M, N and O linkage groups respectively. Five QTLs for MRL were identified on linkage groups B1, L, N and O, respectively. The variation accounted for by each of these five QTLs ranged from 7.05% to 13.18%. Four QTLs for LRN were identified on linkage groups A1, D1b, I and L. The phenotype variation accounted for by each of these four QTLs ranged from 8.21% to 16.43%. Three QTLs for RW were identified on linkage groups F_1, G and N, respectively; the phenotype variation accounted for by each of these three QTLs ranged from 7.55% to 10.85%. Three QTLs for RV were identified on linkage groups K_2 and M based on the data from 27 May. The phenotype variation accounted for by each of these three QTLs ranged from 8.44% to 12.39%, but the main QTL was not detected in the experiment on 28 June. Five QTLs for SW were identified on linkage groups A1, A2 and N. They accounted for phenotype variation ranging from 11.43% to 38.91%. qSW1-a2-1, qSW2-a2-1 and qSW2-a2-1 were all located on chromosome A2. Based on the data for HL on 27 May, the QTL was located in linkage group H_1. It explained 7.86% of the phenotype variation, but the main QTL was not found on 28 June. Three QTLs for HW were identified on linkage groups B2, C2 and H_2. They accounted for phenotype variation ranging from 7.70% to 12.48%.【Conclusion】The inheritance control of root traits is very complicated. Root traits have complex genetic mechanisms at the seedling stage. This study has provided a foundation for further research on root genetic regulation and molecular breeding with emphasis on correlations among root traits to ensure robust root growth and well-developed root systems.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Relationship Between Water Storage Conservation in Fallow Period and Grains Protein Formation in Dryland Wheat

SUN Min, GE Xiao-Min, GAO Zhi-Qiang, REN Ai-Xia, DENG Yan, ZHAO Wei-Feng, ZHAO Hong-Mei

Scientia Agricultura Sinica. 2014, 47(9):  1692-1704.  doi:10.3864/j.issn.0578-1752.2014.09.004

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【Objective】 Rainfall during wheat growth period in dry land of China is less and uneven distribution, how to keep water conservation in fallow period to fulfill wheat production development in growing period is a hot spot of current research. This paper aimed at providing a theoretical basis for improving grain quality and yield based on improving soil moisture level, with the effective utilization of precipitation in fallow period. 【Method】 The experiment was carried out for 3 consecutive years (2009-2012) in Wenxi, Shanxi to study the effect of deep tillage and deep scarification in fallow period on soil water storage at the depth of 0-300 cm and grain protein formation.【Result】The results showed that soil water storage before sowing at the depth of 0-300 cm was improved in fallow period, and it was improved by 63-91 mm in dry year, by 41-70 mm in normal year and by 54-74 mm in humid year. The grain yield was significantly increased by tillage in fallow period, and it was improved by 981-1 330 kg•hm-2 in dry year, by 883-1 089 kg•hm-2 in normal year and by 1 256-1 457 kg•hm-2 in humid year. Deep tillage had better effects in dry and normal year, while deep scarification had better effects in humid year. Tillage in fallow period significantly improved the activity of glutamine synthetase (GS) and glutamate synthase (GOGAT) of post-anthesis 5-15 d in flag leaf in normal and humid year, and the activity of GS and GOGAT in grains under different rainfall years. It also improved grain albumin, gliadin, glutenin, protein content in normal and humid years, glu/gli in dry and humid year, and grain protein yield under different rainfall years. Grain protein yield in dry year and grain albumin, gliadin, protein content and yield in normal year under deep tillage in fallow period were significantly higher than the deep scarification in fallow period. Grain protein and its components content, protein yield and glu/gli in humid year under deep scarification significantly higher than deep tillage in fallow period. Under the condition of tillage in fallow period in different rainfall years, soil water in anthesis influenced the activity of GS and GOGAT in flag leaf, especially the activity of GS in flag leaf, and the activity of GS in flag leaf had a correlation with grain protein and its components content, glu/gli and protein yield, soil water in anthesis had a correlation with grain globulin, gliadin, protein content and yield, especially glu/gli had a better correlation with deep soil water in anthesis. 【Conclusion】 The above results suggested that tillage in fallow period was beneficial to conserve rainfall in fallow period, and improve the soil water storage before sowing, especially the deep soil water in dry year. It benefited the improvement of soil water storage in anthesis, especially in dry year, which had better effects on soil moisture conservation. Fallow tillage could improve grain yield. Soil water at anthesis influenced the activity of GS and GOGAT in flag leaf, especially the activity of GS in flag leaves, consequently improved grain protein content, and glu/gli had a close connection with deep soil water at anthesis, so it improved grain protein quality. In a word, tillage in fallow period was beneficial to improve soil moisture reserve level, thus improving yield and optimizing quality, and deep tillage in fallow period was better in dry and normal year, while in humid year, deep scarification in fallow period was better.

Characters and Comprehensive Evaluation of Nutrient Quality of Sweetpotato Storage Root with Different Flesh Colors

TANG Zhong-Hou-1, 2 , WEI Meng-1, CHEN Xiao-Guang-1, SHI Xin-Min-1, ZHANG Ai-Jun-1, LI Hong-Min-1, DING Yan-Feng-2

Scientia Agricultura Sinica. 2014, 47(9):  1705-1714.  doi:10.3864/j.issn.0578-1752.2014.09.005

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【Objective】Sweetpotato (Ipomoea batatas (l.) Lam) is one of the most important starchy root crops, also a kind of raw material for staple food, animal feed and industrial products in China. Nutritional quality analysis and evaluation is a significant procedure of sweetpotato variety breeding and comprehensive utilization. This study was conducted to analyse nutrient quality of different color-fleshed sweetpotato storage root, establish a suitable and cost-effective evaluation method for sweetpotato nutrient quality, discuss the key factors influencing the comprehensive evaluation of sweetpotato nutrient quality, and provide a basis for sweetpotato breeding and its utilization. 【Method】Thirty sweetpotato materials with different flesh colors were chosen to measure their starch, protein, glucose, sucrose, fructose, polyphenols, flavonoids, anthocyanin, carotene, iron (Fe), calcium (Ca), magnesium (Mg), zinc (Zn), phosphorus (P) and potassium (K) contents by using conventional chemical method and near infrared spectroscopy analysis (NIRS). A comprehensive evaluation on nutritional quality of sweetpotato root was obtained with subordinate function and factor analysis.【Result】The results showed that some differences of nutrient quality existed in thirty sweetpotato storage root materials with different fresh colors. High CV values (coefficient of variation) appeared in carotene, anthocyanin, flavonoids, polyphenols and fructose content, yet little in protein and carbohydrate content. Purple flesh sweetpotato had much Fe, Zn, Mg, flavonoids and polyphenols, more than white flesh and yellow flesh, but its starch rates were lower than the other two kinds. There was a certain correlation among most nutrient quality indexes. From sixteen nutrition quality indexes converted data matrix and factor analysis, the cumulative variance contribution of eight principal factors extracted was 92.36% approximately. The primary nutrition quality indexes can be classified as functional material factor, carbohydrate factor and fundamental nutritional quality factors, and the variance contribution rate and weight index of three common factors were 33.34%, 38.7%, 20.32% and 0.36, 0.42, 0.17, respectively. The results showed that factor analysis is a good statistical method for describing nutritional quality variability among similar, correlated variables. According to weight index and subordinate function value of eight principal factors, the comprehensive evaluation of nutritional quality showed a significant difference in different color-fleshed sweetpotato varieties (or lines) (P＜0.05). Purple flesh type had highest value, which was attributed mainly to much flavonoids, polyphenols, anthocyanin and other functional materials in storage root. The white was the lowest. Hence, functional materials can be used as an important index to assess the comprehensive evaluation on sweetpotato nutritional quality. Nevertheless, some fundamental nutritional quality factors including carbohydrate and protein had no obvious impacts on comprehensive evaluation. 【Conclusion】There were some differences of nutrient quality characteristics among different color-fleshed sweetpotato storage roots. Meantime, evident difference existed in comprehensive evaluation on different nutrient qualities using factor analysis. The comprehensive evaluation value of purple flesh sweetpotato varieties was more than white and yellow ones. The key factors affecting nutrient quality evaluation were successively functional substances factor, carbohydrate factor and fundamental nutrient quality. This study has enriched and perfected the evaluation system of sweetpotato storage root nutrient quality.

