大豆翻译起始因子4E 干扰片段克隆及其 RNAi载体构建

摘要

Soybean transformation based on RNA interference ( RNAi) can specifically silence the homologous target genes in mRNA level ,providing a new strategy for disease-resistant breeding .In this study ,we made the ami-no acid sequence alignment of eIF4E from ten different kinds of plants which have been identified to interact with the virus VPg ,then we determined the conserved interval of soybean eIF 4 E was 62-237 amino acid sequence and cloned the 406 bp interference fragment named eIF4Ei (including 58 bp specific recombination sequence attB ).U-sing the GATEWAY technology ,the RNAi vector pB7GWIWG2(Ⅱ)-eIF4Ei was constructed,which was identified by PCR amplification ,sequencing and restriction digestion .The identification of the recombinant expression vector ensured the eIF4 Ei interference fragment no mutation in the process of vector construction and two ccdB sites were all replaced by eIF4Ei,in addition,the insert direction of the two interference fragments in the terminal plasmid was conversed.Overall,the RNAi vector was successfully constructed to be an inverted repeat structure ,providing the basic materials for cultivating new germplasm of soybeans resistant to SMV using the soybean transformation technol -ogy based on RNAi .%应用基于RNA干扰（ RNA interference，RNAi）原理的大豆转基因技术，能够在转录水平沉默同源靶基因，这为转基因抗病毒作物的培育提供了新的策略。通过比对已报道的能与病毒VPg发生互作的10种植物的eIF4E氨基酸序列，确定出大豆eIF4E的保守区间为62～237位氨基酸序列，克隆出406 bp的干扰片段eIF4Ei（含58 bp特异性重组序列attB），进而采用GATEWAY技术构建了干扰表达载体pB7GWIWG2（Ⅱ）-eIF4Ei。之后通过测序证实干扰片段eIF4Ei在载体构建过程中没有发生变异；酶切试验证实终端质粒的2个ccdB位点均被eIF4Ei置换；用启动子和终止子设计的引物对含有重组质粒的菌液进行PCR扩增，证实2个干扰片段以相反的方向插入到终端质粒中。从而证明反向重复的干扰表达载体构建成功，为应用基于RNAi原理的大豆转基因技术培育对大豆花叶病毒具有抗性的大豆新种质提供了基础材料。