【作者】 杨春；

【导师】 高学文；

【作者基本信息】 南京农业大学 ， 植物病理学， 2003， 硕士

【摘要】 大豆是一种重要的经济作物。大豆灰斑病(soybean frogeye leaf spot)是大豆上的一种严重病害。其病原为尾胞属大豆尾孢菌(Cercospora sojina)。克隆大豆抗灰斑病菌相关基因旨在为大豆抗病基因工程提供材料，并有助于我们深入了解大豆抗灰斑病的分子机制；而利用转基因技术将具有抗病、抗虫和促生长作用的hrf1基因导入大豆可为我们提供抗灰斑病的大豆品种。 大豆抗灰斑病菌相关基因的克隆 1．大豆抗灰斑病菌相关基因cDNA片段的克隆及序列分析 以大豆灰斑病菌亲和与非亲和小种接种大豆，利用mRNA差异显示技术分离了大豆抗灰斑病相关基因cDNA片段共74个。通过反向northern，Southern杂交，northern杂交等检测，最后确定了33个抗灰斑病相关cDNA片段。初步分析表明，片段DN23M是Glycine max major latex protein homologue(msg)3′端序列，可能编码了一个PR10/Bet v 1/MLP蛋白家族的病程相关蛋白或抗病应答蛋白；片段DN31M与G.max glycine-rich RNA-binding protein(GRP)有较高同源性，可能是涉及依赖于GRP的mRNA剪切水平上的一种抗性机制；片段DN32M、DN40M和DN43N则显示了与植物凝集素基因的高度同源，其二级结构的分析也表明它们含有一个保守的豆科凝集素(Legume Lectin)的结构域Lectin_legA，但又与已知的大豆凝集素有很大差异，可能是在大豆中存在一种新的凝集素，并参与了大豆对灰斑病菌的抗性机制。 2．大豆抗灰斑病菌相关基因cDNA片段反向PCR研究 对已知的大豆抗灰斑病菌相关基因cDNA片段DN45N进行反向PCR分析，从大豆基因组中扩增出该片段两侧序列，得到8425 bp的克隆IPPDN45N-SacI。经测序和序列分析，得到完整的片段IPPDN45M(8575 bp)。生物信息学分析(基因预测)表明IPPDN45M中包含了一个完整的基因IPPDN45M-G，其编码的蛋白质IPPDN45M-P可能是一种植物凝集素蛋白或类似物，并包含一个GAL_Lectin活性结构域。大豆抗灰斑病菌相关基因克隆与分析及大豆转h厅1基因相关研究令h厅1墓因在大豆中的表达引发抗病性 力rP基因是植物病原细菌中编码非特异性激发子的一类基因，其产物HarPins可以激发烟草过敏性细胞坏死(hyPersensitive eell death，HCD)，诱导植物抗病性、抗虫，促进植物生长。本研究利用花粉管通道法，将来自Xan动。monas。厂尸aepv.on亿日e的编码harPinxo。的基因h成转入大豆，得到了不同的转基因株系，检测表明转力成基因的大豆对灰斑病有显著抗性，且抗病防卫反应基因IOxZ、pall、Chss组成性表达。更多还原

【Abstract】 Soybean plant is an important economic crop. Soybean frogeye leaf spot is one of serious disease during soybean growing stage. The pathogen of this disease is Cercospora sojina. Cloning of soybean resistance C.sojina relevant genes should provided materials for gene engineering of soybean resistance disease and helped us to understand the molecular mechanism of soybean resistance against C.sojina. Soybean transgened with hrfl gene, which has functions of inducing resistance to pathogen and insects, enhance plant growth, should offered high resistance levels soybean varieties.Cloning of soybean relevant genes to resistance against C.sojina 1. Cloning and sequence analysis of soybean cDNA relevant to resistance against C.sojina and specific induced by the pathogenBy an mRNA differential display protocol, 74 cDNA fragments were obtained from soybean plants inoculated with two different races of C.sojina. Thirty-three fragments were subsequently determined to be associated with resistance to the pathogen in the plant, based on Southern, reverse northern, and northern blot hybridization. Advanced analyses of these cDNAs revealed several ones that are implicated in resistance. The cDNA DN23M shows partial homology with the 3’-terminus of Glycine max major latex protein homologue (msg), which appears to be a pathogenesis related or resistance-related protein and belongs to the PRIO/Bet v 1/MLP protein family. Secondly, cDNA DN32M has a high homologue with G.max glycine-rich RNA-binding protein (GRP), which was considered involved in a resistance mechanism based on mRNA splicing.Moreover, cDNAs DN32M, DN40M and DN43M contain a region highly homologous with lectins. Deduced partial proteins possess a conserved domain of the Legume lectin, LegA, but are distinct from known soybean lectins. Therefore, we propose that these might be an undiscovered type of lectins in soybean, which involved in the mechanism of soybean resistance C.sojina.2.Reverse PCR analysis of soybean cDNA relevant to resistance against C.sojina and specific induced by the pathogenReverse PCR programs were conducted to amplify 5’ and 3’-terminal sequences of the partial cDNA sequence of soybean cDNAs relevant to the resistance against C.sojina and specific induced by the pathogen, as described in the last chapter of this thesis. Primers used were synthesized based on sequences of cDNA clones we got and allowed for amplification of sequences flanking sufficient length of the cloned cDNAs and anticipated termini. By the protocols, two terminal parts of the DN45M clone were obtained. Complete sequence of the positive clone IPPDN45M has been obtained, as revealed by assembling these mutual flanked sequences and analyses of IPPDN45M-SacI fragments. Bioinformatical analyses suggest that IPPDN45M contain a complete gene that encodes a lectin or a lectin-like protein named IPPDN45M-P. Amino acid components and orders in the putative IPPDN45M-P indicate that the protein contains a functional domain GAL-Lectin found in other lectins.Transgenic expression of the hrfl gene in soybean confers pathogen defense.Some hrp genes in the hrp gene cluster in plant pathogenic bacteria encode nonspecific elicitors named harpins. Harpins elicit hypersensitive cell death (HCD) in nonhost plants of bacteria and induce resistance to pathogens and insects in many plants. In this study, hrfl gene, which encodes harpinXoo of Xanthomonas oryzae pv. oryzae, was transferred into soybean by pollen tube pathway. Transgenic expressing of the hrfl gene in soybean confers resistance to C. sojina. Genes LOX2, PAL1, CHS8, which are involved in pathogen defense, were expressed to various levels inhrf1-expressing transgenic soybean lines.更多还原