PLANT PROTECTION

Genome-Wide Identification MAPK Superfamily and Establishment of the Model of MAPK Cascade Pathway in Setosphaeria turcica

GONG Xiao-Dong-1, ZHANG Xiao-Yu-1, TIAN Lan-1, WANG Xing-Yi-1, LI Po-2, ZHANG Pan-1, WANG Yue-1, FAN Yong-Shan-3, HAN Jian-Min-1, GU Shou-Qin-1, DONG Jin-Gao-1

Scientia Agricultura Sinica. 2014, 47(9):  1715-1724.  doi:10.3864/j.issn.0578-1752.2014.09.006

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【Objective】 Identification of MAPK superfamily genes from wide genome, and analysis of phylogeny, gene structure, multiple sequence alignment and conservative sites in Setosphaeria turcica will be useful for further research on the functions of MAPK cascade pathway in plant pathogenic fungus.【Method】Based on S. turcica genome database and genome searching with HMMER 3.0, MAPK superfamily genes were identified and their gene sequences, and genome location were obtained. The conservative protein kinase domains and sites were analyzed with ClustalX, MEME tools.【Result】Four MAPK genes, three MAPKK genes and three MAPKKK genes in S. turcica genome were found, and the MAPK was classified into Kss1/Fus3, Slt2, Hog1 and Ime2 four classes, MAPKK was classified into Pbs2, Ste7 and Mkk1 three classes, MAPKKK was classified into Ste11, Bck1 and Ssk2 three classes. Genome location and gene structure analysis showed that MAPK superfamily genes extensively distributed in the genome, and the structure of MAPK genes was the most complicated, MAPKK taken the second place, and MAPKKK structure was the simplest. Multiple sequence alignment and conservative sites analysis showed that the MAPK superfamily of kinase domain structure was highly conserved in S. turcica, and MAPK contained phosphorylation sites (-TxY-), MAPKK contained conservative phosphorylation sites (-SD[V/I]WS-), MAPKKK contained specificity conservative sites (-G[S/T] [V/P][F/M][W/Y]M[A/S]PEV-).【Conclusion】Genome-wide analysis showed that there were four MAPK genes, three MAPKK genes and three MAPKKK genes in S. turcica. Phylogenetic, gene structure, multiple sequence alignment and conservative site analysis showed that there were Fus3/Kss1-homolog, Slt2-homolog, Hog1-homolog and Ime2-homolog four MAPK cascade pathways, and Ime2 homolog may be a newly discovered MAPK cascade pathway in S. turcica. These results are helpful for the further functional research of MAPK superfamily in plant pathogenic fungus.

Identification and Analysis of Differentially Expressed Proteins of Host Rice (Oryza sativa) Infected with Rice Grassy Stunt Virus

DING Xin-Lun, XIE Li-Yan, WU Zu-Jian

Scientia Agricultura Sinica. 2014, 47(9):  1725-1734.  doi:10.3864/j.issn.0578-1752.2014.09.007

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【Objective】The objective of this study is to screen, identify and analyze the differentially expressed proteins in rice seedlings after infection with Rice grassy stunt virus (RGSV), and to shed new lights on the understanding of molecular mechanisms of RGSV pathogenesis.【Method】Two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) was used to analyze and identify differentially expressed protein spots. BioTools was used to search the matched proteins in NCBI database. Bioinformatics included GO (Gene ontology) terms and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis.【Result】The proteomic profile of the leaves of rice cultivar Xianyou 63 infected with RGSV compared with the control healthy plants was explored by 2D-DIGE. One-hundred and seventy-three protein spots (differential ratio＞1.5) were differentially expressed by RGSV infection over the healthy rice plants, including 72 up-regulated and 101 down-regulated protein spots. Among them, 25 differentially expressed protein spots were identified, including RuBisCO large subunit, sedoheptulose-1,7-bisphosphatase precursor, putative tyrosine phosphatase, HAD-superfamily hydrolase, subfamily IA, variant 3 containing protein, chain S, crystal structure of activated rice Rubisco complexed with 2-carboxyarabinitol-1,5-bisphosphate, C1-like domain containing protein, 20.6 K nonstructural protein (RGSV), P5 protein (RGSV) and some hypothetical proteins. GO function analysis revealed that the proteins’ biological processes were mainly involved in biological process, metabolic process, single-organism metabolic process, nitrogen compound metabolic process, nitrogen cycle metabolic process and nitrogen fixation, and the proteins’ molecular functions included molecular function, catalytic activity, ligase activity, ligase activity-forming carbon-nitrogen bonds, acid-ammonia ligase activity, ammonia ligase activity and glutamate-ammonia ligase activity. The proteins located different cell parts including cell, cell part, cellular component, cytoplasm, cytoplasmic membrane-bounded vesicle, cytoplasmic part, cytoplasmic vesicle, intracellular, intracellular membrane- bounded organelle, intracellular organelle, intracellular part, membrane, membrane-bounded organelle, membrane-bounded vesicle, mitochondrion, organelle, plastid and vesicle. KEGG pathway analysis suggested that the metabolic pathways of these proteins were those of metabolic pathways, carbon fixation in photosynthetic organisms, carbon metabolism, glyoxylate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, biosynthesis of amino acids, pyruvate metabolism, arginine and proline metabolism, glutathione metabolism and nitrogen metabolism.【Conclusion】According to the proteomic profile of rice seedling infected with RGSV, 24 proteins identified might be related to the pathogenicity of RGSV which provided an insight into the interactive relationship between RGSV and the host rice. The biological process and photosynthesis process related to nitrogen changed more obviously.

