茶树CsPLK基因的鉴定与表达分析

Identification and expression analysis of CsPLK gene in tea plant

关键词:  茶树, 基因鉴定, 逆境胁迫, 组织特异性

Key words:  tea plant, gene identification, abiotic stress, tissue specificity

摘要:

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B₆(VB₆)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB₆补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员; 通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性; 组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低; 荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。

Abstract:

Tea plant is rich in important functional ingredients such as theanine, catechin and caffeine, with high value and efficacy. Tea plants are often subjected to stress in their life cycle, and VB₆ is involved in the stress response in plants, Pyridoxal kinase(PLK)is a key enzyme in the vitamin B₆(VB₆)salvage pathway. In order to further understand the function and mechanism of PLK in tea plant biosynthesis, this study based on the tea plant genome database, and using longjing 43 as material, the CsPLK gene was cloned from tea plant by reverse transcription polymerase chain reaction(RT-PCR). The results were as follows: The sequence of CsPLK was 1 179 bp in length, encoding 393 amino acids; CsPLK protein and PLK protein of known species had high homology, both were members of the ribokinase superfamily; The pET-CsPLK vector was constructed for prokaryotic expression, and the recombinant protein was identified to have strong catalytic activity, indicating that the gene cloned from tea tree is PL kinase; Tissue expression specificity analysis showed that the expression level in leaves was higher than that in stems and roots, and the lowest in roots; Real-time PCR showed that CsPLK was up-regulated expressed by low temperature and CsPLK was down-regulated by drought. This study reveals that CsPLK had an obvious stress response in tea plants, suggesting that CsPLK played an important role in the growth and development of tea plants and the stress resistance.