小蓬NeMT2基因的克隆及其植物表达载体的构建

Cloning and construction of plant expression vector of NeMT2 gene in Nanophyton erinaceum

关键词:  小蓬, NeMT2, RACE, 序列分析, 植物表达载体

Key words:  Nanophyton erinaceum, NeMT2, rapid amplification of cDNA ends, sequence analysis, plant expression vector

摘要:

该研究利用RACE(Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白(Metallothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基(Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.603 6 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子(AEF01492)和盐穗木(AHI62953)同源性最高,其次是甜菜(XP_010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白; 疏水性分析表明,NeMT2蛋白的35～45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444); 结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现,NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300+NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。

Abstract:

Nanophyton erinaceum has a long living history at Aletai Copper Mine in Xinjiang. In order to analyze the relation between metallothionein gene and heavy metal response stress, a new heavy metal-responsive gene cDNA sequence was successfully cloned by RACE(Rapid amplification of cDNA ends)from N. erinaceum. The gene was named as NeMT2, and its accession number in GenBank was KT835290. The metallothionein gene NeMT2 was 590 bp in full length, and it had 237 bp ORF(open reading frame)which encoded 78 amino acid residues. There were 14 Cys residues, arranged in the form of C-C, C-X-C and C-X-X-C, in the 78 amino acid residues, and these Cys residues distributed in N terminal and C terminal of peptides. The protein encoding by gene NeMT2 molecular weight was 7.603 6 kD and its isoelectric point was 4.71. Phylogenetic analysis results demonstrated that the deduced amino acid sequence of gene NeMT2 was the highest homology as the Salicornia brachiata(AEF01492)and the Halostachys caspica (AHI62953), secondly the Beta vulgaris (XP_010667708.1), but the lowest similarity with the Nelumbo nucifera (XP_010253171). Bioinformation analysis showed that metallothionein NeMT2 had no signal peptide and belonged to the hydrophilic non-transmembrane protein. Hydrophobicity analysis was carried out and the results showed that there was a strong hydrophobicity region between 35 to 45 amino acids, and the 41 Asp had the strongest hydrophobic property(1.444). Predication of structure indicated that random coil was the major components of its secondary structure. Expression analysis of NeMT2 gene was carried out by RT-PCR. The results showed that expression of NeMT2 gene was detected both in Copper Mine and nor Copper Mine in Nanophyton erinaceum leaves, but the former was more stronger than the latter, which indicated that NeMT2 gene was responsive to the heavy metal stress. Then NeMT2 gene in Nanophyton erinaceum was cloned into 35S promoter downstream of plant over-expression vector pCAMBIA1300 in orientation. The plant overexpression vector pCAMBIA1300+NeMT2 was successfully constructed. These findings will provide information for the functional study of NeMT2 and its molecular mechanism in repnse to the heavy metal stress.