Gene Cloning and Expression Analysis of Three Odorant Receptors in the Diamondback Moth (Plutella xylostella)

KONG Chang-Yi-1, WANG Gui-Rong-2, LIU Yang-2, YAN Shan-Chun-1

Scientia Agricultura Sinica. 2014, 47(9):  1735-1742.  doi:10.3864/j.issn.0578-1752.2014.09.008

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【Objective】The objective of this study is to clone three odorant receptor genes and investigate their expression in different tissues of Plutella xylostella adults, and to provide basic knowledge for further functional studies of these three odorant receptor genes. 【Method】Antennal transcriptome was successfully used to identify candidate odorant receptors (ORs) by the next-generation sequencing. The gene was identified and the function was annotated by assembling the adult male and female antennal transcriptomes. Based on the database, the similarity was analyzed and comparison with Blastx and the candidate ORs of P. xylostella were identified. Based on the transcriptome sequencing results, the full length sequence of the odorant receptor genes were cloned. The expression profiles of these genes in different nine tissues were investigated by using semi-quantitative real-time PCR.【Result】Based on the predicted gene sequences, special primers were designed to clone the full length of odorant receptor genes. Three odorant receptor genes PxylOR16, PxylOR17 and PxylOR18 (GenBank accession number: KF717601-KF717603) were cloned. The open reading frame (ORF) of PxylOR16, PxylOR17 and PxylOR18 was 1 218, 1 200 and 1 191 bp, which encoded a polypeptide of 406, 400 and 397 amino acids, respectively. The sequences were aligned and the phylogenetic tree of the three odorant receptors was analyzed with the selected sequences of Lepidopteran insects, Bombyx mori, Heliothis virescens and Helicoverpa armigera odorant receptors which have been reported, and the reported P. xylostella six sex pheromone receptors and an olfactory receptor. The results showed that there were obvious differences between the three odorant receptor gene and atypical odorant receptors or sex pheromone receptor, and clustered together with other general odorant receptor. The three sequences had a low homology with each other and low sequence conservation. The results of semi-quantitative PCR showed that the three odorant receptor genes expressed in the antennae, heads, proboscises and labial palps and had no sexual specificity. The slight expression of PxylOR17 was detected in the female genitals. The expression of PxylOR18 could also be detected in female abdomens. No expression was observed between male and female. The three odorant receptor genes had no expressions in other tissues such as thoraxes, legs, abdomen and genitals.【Conclusion】Three odorant receptor genes were cloned in P. xylostella and the expression levels of them in different tissues were verified. It was identified that PxylOR16, PxylOR17 and PxylOR18 are three general odorant receptors by phylogenetic tree analyses and the sequences analyses. The three ORs are divergent in function. According to the results of RT-PCR, it was speculated that these three genes might be involved in the common odor molecules recognition process. In addition, it was also speculated that PxylOR17 and PxylOR18 might also participate in the process of identifying the male and female moths.

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

The Nodule Characteristics and Nitrogen Fixation of Soybean in Maize-Soybean Relay Strip Intercropping

YU Xiao-Bo-1, 2 , SU Ben-Ying-1, GONG Wan-Zhuo-1, LUO Ling-1, LIU Wei-Guo-1, YANG Wen-Yu-1, ZHANG Ming-Rong-2, WU Hai-Ying-2, ZENG Xian-Tang-3

Scientia Agricultura Sinica. 2014, 47(9):  1743-1753.  doi:10.3864/j.issn.0578-1752.2014.09.009

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【Objective】Shoot and root biomass, nodule characteristics, nitrogenase reductive ability and ureide content were studied to investigate the biological nitrogen fixation of soybean under relay-intercropping system.【Method】Three varieties, super-nodulating mutant NTS1007, BRAGG and ND12, were used in this field experiment to investigate the growth characteristics of root nodule, nodule nitrogenase reductive ability and ureide content of plant as well as root and shoot biomass accumulation. 【Result】Compared with monoculture, the results showed that root and shoot biomass decreased significantly in relay strip intercropping system. Nodule biomass was regulated by shoot and root, however, nodule number was correlated with the root biomass. The decreasing amplitude of root biomass (33.15%) was higher than that of shoot (20.84%) in relay strip intercropping system, as a result , the root/shoot ratio was decreased significantly. Furthermore, the nodule nitrogenase reductive ability and the ureide content of plant were also decreased significantly. Compared with monoculture, nitrogenase ability per plant and per weight all decreased, and the declining speed was slower after peak value. The fastest increasing stage of nitrogenase ability per plant came later. Among the three soybean varieties, NTS1007 (52.42%) had a bigger decreasing amplitude in nodule number than others, BRAGG had the biggest decreasing amplitude in root biomass, shoot biomass and nodule weight (37.73%, 31.28%, 43.25%). In maize-soybean relay strip intercropping system, NTS1007 had the biggest decreasing amplitude in nitrogenase activity per plant and ureide content per weight (64.41%, 21.72%), as well as the nitrogenase activity per weight of BRAGG (21.46%), nitrogenase activity and ureide content per plant of ND12 was higher than that of NTS1007 and BRAGG, but the decreasing amplitude compared to monoculture was the lowest among the three soybean varieties (10.01%, 45.81%, 17.88%).【Conclusion】In relay strip intercropping system, shoot biomass, root biomass and root/shoot ratio decreased significantly, as well as the nodule number and nitrogenase ability. The declining speed of nitrogen fixing ability was slower, and this will be favorable to protein accumulation. The decreasing amplitude was different among the three soybean varieties. The biomass accumulation and nodule formation of super-nodulating mutant NTS1007 were sensitive to relay strip intercropping system, but ND12 showed a better adaptability.

Meteorological Drought Trend in Winter and Spring from 1961 to 2010 and Its Possible Impacts on Wheat in Wheat Planting Area of China

FANG Shi-Bo-1, QI Yue-1, HAN Guo-Jun-1, ZHOU Guang-Sheng-1, DavideCammarano2

Scientia Agricultura Sinica. 2014, 47(9):  1754-1763.  doi:10.3864/j.issn.0578-1752.2014.09.010

