白及小分子热激蛋白BsHsp17.3基因的克隆与表达分析

Cloning and expression analysis of small heat shock protein gene BsHsp17.3 in Bletilla striata

关键词:  白及, BsHsp17.3, 克隆, 荧光定量PCR

Key words:  Bletilla striata, BsHsp17.3, clone, quantity RT-PCR

摘要:

为了探索人工栽培白及的适宜条件,该研究以湖北省十堰市野生白及为对象,采用同源克隆和3'RACE技术,从白及(Bletilla striata )中获得与热激蛋白合成有关的BsHsp17.3基因,并分析BsHsp17.3基因对不同胁迫的响应。结果表明:BsHsp17.3基因开放阅读框长度为453 bp,编码150个氨基酸; 蛋白的分子量为17.42 kD,等电点为6.33。进化树分析表明BsHSP17.3蛋白与同为兰科的铁皮石斛进化关系较近,同在一分支上。半定量RT-PCR分析显示BsHsp17.3基因在白及根、叶、鳞茎及花组织中的表达具有特异性,且BsHsp17.3基因在叶中的表达量较高,在鳞茎及花中不表达。实时荧光定量PCR检测显示BsHsp17.3对非生物胁迫高温、低温具有明显应答反应,20%PEG模拟干旱胁迫不诱导该基因表达,推测该基因在白及防止倒苗过程中可能发挥一定作用。

Abstract:

BsHsp17.3 gene was isolated from Bletilla striata using homologous cloning and 3'RACE methods. In this study, the expression profiles of BsHsp17.3 under different stresses were analyzed and suitable conditions were explored for artificial cultivation of B. striata. The results were as follows: The opening reading frame of BsHsp17.3 was 453 bp, which encoded a 150-amino acid peptide. Its protein molecular weight and isoelectric point were 17.42 kD and 6.33. Phylogenetic analysis demonstrated that BsHsp17.3 was closed to the Hsp17.3 from Dendrobium catenatum. Quantitative real-time PCR analysis showed that the expression of BsHsp17.3 was different in leaf, root, bulb and flower of B. striata. The expression level of BsHsp17.3 gene was the highest in leaf and root,respectively, but was absent from the bulb and flower. The expression of BsHsp17.3 was analyzed by quantity RT-PCR under cold, hot and drought treatments. The results showed that the expression of BsHsp17.3 quickly induced by high temperature and cold, but drought stress simulated by 20% PEG did not regulate the gene expression. These results suggest that BsHsp17.3 may be involved in regulating sprout tumble of Bletilla striata.