海南龙血树金属硫蛋白基因DcMT2的克隆和表达分析

Cloning and Expression Analysis of Metallothionein Gene DcMT2 in Dracaena cambodiana in Hainan

金属硫蛋白是植物金属离子代谢及胁迫应答的关键蛋白,而且通过清除多余的活性氧调控着植物抗性的形成过程。MT2蛋白是重要的植物金属硫蛋白之一。本研究利用海南龙血树转录组数据,在注释拼接的基础上首次克隆了海南龙血树金属硫蛋白MT2基因(命名为DcMT2,Gene Bank登录号为MF594215),DcMT2基因序列全长为237 bp,编码79个氨基酸。序列分析发现DcMT2具有典型的植物金属硫蛋白MT2的结构特征,包含2个富含半胱氨酸的保守基序。DcMT2蛋白与百合目植物芦笋、水仙和马蔺具有较高的相似度,且在系统进化上划为同一分支。生物信息学分析显示该编码蛋白包含6个磷酸化位点,为非分泌型蛋白,主要在细胞外发挥作用,相对分子量为7.81 kD,理论等电点为4.86;二级结构主要由α螺旋(20.25%)和无规则卷曲(56.96%)构成。实时荧光定量PCR结果表明,DcMT2在海南龙血树的各组织中均有表达,在果实中的表达量最高,而根、茎、叶中的表达量较低。此结果为研究金属硫蛋白DcMT2在海南龙血树发育和血竭形成过程中的作用奠定了基础。

Metallothionein （MT） is a key protein in the metabolism and stress response of plant metal ions. It also regulates the formation of plant resistance by scavenging excess reactive oxygen species. MT2 is one of the most critical NiT proteins in plant. In this study, we used the transcriptome data of Dracaena cambodiana in Hainan to clone MT2 gene ofD. cambodiana for the first time on the basis of annotation stitching. It was named as DcMT2, and the accession number of it in GeneBank was MF594215. DcMT2 was 237 bp long and encoded 79 amino acid. Sequence analysis revealed that DcMT2 had typical structural features of metallothionein MT2, and contained 2 conserved conserved motifs with cysteine. DcMT2 protein was similar with the MT2 of Asparagus officinali, Narcissus pseudonarcissus and Iris lacteal, and they were classified in the same branch. Bioinformatic analysis showed that the encoded protein contained 6 phosphorylation sites, which was a non secretory protein. It mainly acted outside the cell, with a relative molecular weight of 7.81 kD and a theoretical Pi of 4.86. The secondary structure consisted of a helix （20.25%） and random coil （56.96%）. RT-qPCR results indicated that DcMT2 was expressed in all the tissues ofD. cambodiana in Hianan, with the highest expression in fruit and the lowest in root, stem and leaf. This study laid the foundation for the research on the effect of metallothionein DcMT2 protein in the development and dragon＇s blood formation ofD. cambodiana.