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【Objective】Continuous occurrence of winter and spring drought events in recent years has seriously threatened the safety production and grain increase of winter wheat in the north of China. This study focused on the main wheat growing areas of China, precipitation, no precipitation days and frequency of extreme meteorological drought trend in winter and spring from 1961 to 2010 and its possible impacts on wheat in wheat planting area in north of China.【Method】Trend analysis of precipitation, no precipitation days and frequency of extreme meteorological drought for the 1961-2010 period using data from 329 meteorological stations located in the wheat growing regions in the north of China. The linear trend and significance of precipitation and no precipitation days in different regions in north of China was analyzed using the methods of regression analysis, inverse distance weighted and robust F linear significant test. The precipitation, no precipitation days and frequency of extreme meteorological drought of interannual and decadal variation trend was analyzed. Based on the sliding average precipitation anomaly, the linear tendency of annual extreme drought frequency in winter from 1961 to 2010 was calculated, and the interannual variation trend of extreme drought frequency and changing trend of precipitation in different regions were analyzed for exploration of the temporal dynamic trend of drought. According to the GB/ T20481-2006 national standards of meteorological drought classification, the severe drought and specially drought of drought grade were defined as extreme drought. According to the different climate characteristics of wheat growing regions in China, the main wheat growing area is divided into six zones as follows in order to quantify the different drought trends in the north of China. ① The Northeast China spring wheat region: Heilongjiang Province, Jilin Province and Liaoning Province; ②The Inner Mongolia spring wheat region including the Inner Mongolia Autonomous Region. ③ The Northwest China spring wheat region including Gansu Province and Ningxia Autonomous Region. ④ The North China winter wheat region including Hebei Province, Shanxi Province, Shaanxi Province, Beijing and Tianjin. ⑤The Huang-Huai Plain winter wheat region including Shandong Province, Henan Province, Jiangsu Province and Anhui Province. ⑥ The middle and lower Yangtze River winter wheat region including Hubei Province, Hunan Province, Jiangxi Province, Zhejiang Province and Shanghai.【Result】 In the winter wheat growing region of North China and north of the Huang-Huai Plain, as well as in spring wheat area of Northwest China, the precipitations in winter and spring decreased in the last 50 years with a particular sharp decline after 1990, the frequency of extreme drought showed an increasing trend. The precipitations in winter and spring decreased in the last 50 years in the east Shaanxi and northwest Hubei, the frequency of extreme drought in Hebei province, Beijing, Tianjin, Shanxi province and west of Shandong increased in 1961-2010. Winter and spring precipitation and the number of precipitation days showed an increasing trend in the Northeast China, Inner Mongolia and Northwest China's southern spring wheat region. Winter and spring precipitation and the number of precipitation days showed a decreasing trend in a part of Huang-Huai Plain winter wheat area, the southern part of the North China winter wheat region, the southeast and northwest spring wheat regions and the middle and lower Yangtze River winter wheat region. A sharp decline in winter precipitations were observed in the last 50 years in North China, and no precipitation days showed an increasing tendency, and the meteorological drought aggravated. The winter precipitation decreased and no precipitation days increased in Hebei Province, Beijing, Tianjin, Shanxi province and the western Shandong Province in 1961-2010. The winter precipitations increased in other areas, and no precipitation days decreased. The spring precipitations showed a decreasing tendency in North China, Central and southern Huang-Huai Plain and southern of Northwest China in 1961-2010, the largest decline up to 20 mm/10a. The no precipitation days showed an increasing tendency, the largest increased up to 1-1.5 d/10a. Among the seven sites, precipitation was significantly decreased in Shaanxi, the decrease rate up to 10-20 mm/10a. The no precipitations days showed a significant decreasing tendency in most areas in Shaanxi, the west of Henan, Jiangxi and Zhejiang, the decrease rate up to 1-1.5 d/10a. The spring precipitations increased in other areas, and no precipitation days decreased in the last 50 years. Temporal dynamics of key regional precipitation showed a sharp decline in winter and spring precipitations in the last 20 years in North China and no precipitation days showed an increasing tendency, the winter and spring meteorological drought aggravated. The winter precipitations increased in the Huang-Huai Plain winter wheat region and the middle and lower reaches of the Yangtze River, and the no precipitation days decreased in the last 50 years. But the spring precipitation decreased and the no precipitation days increased after the mid 1970’s.【Conclusion】Dramatically decline of winter and spring precipitations could have a potential negative impact on future winter wheat production in North China because of the shortage of water resources available to wheat growth. The decline of precipitations during spring would not affect growth of wheat in south of the Huang-Huai Plain and the middle and lower zones of the Yangtze River. This is because the spring precipitations are still enough for wheat to avoid drought stress. The winter and spring precipitation patterns in Northeast China, Inner Mongolia, and Northwest China increased over the 1961-2010 period, which would benefit spring wheat production.

Advances in Research on the Effects of Irrigation on the Greenhouse Gases Emission and Soil Carbon Sequestration in Agro-ecosystem

QI Yu-Chun-1, GUO Shu-Fang-1, 2 , DONG Yun-She-1, PENG Qin-1, JIA Jun-Qiang-1, 2 , CAO Cong-Cong-1, 2 , SUN Liang-Jie-1, 2 , YAN Zhong-Qing-1, 2 , HE Yun-Long-1, 2

Scientia Agricultura Sinica. 2014, 47(9):  1764-1773.  doi:10.3864/j.issn.0578-1752.2014.09.011

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Agricultural carbon (C) pool is one of the most active parts in global terrestrial ecosystem C pool and is very sensitive to change in management measures. As an important means for dry farming in China to get high yield, irrigation and its change in patterns certainly will have a significant impact on the ecosystem C budget and its atmospheric greenhouse effect, but the relative data and corresponding mechanism analysis are rarely known. This paper reviews research progresses in the quantitative impacts of irrigation on soil respiration and its different components, the fluxes of nitrous oxide and methane as well as soil C storage, and compares the differential responses of the above-mentioned processes to different irrigation patterns, amounts, frequencies, lasting periods and irrigation water quality, etc. The results indicate that irrigation generally will increase soil C emission, but the relationship between irrigation quota and soil carbon dioxide (CO2) effluxes is nonlinear. Water additions stimulate soil CO2 emission when soil is under water deficit condition, whereas excessive irrigation would decrease soil respiration rates. Irrigation increases the sensitivity of soil respiration to temperature change (Q10), and the response of Q10 to the irrigation amounts can describe as a parabola. The Q10 values under drip irrigation are larger than those under flood irrigation. There is a great difference in the response of different respiration components to the change of soil water under different irrigation methods, and the effect of irrigation on root respiration is more significant than on soil microbial respiration. The ratios of root respiration contribution to soil respiration under drip irrigation are much larger than those under flood irrigation. Irrigation water quality and irrigation depth also have an important impact on total emission amount of greenhouse gases, and full municipal wastewater treatment can obtain much more carbon emission credits for countries and regions. Water management is one of the important measures that mitigate the emissions of nitrous oxide (N2O) and methane (CH4). Both the emission amount and the emission pathway of N2O and CH4 are significantly affected by the soil water condition. There usually exists marked trade-off relationship between CH4 effluxes and N2O effluxes when soil moisture changes, so the comprehensive evaluation indicators such as global warming potentials (GWPs) are more conducive to reflect the actual change in greenhouse effect contribution of agricultural soil exactly. There exist various possibilities in the effect of irrigation on soil organic C (SOC) pool, such as increase, decrease, or no significant changes, and the responses of SOC to irrigation also vary a lot under different climate and soil conditions. In general, the stimulated effects of irrigation on SOC are more significant in relatively dry regions than in humid regions. Meanwhile, SOC in different forms often shows different response sensitivities and variation trends to irrigation. The effect evaluation of irrigation measures should be considered from the multiple perspectives of water-saving, increasing SOC pool and enhancing the utilization efficiency of liable organic C. To sum up, there still exists large uncertainties in the effects of irrigation on soil greenhouse gas emission and SOC in agro-ecosystem up to now. The future study should be focused on following contents: (1) to strengthen the comparison study of different irrigation patterns, especially the study on the spatial differences of greenhouse gases emission under different irrigation patterns; (2) to pay more attention to the comprehensive effect of different greenhouse gases and the ecosystem carbon budget; (3) to develop in-situ field research for both long period and short period; (4) to probe into the microbiology driving mechanism of irrigation on farmland greenhouse effect and soil C sequestration.

HORTICULTURE

Progresses in Research of Litchi Floral Differentiation

CHEN Hou-Bin, SU Zuan-Xian, ZHANG Rong, ZHANG Hong-Na, DING Feng, ZHOU Bi-Yan

Scientia Agricultura Sinica. 2014, 47(9):  1774-1783.  doi:10.3864/j.issn.0578-1752.2014.09.012

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The commercial production of litchi (Litchi chinensis Sonn.), a subtropical evergreen fruit tree, has been restricted in the two narrow areas of 17o-32o latitude in the north and south hemispheres. Within the multiple cultivars-based industrial structures, unreliable flowering has taken place in almost all litchi cultivars. The flowering status of varieties, flower bud differentiation stage performance, influence factors, flower bud differentiation mechanism and flowering regulation are reviewed in this paper. It is considered that, litchi trees must have fully matured terminal shoots in autumn and be florally induced under chill conditions for a quite long period during the winter. Primordial inflorescence initiated upon the ascending temperatures and with release of water stresses during late winter or early spring, the inflorescence and flowers developed at mild conditions with warm days and cool nights. Any one unsatisfactory factor during the phase changes might cause failure of flower formation. The major factors influencing litchi floral development are state of terminal shoots, temperatures and water. Carbohydrate accumulation in shoots and the endogenous hormones affect flowering. In order to solve the instability problem in production of flowering and ensure good flowering under extreme and marginal conditions, the molecular mechanism about litchi floral differentiation and why young shoots and leaves could not be florally induced with chills, how the chill signals perceived and transduced, stimulate the floral transition of the terminal buds, when and how the flower sex is determined should be studied, and to explore nutrient, water management, growth regulators and other mechanical measures according to the phase changes of floral differentiation.

