PCR primers were designed baaed on RS gene coding sequence, which was screened from Arabidopsis cDNA library. The 476 bp cDNA fragment of RS was cloned from pGADT7-AS recombinant plasmid using PCR. The RNA interference technology was used to construct gene silencing vector containing AS gene. This study may provide a basic material for further studies on the bio-function of RS and the mechanism of signal transduction induced by HpaGxoo in plant. Two 476 bp fragment, of RS with Pst I/BamH I and Pst I/Xho I were obtained through digesting pUCm-RS, the combination of vector pUCm-T and gene AS, were connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two fragments were invent duplication. The transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I/Kpn I, was connected into expression vector pCAMBIA1301. Gene silencing vector containing RS gene was constructed, and the combined vector could be employed.%根据拟南芥RS基因的编码序列设计引物,利用PCR技术从pGADT7-RS重组质粒上扩增到RS基因编码区476 bp片段.利用双链RNA介导的基因沉默技术,构建RS基因沉默表达载体,为进一步研究该基因在植物与蛋白激发子HpaGx(oo)互作中的功能及其作用机制奠定基础.首先将RS基因片段连接到pUCm-T载体上,用Pst I/BamH I和Pst I/Xho I分别对pUCm-PS重组载体进行酶切,得到2个RS基因片段;先后将其连接到用相应限制酶酶切的pBSSK-in载体上,构建成pBSSK-PS-in-RS重组载体,该重组载体中的2个RS片段大小一致,反向重复;最后用Sac I/KpnI酶切pBSSK-RS-in-RS载体得到RS-intron-RS片段,连入表达载体pCAMBIA1301中,构建成该基因的沉默表达载体.
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