辣椒CaDREB3基因的克隆和表达特性分析

Cloning, Expression, Subcellular Localization and Transient Expression of CaDRERB3 Gene in Hot Pepper(Capsicum annuum L.)

从构建的辣椒c DNA文库中筛选阳性克隆,测序并获得辣椒CaDREB3基因,研究该基因的亚细胞定位、瞬时表达及组织表达特性,揭示CaDREB3的功能及其在植物抗逆过程中的分子机制。应用预测软件对其进行功能及生物信息学分析,利用q RT-PCR分析CaDREB3基因的瞬时表达及在不同组织中的表达特性,并以基因枪轰击洋葱表皮的方法,对CaDREB3基因进行亚细胞定位分析。结果表明,辣椒CaDREB3长度为1 997 bp,含有1071bp的完整的开放阅读框,编码357个氨基酸,含有ERF亚族的AP_2/ERF保守结构域,与西红柿(NP_001234707.1)、马铃薯(AET99101.1)及拟南芥(NP_177931.1)具有较高的氨基酸相似性,其中与西红柿的相似性最高,达83%。亚细胞定位表明该基因位于细胞核,瞬时表达表明CaDREB3可以和GCC-box结合,q RTPCR检测表明,CaDREB3基因的相对表达量在辣椒茎、叶和果实中表达量较高。实验明确了辣椒CaDREB3基因亚细胞定位信息和组织表达情况,为进一步研究其功能及分子机制提供依据。

The present study was to screen and clone the full length CaDREB3 gene from the cDNA library of hot pepper, and analyze its subcellular location, transient expression and its expression in different tissues of hot pepper, to reveal the function of CaDREB3 and its role in stress tolerance. The protein structure of CaDREB3 was analyzed based on bioinformatics using a prediction software. The transient expression of CaDREB3 and its expression pattern in different tissues were detected by qRT-PCR. Sequence analysis showed that the CaDREB3 was 1 997 bp in length, encoded a polypeptide of 357 amino acid residues and contained an AP2/ERF domain, and the amino acids of CaDREB3 shared identity of more than 83% with tomato（NP_001234707.1）. Subcellular localization analysis in onion epidermal cells suggested 35S：：CaDREB3-GFP fusion protein was localized in the nucleus, and transient expression analysis of CaDREB3 could bind to the GCC-box. Real time PCR results showed that the relative expression of CaDREB3 was higher in stems, leaves and fruits of hot pepper. This study would lay a good foundation for the research of the function of CaDREB3 and response mechanisms of stress in hot pepper.