Effects of Different Irrigation Treatments on the Growth and Fruit Quality of Himrod in Protected Cultivation

LI Ya-Shan-1, LI Hua-1, 2 , 3 , WANG Hua-1, 2 , 3 , Nan Li-Jun-1

Scientia Agricultura Sinica. 2014, 47(9):  1784-1792.  doi:10.3864/j.issn.0578-1752.2014.09.013

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【Objective】 Under the background of severe shortage of water resource and continuous development of facility cultivation, there is of important practical significance for studying the efficient use of water resource under protected cultivation condition. Based on this reason, the objective of this article was to select an optimal irrigation method and supply a substantial theoretical support for actual production application. 【Method】 Under the protected cultivation condition, the table grape variety Himrod was used as test materials. There were four different irrigation methods, including full irrigation, alternate partial root-zone irrigation, water deficit pre- or post-veraison irrigation methods, which were used for the grapevine. Then the effects of different irrigation methods on the number of axillary shoots, leaf area, stem diameter, internodes length, weight of summer pruning, particle size, length of cluster, weight of cluster, berry weight, fruit quality, photosynthetic parameter of leaf, water use efficiency, leaf water potential, and fruit yield were analyzed. Based on these analyses, the best irrigation method could be screened out through comprehensive assessment. 【Result】 The three different patterns of water deficit could not only reduce the number of axillary shoots, leaf area, stem diameter, internodes length, weight of summer pruning, but also decrease berry length, berry width, berry weight, cluster length, and cluster weight. However, they increased reducing sugar, soluble solids, Vc of berry. The effects of different patterns of water deficit on scores internal quality of berry were different. Alternate partial root-zone irrigation and water deficit after veraison both enhanced the scores significantly, but water deficit during pre-veraison made the scores below the contrast treatment. Water deficit lowered the net photosynthetic, stomatal conductance and transpiration rate of leaf, while increased the intercellular CO2 concentration and water use efficiency of it. In water deficit treatments, initial quantum efficiency, light compensation point, dark respiratory rate were higher than contrast treatment, but the maximum net photosynthetic rate was lower than the latter. The leaf water potentials of contrast treatment were higher than the others. Alternate partial root-zone irrigation method and water deficit during pre-veraison reduced yield significantly, however, the yield decrease caused by regulating deficit irrigation of water deficit occurred post-veraison was not obvious as compared with the contrast treatment. 【Conclusion】 Water deficit could be as a strategy to regulate the vegetative and reproductive growth, improve fruit quality and water use efficiency, thus increasing the economic and ecological benefits. Water deficit could also reduce the net photosynthetic rate and leaf water potential, which had a bad effect on the berry growth. Water deficit during post-veraison could reduce the plant’s vigour, maintain yield and improve fruit quality and water use efficiency, it is a better strategy compared with the other irrigation methods.

STORAGE·FRESH-KEEPING·PROCESSING

Selection and Identification of Critical Amino Acids in Epitope 187-202 of Pen a1

MOU Hui-1, GAO Mei-Xu-1, PAN Jia-Rong-2, ZHI Yu-Xiang-3, ZHAO Jie-1, LIU Chao-Chao-1, LI Shu-Jin-1, ZHAO Xin-1

Scientia Agricultura Sinica. 2014, 47(9):  1793-1801.  doi:10.3864/j.issn.0578-1752.2014.09.014

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【Objective】Pen a1 is the major allergen in shrimp, and the sensitization mechanism is related with epitopes. The epitope (187-202) of Pen a1 was chosen as the research material, the amino acids’ frequency of occurrence and their conservative property were analyzed, the mutants were synthesized and the IgE capacity of the mutants were analyzed. The critical amino acids of the epitope of Pen a1 were screened out in order to study the shrimp sensitization mechanism and provide a theoretical basis for desensitization. 【Method】 The amino acid composition and frequency of occurrence in Pen a1, all epitopes and the studied epitope were analyzed using MEGA5, and the amino acids with the highest frequency were chosen. And then the amino acids of tropomyosin in all the allergenic foods in SDAP were also investigated, and the high conservative amino acids were chosen. The common amino acids in the results of both methods were considered to be the potential critical amino acids. Then these amino acids were substituted by alanine, the wild-type peptide and the mutant peptides were then synthesized with solid phase synthesis method. The tested serum was prepared by immuning New Zealand rabbits with wild-type peptide. The capacity of IgE-binding between the wild-type peptide and the mutants were compared with iELISA and competitive Dot-blot methods. The mutant peptides whose capacity of IgE-binding showed a dramatic decline were selected, and the replaced amino acids of the peptides were recognized as the critical amino acids. 【Result】 The amino acids of glutamic acid (E), leucine (L), arginine (R), glutamine (Q), valine (V), serine (S), aspartic acid (D) were found with higher frequency occurrence in epitope than in Pen a1, and were considered as the active amino acids. E, V, L in epitope (187-202) were found with higher frequency occurrence, and were inferred to be the potential critical amino acids of studied epitope. The multiple sequence alignment of tropomyosin from allergenic foods in SDAP and Pen a1 with DNAMAN discovered that K, L, E, V, G existed in all the sequences, which indicated that the five amino acids were conservative. E, V and L which appeared in the results of both methods were selected to be substituted by alanine to synthesize the mutants 1, 2 and 3, respectively. The mutants were interacted with epitope sera to detect the IgE reactivity by competitive Dot-blot. QCA-OVA were the purified Pen a1, and the mutated peptides were competitors. The IgE reactivity against wild type and mutants were then compared. The wild-type and mutant 1 showed a similar evident inhibiting effect, mutants 2 and 3 showed a decrease in inhibiting effect. The mutant 1 with Glu to alanine showed a similar IgE reactivity to wild-type, which meant that Glu should not be the critical amino acid. Peptides with Leu (mutant 2) or Val (mutant 3) to alanine exchange showed a decrease in IgE-binding, and Leu and Val were deduced the critical amino acids of the studied epitope. The mutants were interacted with epitope sera by ELISA to compare the OD450 value with the wild-type as control. Mutant 1 showed a decrease in IgE-binding in tested epitope sera, its OD450 value was 1/2.1 of control. OD450 value of mutant 2 decreased to 1/2.6 of control, and mutant 3 decreased to 1/3.2 of control. These three mutants’ IgE-binding capacity decreased in different degrees. Combined with the results of competitive Dot-blot, Leu and Val were considered to be the critical amino acids of studied epitope. 【Conclusion】 The method for selection and identification of critical amino acids was established, L and V were the critical amino acids of epitope (187-202) in Pen a1. This method for screening the potential critical amino acids could also be used in identifying critical amino acids of other epitopes, and exploring the influence of amino acids on the IgE capacity of epitope, thus further studying the mechanism of desensitization, and can also be used in genetic engineering or amino acids modification to reduce the sensitization of allergen.

