茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达

Cloning and Expression Analysis of Tea Hydroperoxide Lyase Gene CsiHPL1

根据茶树基因CsiHPL1的cDNA序列设计引物,采用RT-PCR方法从茶树品种龙井43'中克隆了CsiHPL1序列,并分析了CsiHPL1在生物和非生物胁迫下的诱导表达情况及亚细胞定位。结果表明,CsiHPL1包含一个1 476 bp的最大开放阅读框,编码491个氨基酸,预测为13-HPL基因。Real-Time PCR分析结果表明,CsiHPL1的表达受到茶尺蠖取食、机械损伤和茉莉酸的诱导;构建了CsiHPL1与绿色荧光蛋白(GFP)基因的重组表达载体,经农杆菌瞬时转化烟草叶片,利用激光共聚焦显微镜在叶绿体中观察到GFP荧光,表明CsiHPL1编码的蛋白质为叶绿体蛋白质。

Primers were designed according to the cDNA sequence of tea CsiHPL1.RT-PCR method was used to clone CsiHPL1 from Longjing 43′.The expression of CsiHPL1 under biotic and abiotic stress and subcellular localization were analyzed.The results showed CsiHPL1 contains an open reading frame of 1 476 bp which encodes a protein of 491 amino acids.This gene was predicted as a 13-HPL gene.Tea geometrid feeding,wounding and JA treatment up-regulated the expression levels of CsiHPL1.The total sequence of this gene was fused with GFP to construct a binary vector for tobacco transient transformation.Under confocal laser-scanning microscopy,green fluorescent signals were localized in chloroplasts in transgenic tobacco plants,suggesting that the gene encodes a protein targeting to chloroplast.