番茄Sly-miR393基因超表达载体的构建及其靶基因鉴定.docVIP

 番茄 Sly-miR393 基因超表达载体的构建及 其靶基因鉴定# 林冬波，杨迎伍，李正国** 5 10 15 20 25 30 （重庆大学生物工程学院基因工程研究中心，重庆 400030） 摘要：为了研究番茄 Sly-miR393 的功能，通过生物信息学方法从番茄基因组数据库中分析 和预测 Sly-miR393 的前体序列及其潜在的靶基因，以基因组 DNA 为模板，克隆了番茄 Sly-miR393 前体基因并将其亚克隆到 pLP35s-100 植物表达载体；同时，采用 5’RACE 技术 验证 Sly-miR393 对预测靶基因 mRNA 的剪切作用及其剪切位点。结果表明，Sly-miR393 的 前体序列含有完整的茎环结构；成熟 Sly-miR393 与 TIR1/AFB 生长素受体同源基因之间具有 识别位点。Sly-miR393 在番茄体内对靶基因 SlTIR1，SlTIR1-like1 和 SlAFB 的转录本通过 一个切割位点进行剪切降解；从而证实了生长素受体同源基因为 Sly-miR393 的靶基因。成 功构建了以 CaMV35S 为启动子的植物表达载体 pLP35S-pre-SlymiR393, 为深入研究 Sly-miR393 在番茄生长素信号转导中的功能奠定了基础。 关键词：Sly-miR393；靶基因；生长素受体基因；载体构建 Construction of Sly-miR393 Over-expression Vector and Verification of its Target Genes in Tomato Lin Dongbo, Yang Yingwu, Li Zhengguo (Genetic Engineering Research Center, Bio-engineering College,Chongqing University, ChongQing 400030) Abstract: To understand the function of Sly-miR393 in tomato, the precursor sequences and potential target genes of Sly-miR393 were identificated from tomato genome database by computational homology search method. The Sly-miR393 gene was amplified from the genomic DNA by PCR and cloned into plant expression vector pLP35s-100. Sly-miR393 guided-cleavage to putative target transcripts was validated using 5’RACE RT-PCR. In this study, our results indicated that the precursor sequence of Sly-miR393 contains the complete hairpin structure. TIR1/AFB auxin receptor genes contain recognition sites with high complementarities to Sly-miR393 sequence. In tomato, Sly-miR393 directs the cleavage of SlTIR1，SlTIR1-like1 and SlAFB mRNA, then auxin receptor homologous was validated to be as target of Sly-miR393. The pLP35s-pre-SlymiR393 vector containing Sly-miR393 gene was successfully constructed, which would provide significant evidence for further study of Sly-miR393 function in auxin signaling pathway in tomato. Keywords: Sly-miR393; Target genes; Auxin receptor gene; Construction 35 0 引言 MicroRNA(miRNA)是一类内源性的、长度为 21~24 个碱基的小分子非编码 RNA，它通 过对靶基因 mRNA 的降解或者抑制其翻译等方式，在转