Selection and Identification of Anti-Cry1Ac scFv from a Phage Display Antibody Library by Magnetic Beads and Sandwich ELISA Immunoassay

ZHANG Liu-Quan-1, 2 , ZHANG Xiao-1, LIU Yuan-1, XU Zhong-Xin-1, ZHANG Cun-Zheng-1, XIE Ya-Jing-1, KOU Li-Ping-2, LIU Xian-Jin-1

Scientia Agricultura Sinica. 2014, 47(9):  1802-1810.  doi:10.3864/j.issn.0578-1752.2014.09.015

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【Objective】By using the biotin-avidin labeled magnetic bead screening strategy, this paper aims at establishing a new type of double-antibody sandwich ELISA detection method for Cry1Ac toxin, so as to build a new detection mode to analyze the sensitive and rapid Bt toxin detection method. 【Method】 The enrichment of Cry1Ac phage-scFvs was investigated after each round of panning (total four rounds) by using magnetic beads with subtractive panning. Positive clones as specific binding to Cry1Ac were selected from the secondary library, which was gained from the fourth round of panning by the method of monoclonal ELISA, and after the PCR identification and DNA sequencing of these positive clones, post-relevant tests were conducted when confirming the complete insertion of the scFvs. Meanwhile, the positive clone, scFv-A12, with relatively best binding activity, was chosen to replace the host strain HB2151 for the purpose of induced-soluble expression and purification. And the purified scFv-A12 was regarded as “capture antibody”, the anti-Cry1Ac rabbit polyclonal antibody as “detection antibody”, thus to establish the detection method of double sandwich ELISA for Cry1Ac.【Result】With the coating antigen of biotinylated Cry1Ac protein, four rounds of panning were conducted by using the antibody phage library. The results show that, along with the increase of panning rounds, the relative productivity was enhanced, and the data of the fourth round was increased by 42 times compared with that of the first round. The positive standard for monoclonal phage ELISA to identify anti-Cry1Ac phage antibody was decided by the figure gained from dividing test hole OD450 by negative hole OD450, which should be over 2.1. Three hundred bacterial clones were selected randomly from the Petri dish after the fourth panning, in which six positive clones were identified by monoclonal ELISA, being respectively named as hsCry1Ac-C5, hsCry1Ac-D8, hsCry1Ac-C12, hsCry1Ac-A12, hsCry1Ac-F9 and hsCry1Ac-F4. Among them, hsCry1Ac-A12 had a relatively high affinity. After the bacteria PCR detection upon above six positive clones, the results show that the target band was obvious in the place of 935 bp, and with the sequencing and amino acid sequence alignment on the target band, four positive clones with different amino acid sequences were gained. After successfully replaced the host strain HB2151, hsCry1Ac-A12 was cultured at 30℃ with 1 mmol•L-1 IPTG for inducing expression. Through the His Trap HP nickel affinity column purification and the SDS-PAGE identification of its expression product, the molecular weight of hsCry1Ac-A12 protein was approximately 30 kD. After optimizing the antibody concentration by titration test and taking advantage of the single chain antibody as “capture antibody”, polyvalent antibody as “detection antibody”, the detection method of double antibody sandwich ELISA was established. Within the linear range of 1.25-2.43 μg•mL-1, the linear regression equation was Y=0.2522X+1.2832(R2=0.9564), with the limit of detection 1.08 ng•mL-1.【Conclusion】This paper adopted the method of magnetic beads with subtractive panning, the detection method of double-antibody sandwich ELISA for Cry1Ac toxin was established after the expression and purification of the selected clone A12 with best binding activity which provide a new detection mode for detecting the Cry1Ac contained in GMF rapidly and accurately.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

The Genetic Polymorphism, Expression of BMP7 Gene and Its Relationship with Lamb Skin Follicle Traits in Hu Sheep

YIN Jin-Feng-1, NI Rong-1, WANG Qing-Zeng-1, SUN Wei-1, DING Jia-Tong-1, ZHANG You-Fa-2, CHEN Ling-2, WU Wen-Zhong-2, ZHOU Hong-3

Scientia Agricultura Sinica. 2014, 47(9):  1811-1818.  doi:10.3864/j.issn.0578-1752.2014.09.016

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【Objective】 The article aims to study the influence of BMP7 on the formation of Hu sheep’s different patterns and the possible regulatory mechanisms on the development of hair follicles. 【Method】 BMP7 gene in a sample of 205 single nucleotide polymorphisms in exons 1,2 and 3 were detected by PCR-SSCP. Hu sheep’s hair follicles were observed by histology and microscopic examination, statistical analysis on the diameter of primary and secondary hair follicles, the number of primary and secondary hair follicles were statistically analyzed. The relative expression of BMP7 among large, medium and small waves was detected by fluorescence quantitative PCR, and SPSS 17.0 software was used to conduct the significant difference analysis, to investigate the BMP7 gene’s regulatory mechanisms of the formation of Hu sheep’s different patterns and hair follicles traits. 【Result】 The results showed that by using PCR-SSCP method for detecting the single nucleotide polymorphisms of BMP7 gene’s 3 exons and no polymorphism was found. In histological analysis, the diameter of large waves primary hair follicles was more than medium and small waves, the diameter of small waves primary and secondary hair follicles was between large and medium waves. The number of large waves secondary hair follicles was more than medium and small waves, while the small waves were between large and medium waves. Through significant difference analysis, the difference in diameter of primary follicles in large waves was highly significant with medium and small waves (P＜0.01), the difference in diameter of primary follicle between large and small waves was not significant (P＞0.05). The difference in diameter of secondary follicles in medium waves was highly significant with large and small waves (P＜0.01), but the difference in diameter of secondary follicle between large and small waves was not significant (P＞0.05). The difference in number of primary follicles among large, medium, and small waves was not significant (P＞0.05), but in the same vision, the number of primary follicles among large waves was higher than medium and small waves. The difference in number of secondary follicles in medium waves was highly significant with large and small waves (P＜0.01), but the difference in number of secondary follicles between large and small waves was not significant (P＞0.05). qRT-PCR results showed that in the same period, the expression of BMP7 in small waves was higher than large waves, and showed a significant difference between large and small waves (P＜0.05). The relative expression of BMP7 was significantly correlated with the primary follicle diameter in small waves, and showed a highly significant positive correlation (P＜0.01)with the number of small waves’ secondary follicles, and negatively correlated with middle waves’ primary follicle diameter, the number of secondary follicle (P＜0.05). The expression was consistent with biopsy results. 【Conclusion】 Single nucleotide polymorphism was not found in BMP7 gene, however, the expression of BMP7 was correlated with hair follicles indexes and consisted with the histological observation. It is speculated that BMP7 gene may be involved in the development of hair follicle and regulation of hair growth.

