湖北海棠植物络合素合酶MhPCS基因克隆及表达分析

Isolation and expression analysis of a phytochelatin synthase gene (MhPCS) from Malus hupehensis

【目的】为了探明植物络合素酶参与植物解毒重金属的作用,【方法】以湖北海棠(Malus hupehensis)为试材,克隆其植物络合素合酶基因(MhPCS)编码区序列,并研究该基因的表达特点。【结果】结果表明MhPCS基因编码区全长为1 494 bp,编码497个氨基酸。序列分析表明,MhPCS编码的氨基酸序列由2个典型的植物络合素合酶亚家族结构域组成,具有3个相邻的Cys-Cys元件(331-332、351-352和369-370位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91和Cys-109)。【结论】湖北海棠MhPCS与杜梨PbPCS蛋白同源性最高(94%),属于系统发生树的同一分支。MhPCS基因为组成型表达,各器官表达水平存在差异,其中根的表达量最高;100μmol.L-1CdSO4处理48 h内,MhPCS基因的表达量迅速上升;不同金属离子对其上调表达的诱导能力为:Cd2+>Cu2+>Pb2+。这为揭示重金属胁迫下湖北海棠环境适应的分子机制奠定了基础。

[Objective]The objective of the study is to elucidate the mechanism of phytochelatin synthase involved in theplant resistance to heavy metal. [Method]The phytochelatin synthase gene （MhPCS） was isolated from the roots of Malus hupehensis, which was used as one of apple rootstocks, and its expression characterization was researched via reverse transcription-PCR （RT-PCR） and semi quantitative reverse transcription PCR （semi RT-PCR） methods. [Result]The intact open reading frame （ORF） length of MhPCS gene was 1494 bp in length, which encoded a 54.9 kD protein of 497 amino acids. The MhPCS protein was constituted of two typical phytochelatin subfamily domains, and included three adjacent Cys- Cys elements （331-332, 351-352 and 369-370 amino acid） in the C-terminal region and all characteris- tics sites （Cys-56, Cys-90/91 and Cys-109） of phytochelatin synthase protein. [Conclusion]MhPCS had the highest homology （94%） with PbPCS from Pyrus betulifolia, and belonged to the same branch in PCS phylogenetic tree. Semi quantitative RT-PCR results showed that MhPCS gene was constitutively expres- sion and its expression level was different in various organs, while the expression level was highest in roots. Furthermore, MhPCS transcription level was increased after 100 Ixmol L-1 CdSO4 treatment for 48 hours. It was significant that three heavy-metal ions had different abilities to up-regulated MhPCS expres-sion level, and the order is Cd2＋〉Cu2＋〉Pb2＋. These results laid the foundation for the molecular mechanism of adaptation to environment of Malus hupehensis under heavy metal stress.