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大豆花叶病毒(soybean mosaic virus,SMV)病是全球最主要的大豆病害之一,严重危害大豆的产量和品质。RNA干扰(RNA interference,RNAi)能够在mRNA水平特异性沉默同源靶基因,为抗病毒作物的培育提供了新的途径。本研究对国内外11个不同SMV流行株系的HC-Pro基因进行了核苷酸序列比对,并以SC3株系的cDNA为模板克隆出高度保守的268 bp的基因片段,进而利用GATEWAY技术构建了适合农杆菌介导大豆转化的RNAi表达载体pB7GWIWG2(II)-HC-Proi。并对BP反应和LR反应的纯化产物进行了测序鉴定,测得序列在NCBI上进行比对,匹配度为100%;以35S启动子和终止子设计引物,扩增得到464和478 bp两个片段,说明HC-Proi基因与pB7GWIWG2(II)重组形成反向重复结构,载体构建成功。结果为利用RNA干扰技术改良大豆对大豆花叶病毒的抗性奠定了基础。
Soybean mosaic virus (SMV) is one of the most important soybean diseases in the world and seriously endangers soybean yield and quality. RNA interference (RNAi) can silently silence homologous target genes at the mRNA level, which provides a new way for the cultivation of virus-resistant crops. In this study, nucleotide sequences of HC-Pro genes in 11 different SMV strains at home and abroad were aligned and a highly conserved 268 bp gene fragment was cloned using the cDNA of SC3 strain, and then GATEWAY technology The RNAi expression vector pB7GWIWG2 (II) -HC-Proi was constructed for Agrobacterium-mediated soybean transformation. The purified products of BP reaction and LR reaction were identified by sequencing. The sequences were aligned on NCBI and the match degree was 100%. The primers of 35S promoter and terminator were designed to amplify 464 bp and 478 bp Fragment, indicating that the HC-Proi gene and pB7GWIWG2 (II) recombination to form an inverted repeat structure, the vector was successfully constructed. The results laid the foundation for improving the resistance of soybean to soybean mosaic virus by using RNA interference technology.