RESEARCH NOTES

Identification and Gene Mapping of a Dominant Narrow Leaf Mutant Dnal1 in Rice (Oryza sativa)

SANG Xian-Chun, LIN Ting-Ting, HE Pei-Long, WANG Xiao-Wen, LIAO Hong-Xiang, ZHANG Xiao-Bo, MA Ling, HE Guang-Hua

Scientia Agricultura Sinica. 2014, 47(9):  1819-1827.  doi:10.3864/j.issn.0578-1752.2014.09.017

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【Objective】Leaf blade is the main location of photosynthesis in high plants. A moderate narrowing could enhance crop quality and yield by fascinating the utilization of solar energy and has been paid wide attentions in crop breeding. A novel dominant narrower leaf mutant has been identified and the results provided a foundation in the research of leaf morphological formation and plant type breeding in Oryza sativa.【Method】A narrow leaf mutant (Dnal1) was discovered from the progeny of indica restorer line Jinhui10 with seeds treated by ethyl methanesulfonate and the trait of narrow leaf has inherited steadily after seven generations’ self-fertility. The flag, second and third leaf blades were utilized for detecting leaf widths and rolled indexes at the blooming stage, meanwhile, the middle position of flag leaf was used for paraffin section. At the filling stage, the characteristics of chloroplast pigment, photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were measured. The Dnal1 was crossed with indica line Xinong1A and japonica line Zhonghua11, respectively, and the F1 and F2 generations were used for genetic analysis. Additionally, gene mapping was performed based on the F2 recessive individuals of Dnal1/Zhonghua11. The Dnal1 and the wild type were cultivated in paddy field with the spacing in the rows and between the rows 13.36 cm×20.04 cm and 16.7 cm×26.72 cm, respectively. At the maturity stage, agronomic traits were analyzed and they were plant height, efficient panicle per plant, filled grain number per panicle, seed setting ratio, 1000-seed weight, seed length and seed width. At last, theory yield per 667m2 was estimated.【Result】The Dnal1 displayed narrower leaf blades in the life and the width of flag leaf, second leaf and third leaf were 0.96, 0.89 and 0.88 cm, respectively; correspondingly, those of the wild type were 19.6, 19.41 and 18.42 cm, respectively. The number of large vascular bundles in the mutant had no significant difference with those of the wild type and the number of small vascular bundles decreased by 44.13%, leading to the significant difference. In detail, the number of small vascular bundles between the large vascular bundles was six in the wild type, and three in the Dnal1. The leaf blades of Dnal1 displayed slightly curling and the rolled indexes of flag, second and third leaf blades were 11.2, 12.1 and 11.8, respectively, while those of the wild type were zero. In addition, the colour of leaf blades of Dnal1 was darker than those of wild type with a slight higher content of chloroplast a. The increases were detected for the values of the water use efficiency and the decrease was identified for the parameters of the photosynthetic rate, transpiration rate and stomata conductance in the Dnal1, while only the alteration of stomata conductance led to the level of significant difference between the Dnal1 and the wild type. The culm and seed of Dnal1 were narrower than those of the wild type, while no changing in plant height and seed length was detected. The seed width of Dnal1 was 2.33 mm, which was significantly lower than those of the wild type (2.78 mm), therefore, the 1000-seed weight decreased significantly in the Dnal1. The yield of per unit area in the Dnal1 reduced under the lower plant density while increased under the higher plant density than those of the wild type, which was caused by higher efficient panicle per plant and seed setting ratio. For the combinations of Xinong1A/ Dnal1 and Dnal1/Zhonghua11, the widths of F1 flag leaves were 0.99 cm and 0.94 cm, respectively, both have no significant differences with the wild type’s 0.96 cm; in the F2 generations, the plants could be classified as two groups according to the leaf width, the narrow-leaf individuals to those with broad leaves has no significant difference with the ratio of 3:1, suggesting that the narrow leaf trait of Dnal1 was controlled by a dominant nuclear gene. Finally, the DNAL1 was mapped on chromosome 2 between SSR marker RM13646 and RM1307 with the physical distance 391 kb in this paper. 【Conclusion】 Dnal1 was a dominant narrow leaf mutant coming from EMS-inducement and showed higher yield potential when cultivated under the higher planting density. The DNAL1 was mapped on chromosome 2 with 391 kb physical distance.

Effects of Different Cultivation Patterns on Rice Yield and Crab in Rice-Crab Culture System

XU Min-1, 2 , MA Xu-Zhou-1, WANG Wu-1

Scientia Agricultura Sinica. 2014, 47(9):  1828-1835.  doi:10.3864/j.issn.0578-1752.2014.09.018

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【Objective】Rice-crab culture system is a relatively new paddy cultivation and breeding model, and it has a great advantage in production and development potential. Rice-crab culture system could ameliorate rice field aquatic ecological environment, increase soil effective nutrition, promote rice growth and development, improve resource utilization rate, ameliorate cultivation structure, and increase rural income, etc. Rice-crab ecological breeding model studies were mainly focused on the crab stocking density, rice growth, yield, water environment and plankton, etc., but few of research reports on rice cultivation density. The effects of different rice cultivation patterns on rice tiller, rice biomass production, rice stem characters, rice yield composition, crab and rice paddies comprehensive economic benefits were studied. 【Method】 Four treatments were designed, i.e. one plant per hill in rice-crab (T1), two plants per hill in rice-crab (T2), four plants per hill in rice-crab (T3), two plants per hill in rice (CK), and stock 900 juvenile crabs in rice-crab paddy field. Four replicates were designed for each treatment and cultivated for 137 d. Thereafter, rice growth indices and crab traits were measured. 【Result】 Crab bait protein content was about 32%, crab fed at 4 pm and the feeding amount for crabs was generally 3%-5% of body weight. Compared with conventional paddy fields, rice-crab paddy fields could increase 0.17 tillers per hill, 3.3% of biomass production and 11.5% of rice yield, rice stem characters were not significantly affected, seed setting rate and grain number per panicle were significantly affected. Different cultivation patterns showed no significant effect on rice-crab paddy fields. The size of four plants per hill had the highest biomass production, rice stem characters showed an increasingly decreasing trend with the decrease of plant spacing. The treatment of one plant per hill increased the grain number per panicle by 6-26, seed setting rate by 1.2%-3.6% and 1000-grain weight by 1.5%-3.3%.【Conclusion】Based on rice growth indices and economic benefits, the optimal cultivation pattern was one plant per hill.

Genetic Diversity of Helicoverpa assulta (Lepidoptera: Noctuidae) in Guizhou Province

SHI Pei-Qiong-1, YANG Mao-Fa-1, 吕Zhao-Yun-1 , LI Shang-Wei-1, LIAO Qi-Rong-1, SHANG Sheng-Hua-2, XU Jin-1, WU Yi-Bei-1

Scientia Agricultura Sinica. 2014, 47(9):  1836-1846.  doi:10.3864/j.issn.0578-1752.2014.09.019

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【Objective】 The objectives of the study are to explore genetic differentiation of oriental tobacco budworm (Helicoverpa assulta) among different geographic populations in Guizhou, uncover the differential law and mechanism, and to provide a scientific basis for the pest monitoring and integrated control. 【Method】 PCR amplifications were performed by 6 pairs of SSR primers that were screened out from 43 pairs of primers from closely-related species, using DNA from 30 H. assulta geographic populations in Guizhou as template. The PCR products were subjected to 8% non-denaturing polyacrylamide gelelectrophoresis and visualized by silver staining. Genetic diversity was analyzed by PopGene Version 1.32, and the UPGMA dendrogram was constructed by using MEGA 5.0 based on the Nei’s genetic distance. The correlation between genetic and geographic distances as well as between genetic identity and elevation were analyzed by using Mantel. 【Result】 The observed and effective number of alleles ranged from 3 to 8 with an average of 5 and from 1.4498 to 2.2219 with an average of 1.8594, respectively. Shannon’s information index ranged from 0.5310 to 1.0609 with an average of 0.8423. The observed (Ho) and expected heterozygosity (He) varied from 0.0260 to 0.2672 with a mean of 0.1239 and from 0.3123 to 0.5520 with a mean of 0.4539, respectively. The He was higher than the Ho, suggesting that all populations were mainly in homozygous condition. FIS changed from 0.0798 to 0.7906 with a mean of 0.2801; FIT changed from 0.4842 to 0.9731 with a mean of 0.7809. Both FIS and FIT were positive, indicating that there existed inbreeding in the all populations of H. assulta from tobacco-growing areas of Guizhou. FST ranged from 0.3897 to 0.9256 and gene flow (Nm) was less than 1, which reflected high genetic differentiation and a low level of Nm among all the populations. Genetic distance was in a range of 0.0068-2.5193 and genetic identity was in a range of 0.1051-0.9933. The minimum genetic distance (0.0068) and the maximum genetic identity (0.9933) were observed between Songtao and Yinjiang populations, whereas the maximum genetic distance (2.5193) and the minimum genetic identity (0.1051) were observed between Daozhen and Hezhang populations. The UPGMA phylogenetic analysis showed that the 30 geographic populations in Guizhou were clustered into three groups. The clustering had no significant correlation with the geographic distribution and only in a few populations the genetic differentiation had relationship with geographic distance. Mantel tests demonstrated that there was no significant correlation between genetic and geographic distances as well as between genetic identity and elevation. 【Conclusion】 There exists a high level of genetic diversity among different geographic populations of H. assulta in Guizhou. All the populations are highly differentiated and the genetic differentiation primarily occurred between populations. Geographic isolation has no significant effect on the genetic differentiation.

