参薯金属硫蛋白基因DaMT2a的克隆与表达分析

Molecular Cloning and Expressional Analysis of Metallothionein Gene DaMT2a from Dioscorea alata

金属硫蛋白属于一类富含半胱氨酸,能够与重金属离子结合的低分子量蛋白质,在植物抗逆过程中起着重要作用。本研究从参薯中克隆了一个金属硫蛋白基因,命名为DaMT2a (与金属硫蛋白MT2a同源),序列分析显示克隆获得的金属硫蛋白基因的c DNA长度为540 bp,包含了3个外显子和2个内含子,编码79个氨基酸,具有金属硫蛋白基因家族的保守结构域。进化树分析表明DaMT2a属于金属硫蛋白Type2类成员。利用实时荧光定量PCR技术研究了其在正常组织及胁迫条件下的表达特性。结果表明DaMT2a主要在参薯的茎和雄花中大量表达,机械伤害可显著上调DaMT2a基因表达,高温处理则明显下调DaMT2a基因表达,乙烯利和脱落酸处理上调DaMT2a基因的表达。本研究为参薯抗逆相关金属硫蛋白基因的筛选和功能鉴定提供了参考依据。

Metallothioneins(MTs),a class of small,cysteine(Cys)-rich and metal-binding proteins,play important roles in stress response.In this study,the full-length cDNAs encoding a metallothionein gene was obtained by RT-PCR in Dioscorea alata,which is homologous to MT2a and named as DaMT2a.Sequence analysis revealed that DaMT2a was 540 bp long,contained 3 exons and 2 introns,predicted to encode proteins of 79 amino acids,and had the conserved domains of the metallothionein.Phylogenetic analysis showed that DaMT2a was clustered into Type2 of the metallothionein family.The DaMT2a gene was explored by RT-qPCR for its expressional patterns in different tissues and in tissue-cultured seedling responding to stresses.The results indicated that DaMT2a showed a predominance expression in the stems and male flowers.The treatments of mechanical wounding,ethylene and abscisic acid markedly up-regulated the expression of DaMT2a,whereas the high-temperature treatment down-regulated its expression.This study provides a reference for the screening and functional identification of stress-related MT genes in Dioscorea alata.