大豆疫霉根腐病抗性相关基因SDR1的克隆及功能分析  

Isolation and Functional Analysis of Resistance Gene SDR1 to Phytophthora sojae in Soybean

作　　者：范素杰[1] 吴俊江[2] 陈晨[1] 王欣[1] 姜良宇[1] 王金生[2] 李文滨[1] 徐鹏飞[1] 张淑珍[1] 

机构地区：[1]东北农业大学大豆研究所/大豆生物学教育部重点实验室,哈尔滨150030 [2]黑龙江省农业科学院大豆研究所/博士后工作站,哈尔滨150086

出　　处：《中国农业科学》2012年第11期2139-2146,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30971811;31071439;31171577;31101167);教育部新世纪人才培养计划(NCET-09-164);高等学校博士点基金(20112325120005);黑龙江省教育厅科学技术研究项目(12511041)

摘　　要：【目的】获得大豆疫霉根腐病抗性相关基因,为培育大豆抗病品种提供理论依据。【方法】在以大豆抗病品种绥农10构建的受疫霉菌诱导后差异表达的cDNA消减文库的基础上,选取文库中一条与其它植物的DR1基因具有较高同源性且上调表达的EST序列。通过RT-PCR方法从绥农10中克隆该基因,并构建到植物表达载体pCAMBIA3301上,以感病品种东农50的子叶节为外植体通过根癌农杆菌介导的方法进行大豆遗传转化。【结果】该基因全长805 bp,开放读码框为471 bp,编码156个氨基酸,在此命名为SDR1。遗传转化获得转基因PCR鉴定阳性植株5株,Real-time PCR检测T1转基因植株较非转基因植株SDR1表达量提高20倍以上的有3株,经Southern杂交分析表明,出现杂交信号的有3株。经离体叶片接种大豆疫霉菌,转基因大豆的抗性较非转基因大豆明显提高。【结论】成功克隆了大豆疫霉根腐病抗性相关基因SDR1,并通过对过量表达的大豆转基因植株的抗病性鉴定初步确定了SDR1的抗病功能。【Objective】The objective of this research is to obtain resistance-related genes to Phytophthora sojae in soybean,and to provide a theoretical basis for breeding of resistant soybean cultivars.【Method】In previous study,a cDNA library was constructed from the resistant soybean cultivar Suinong 10 in order to identify resistance-related genes in response to P.sojae infection in soybean.An up-regulated EST sharing high sequence similarities with the DR1 genes in other plants was identified and selected in this study.The gene was cloned from Suinong 10 by RT-PCR and constructed to the plant expression vector pCAMBIA3301/SDR1 and transformed into susceptible soybean cultivar Dongnong 50 via Agrobacterium mediated method.【Result】The full-length of this gene was 805 bp,encoding 156 amino acids,with ORF of 471 bp,defined as SDR1 in this study.Three transgenic plants overexpressing SDR1 were obtained and confirmed by real-time PCR,with an increase of gene expression levels by more than 20-fold compared to that of non-transgenic plants,and dot blot assay of DNA of the plants showed that 3 plants had positive hybridization signals.Detached leaf inoculation assay showed significantly increased resistance in the transgenic plants to P.sojae infection.【Conclusion】The soybean SDR1 was cloned and preliminarily confirmed to confer the resistance to P.sojae by creation and pathogenicity assays of transgenic plants overexpressing the gene.

关 键 词：大豆 大豆疫霉根腐病 抗性相关基因 SDR1 遗传转化 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]