大豆翻译延伸因子GmEF1A干扰载体的构建及大豆遗传转化  

Interference Vector Construction of Soybean Translation Elongation Factor GmeEF1A and Soybean Transformation

作　　者：廖文林[1] 栾鹤翔[1] 高乐[1] 丁雪妮[1] 宋英培 殷金龙[1] 牛浩鹏 智海剑[1] LIAO Wen - lin LUAN He - xiang GAO Le DING Xue - ni SONG Ying - pei YIN Jin - long NIU Hao - peng ZHI Hai-jian(National Center for Soybean Improvement of Nanjing Agricultural University/National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing 210095, China)

机构地区：[1]南京农业大学国家大豆改良中心/作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《大豆科学》2016年第6期902-910,共9页Soybean Science

基　　金：转基因生物新品种培育重大专项(2008ZX08004-004);国家自然科学基金(31371646,31571690);国家现代农业产业技术体系(CARS-004);江苏省现代作物生产协同创新中心(JCIC-MCP)

摘　　要：研究发现GmeEF1A与大豆花叶病毒的P3蛋白存在互作关系,并且参与SMV在大豆体内的繁殖。通过大豆GmeEF1A基因5个拷贝的核苷酸和氨基酸序列比对,确定其保守区间,克隆得到180 bp的干扰片段GmeEF1Ai。利用GATEWAY技术构建RNA干扰(RNA interference,RNAi)表达载体pB7GWIWG2(II)-e EF1Ai,并通过农杆菌介导的子叶节转化法导入到受体大豆品种天隆1号中。测序结果显示重组质粒中插入的干扰片段GmeEF1Ai与目标序列完全符合,通过PCR和酶切验证表明GmeEF1Ai是以反向重复形式插入到表达载体中。经转化获得组培苗8株,PCR、除草剂涂抹和PAT/bar试纸条多重检测确认7株为阳性。绝对荧光定量结果显示,阳性苗中4株为单拷贝。GmeEF1A基因表达量分析结果显示GmeEF1Ai载体对GmeEF1A的5个拷贝基因有不同程度的阻抑。这不仅为验证GmeEF1A基因在大豆受SMV侵染时的功能提供试验材料,也为大豆抗病育种提供新的种质。Previous research indicated the interaction between GmeEFIA and P3 protein of soybean mosaic virus, and it is re- lated to SMV replication in soybean. In order to determine the conserved interval of soybean G meEF1A, we made the nucleotide sequence and amino acid sequence alignment from 5 isoforms of GmeEF1A, cloned the 180 bp interference fragment named GmeEF1Ai. The RNAi vector pB7 GWIWG2 （II） -eEF1Ai was constructed by GATEWAY technology, and transfered into soybean genotype Tianlong 1 through Agrobacterium-mediated system. The sequencing result of GmeEF1Ai fragment in vector matched with the expected sequence completely, and the insert direction was conversed identified by PCR amplification and restriction digestion. We obtained eight transgenic seedlings, seven plants of them were positive detected by PCR, herbicide painting and Liberty Link strip. The data of the fluorescence quantitative PCR showed 4 seedlings contained one copy of bar gene. The resuhs of expression levels of GmeEF1A indicated GmeEF1Ai vector could inhibit the expression of 5 isoforms in different extent. The results of this study provided a material to verify the function of GmeEF1A in SMV resistance and new germplasm resource in soybean mosaic virus resistance breeding.

关 键 词：大豆花叶病毒 GmeEF1A基因 RNAI GATEWAY技术 农杆菌介导大豆遗传转化 

分 类 号：S565.1[农业科学—作物学]