苋菜AmPCS基因的克隆及表达载体构建

合成植物络合素(PCs)是高等植物减轻重金属毒害的重要机制。通过对镉超积累苋菜品种天星米植物络合素合成酶基因(PCS)的克隆及表达载体构建,旨在为植物修复镉污染土壤奠定基础。依据同源克隆原理,通过RT-PCR和RACE方法克隆苋菜PCS基因。序列分析表明:苋菜PCS基因cDNA全长1 770 bp,包含完整的阅读框,编码485个氨基酸;苋菜PCs合成酶与拟南芥、遏蓝菜、芥菜及油菜PCs合成酶相似性为91.96%～95.67%,其氨基酸序列N端有1个Pfam:Phytochelatin结构域(功能为催化合成植物络合素)、C端有1个Pfam:Phytochelatin_C结构域(功能为重金属离子感应器)。因此,推断苋菜PCS基因编码蛋白是PCS-like家族的新成员,具有催化合成植物络合素的功能,将它在GenBank注册,序列号为:JN979370,命名为AmPCS。在AmPCS基因的编码区加入BamH I和Sac I酶切位点,双酶切植物表达载体pBI-121和带有酶切位点的PCR产物,成功获得苋菜PCS基因正义表达载体pBI-PCS。

Cloning of AmPCS Gene from Amaranthus mangostanus and Construction of Its Plant Expression Vectors

The synthesis of phytochelatins(PCs) represents a major heavy metal detoxification mechanism in higher plants.To contribute to the development of the phytoremediation removing Cd from the soil,a PCS gene was cloned according to homologous gene conserved region by RT-PCR and RACE method from Amaranthus mangostanus L.,a typical Cd hyperaccumulator named Tianxingmi.Its full-length cDNA is 1 770 bp and encodes a polypeptide of 485 amino acids with an entire ORF.The AmPCS protein showed high homology to other PCS-like proteins such as Arabidopsis thaliana、Thlaspi caerulescens、Brassica napus and Brassica juncea between 91.96% and 95.67% identity.The AmPCS protein contains a N terminal Pfam:Phytochelatin domain known as a heavy-metal-induced phytochelatin catalysis center and a C terminal Pfam:Phytochelatin_C domain known as a heavy metal sensor.Therefore,the present results suggested that the PCS gene from Amaranthus mangostanu might be a novel member of the PCS-like family with the function of phytochelatins synthesis.This gene named AmPCs has been submitted to GenBank with the accession number JN979370.The plant expression vector pBI121 and AmPCS gene with BamHI和SacI site were digested by these two restriction enzymes,were ligated and recombinant,and then plant expression vector pBI-PCS was successfully constructed.