番茄SlmiR393基因超表达载体的构建及其靶基因鉴定

Construction of SlmiR393 Over-expression Vector and Identification of Its Target Genes in Tomato

摘要:

为研究番茄(Lycopersicum esculentum)的SlmiR393基因功能,采用生物信息学方法从番茄基因组数据库中获得SlmiR393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SlmiR393前体基因并整合到pLP35S-100植物表达载体;采用5' RACE RT-PCR技术验证SlmiR393对预测靶基因mRNA的剪切作用,采用定量PCR 技术检测SlmiR393及其靶标基因在番茄不同组织的表达。结果表明,SlmiR393的前体序列含有完整的茎环结构;成熟miR393与3个生长素受体同源基因(SlTIR1、SlTIR1-like1和SlAFB)之间具有识别作用位点。SlmiR393可对番茄3个靶基因的转录本进行剪切降解;SlmiR393与3个不同靶基因(SlTIR1、SlTIR1-like1和SlAFB)的表达模式在叶和茎、花蕾、花中存在一定的互补关系。因此,推断番茄中的SlmiR393可能在特定的组织或发育时期介导特定的靶基因mRNA的剪切降解, 初步证实生长素受体同源基因为SlmiR393的靶基因。此外,成功构建以CaMV 35S为启动子的植物表达载体pLP35S-pre-SlmiR393, 为深入研究SlmiR393在番茄生长素信号转导中的功能奠定了基础。

Abstract:

In order to understand the function of SlmiR393 in tomato (Lycopersicum esculentum), the precursor sequences and potential target genes of SlmiR393 were obtained by searching tomato genome database with computational algorithms. The SlmiR393 gene was amplified from tomato genomic DNA by PCR and integrated into plant expression vector pLP35s-100. Sly-miR393 guided-cleavage to putative target genes mRNAs was validated using 5' RACE RT-PCR. The expression of SlmiR393 and its target genes in tomato different tissues were determined by Real-time quantitative PCR. The results showed that the precursor sequence of SlmiR393 contains the complete hairpin structure. Three auxin receptor gene homologs (SlTIR1, SlTIR1-like1 and SlAFB) mRNAs contain recognition sites with high complementarities to Sly-miR393 sequence. In tomato, SlmiR393 directs the cleavage of SlTIR1, SlTIR1-like1 and SlAFB mRNA. The expression of SlmiR393 has opposite effects on SlTIR1, SlTIR1-like1 and SlAFB in stem, leaf, bud and flower, respectively. Therefore, it was suggested that SlmiR393 might direct specific target gene mRNA cleavage in tomato specific tissue and developmental stage, and the auxin receptor homologous (SlTIR1, SlTIR1-like1 and SlAFB) were validated to be as target of SlmiR393. Additionally, the pLP35s-pre-SlmiR393 vector containing SlmiR393 gene was successfully constructed with CaMV 35S as promoter, which laid a foundation for further studies of SlmiR393 function in auxin signaling pathway in tomato.