论文部分内容阅读
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。
It has been found that GmeEF1A interacts with the P3 protein of soybean mosaic virus and participates in the propagation of SMV in soybean. Based on the nucleotide and amino acid sequence alignment of five copies of GmeEF1A gene in soybean, the conserved region was identified and a 180 bp GmeEF1Ai fragment was cloned. The RNA interference (RNAi) expression vector pB7GWIWG2 (II) -e EF1Ai was constructed by GATEWAY technology and introduced into the recipient soybean variety Tianlong No.1 by Agrobacterium tumefaciens-mediated cotyledonary node transformation. Sequencing results showed that GmeEF1Ai inserted into the recombinant plasmid was completely consistent with the target sequence. PCR and restriction enzyme digestion showed that GmeEF1Ai was inserted into the expression vector in an inverted repeat manner. After transformation, 8 strains of tissue culture seedlings were obtained, and 7 strains were positive by multiplex PCR and herbicide smear and PAT / bar test strips. The results of absolute fluorescence quantitative analysis showed that four of the positive seedlings were single copy. GmeEF1A gene expression analysis showed that the GmeEF1Ai vector had 5 different copies of GmeEF1A gene repression. This not only provided a test material for verifying the function of GmeEF1A gene when soybean was infected by SMV, but also provided new germplasm for soybean disease-resistant breeding.