Effect of Different Concentrations of Organic Matter Fermentation Fluid on the Young Apple Tree Leaf Photosynthesis Fluorescent Parameters and Root Antioxidant Activity Under Replant Conditions

YIN Cheng-Miao, ZHANG Xian-Fu, HU Yan-Li, SHEN Xiang, CHEN Xue-Sen, WU Shu-Jing, MAO Zhi-Quan

Scientia Agricultura Sinica. 2014, 47(9):  1847-1857.  doi:10.3864/j.issn.0578-1752.2014.09.020

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【Objective】The Fuji/Malus robusta biennial plants were selected as the test materials, and potted with continuous cropping soil. Effect of different concentrations of organic matter fermentation fluid on the young apple tree leaf photosynthesis fluorescent parameters and root antioxidant activity under replant conditions was studied.【Method】Fuji/Malus robusta biennial grafted plants were planted in clay basins. Six organic matter concentrations in the fermentation fluid were designed: CK, CK+1% (T1), CK+3% (T2), CK+5% (T3), CK+7% (T4) and CK+9% (T5). The plant height and basal diameter of young apple tree, superoxide dismutase (SOD) activities, catalase (CAT) activities, peroxidase (POD) activities of roots of young apple tree, the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci), actual PSⅡ efficiency (ΦPSⅡ), efficiency of light energy capture by open PSⅡ reaction centers (Fv′/Fm′), non-photochemical quenching of PSⅡ(NPQ), quantum yield of PSⅡ electron transport (ETR), photochemical quenching of PSⅡ(qP) of leaves of young apple tree were determined.【Result】The main results were as follows: the role of organic matter fermentation on the growth, root antioxidant activity and leaf photosynthesis fluorescent parameters of young apple tree showed a “promotion in low concentration and inhibition in high concentration” effect. Low and middle concentrations of organic matter fermentation fluid (1%, 3%, 5%) watered young apple tree plant height was higher than the concurrent control, and 3% treatment had the most significant growth, were 1.24, 1.12 and 1.23 times higher compared with the concurrent control in spring, summer and autumn. The activities of CAT were increased gradually from spring to autumn. In spring and autumn, root CAT activities of other treatments except 3% treatment were significantly lower than that of the control. The root CAT activity in all treatments increased, in addition to 5% treatment, CAT activity in 1%, 3%, 7% and 9% treatments were significantly higher than the control by 1.54, 1.84, 1.65 and 1.62 times in summer. Applying different concentrations of organic material fermentation fluid also changed root POD activity of young apple tree, and in addition to the 7% and 9% treatments, root POD activities in other treatments were increased first and then decreased according to the season. The activities of SOD were decreased gradually from spring to autumn, and in the spring SOD activities of 1%, 3% and 5% treatments were 1.28, 1.34 and 1.24 times higher than the control, while the 7% and 9% treatments were 91.5% and 93.9% lower than the control. Chlorophyll a, chlorophyll b and carotenoid contents of young apple tree leaves in 1%, 3% and 5% treatments were increased gradually from spring to autumn. Chlorophyll a, chlorophyll b, carotenoid and chlorophyll (a+b) contents in 3% treatment were the highest in spring, summer and autumn, and were 1.17, 1.34, 1.09 and 1.22 times of the concurrent control in spring; and were 1.18, 1.29, 1.11 and 1.21 times of the concurrent control in summer, and 1.52, 2.23, 1.42 and 1.65 times of the concurrent control in autumn. In 3% treatment, the leaf Pn, Tr, and Gs of the continuous cropping apple trees increased by 13.8%, 27.3% and 17.3% compared with the control. 【Conclusion】The effects of different concentrations of organic material fermentation fluid on the growth, leaf photosynthesis fluorescent parameters and root antioxidant activity of young apple tree under replant conditions were different, 1%, 3% and 5% treatments could reduce the apple continuous cropping obstacle, and organic material fermentation fluid concentrations higher than 5% inhibited the growth of young apple trees. Therefore, the organic material fermentation fluid concentration of 3% can more effectively reduce the apple continuous cropping obstacle.

Genetic Polymorphism of the SCD1 Gene and Its Associations with Milking Traits in Chinese Holstein

WANG Xiao-Long, CHANG Ling-Ling, CHEN Ying, CHEN Ren-Jin, LI Yun-Long, MAO Yong-Jiang, JI De-Jun, YANG Zhang-Ping

Scientia Agricultura Sinica. 2014, 47(9):  1858-1864.  doi:10.3864/j.issn.0578-1752.2014.09.021

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【Objective】This experiment was conducted to explore the association between SCD1 gene and milking traits in Chinese Holstein in south China. 【Method】 The polymorphism of SCD1 gene was investigated for 323 Chinese Holstein cattle from the experimental farm of Yangzhou University using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) technique, at the same time, from 140 Chinese Holstein with similar body condition (BCS=3.0±0.5), no clinical mastitis, 2-3 parities and during lactation, 100 mL mixed milk samples a day (morning﹕noon﹕evening=4﹕3﹕3) were collected for determining the content of fatty acid, and then 100 mL mixed milk samples were collected all day (morning﹕mid-day﹕evening), which were used for determining fatty acid, and the effects of alleles on the component of fatty acids were then analyzed using a mixed animal model. 【Result】Two polymorphic sites, SCD1-762 and SCD1-878, were detected with the advantageous allele frequencies of A at SCD1-762 and C at SCD1-878 were 0.7897 and 0.7835, respectively. χ2 test indicated that these two sites were in the Hardy-Weinberg equilibrium. Linkage disequilibrium existed between SCD1-762 and SCD1-878 (r2=0.879) at a very significant level (P＜0.01). Four haplotypes were defined at the two sites, TC, CC, TT, and CT. Effect of SCD1-762, SCD1-878 or certain haplotypes was significant on the ratio of MUFA and the total saturation degree in milk (P＜0.05). The ratio of MUFA and total saturation degree in milk of haplotype TC was significantly higher than CT, while the ratio of SFA was significantly lower than CT (P＜0.05).【Conclusion】The two polymorphic sites had some genetic effects on milking traits, but further researches are needed for its mechanism.