摘要： 为深入研究板栗WRKY转录因子基因在板栗抗病过程中的分子作用机制，根据板栗转录组数据库分析获得EST序列，利用RT-PCR技术，克隆板栗WRKY转录因子cDNA （CmWRKY）序列，分析CmWRKY基因及其编码蛋白的相关信息并进行原核表达研究。结果表明，CmWRKY基因为1 437 bp，编码479个氨基酸，推测蛋白分子质量为52.128 96 ku，理论等电点为9.15，具有2个WRKY保守结构域和2个C2H2锌指结构域，属于WRKY家族的第Ⅰ组，基因登录号为KY312850.1。CmWRKY核苷酸序列与巴旦木等植物的WRKY基因一致性均在80%以上，与西班牙栓皮栎WRKY基因一致性最高，达到97%。同源建模表明，CmWRKY蛋白三级结构与拟南芥AtWRKY1蛋白结构相似，推测其可能与AtWRKY1具有相似的调控功能。分子进化分析显示，板栗CmWRKY与西班牙栓皮栎及核桃WRKY蛋白亲缘关系最近。SDS-PAGE蛋白电泳分析表明，CmWRKY蛋白最佳诱导表达条件为0.2 mmol/L IPTG在30℃下诱导10 h，蛋白分子质量为56 ku，其主要以可溶性蛋白的形式存在。为板栗CmWRKY基因的生物学功能研究及应用奠定基础。

关键词: 板栗, CmWRKY, 基因克隆, 序列分析, 原核表达

Abstract: The EST sequence of WRKY gene was obtained based on the transcriptome database of Castanea mollissima BL, and the cDNA(CmWRKY)sequence of WRKY transcription factor in chestnut was cloned by RT-PCR. The information of CmWRKY gene and its encoding protein were analyzed and the gene expression in the prokaryotic cell was carried out. The results showed that CmWRKY gene was 1 437 bp in length, encoding 479 amino acids with a calculated molecular weight of 52.128 96 ku and theoretical isoelectric point of 9.15. It had two WRKY conserved domains and two C2H2 zinc finger domains, belonging to Group Ⅰ of the WRKY family, and its gene accession number was KY312850.1. At the nucleotide level, the similarity of the CmWRKY gene with the corresponding sequence of Prunus persica was over 80%, and the highest similarity with the Qurcus suber WRKY gene was 97%. Homology modeling indicated that the tertiary structure of CmWRKY protein was similar to WRKY1 protein of Arabidopsis thaliana, suggesting that it might have the similar regulatory function as AtWRKY1 did. Molecular evolution analysis showed that chestnut CmWRKY was closely related to the WRKY of Qurcus suber and Juglans regia. SDS-PAGE analysis showed that the optimal expression condition of CmWRKY protein was 0.2 mmol/L IPTG induced at 30℃ for 10 h, and the molecular weight of the protein was about 56 ku, mainly in the form of soluble protein. The results laid the foundation for further studying the biological function and application of CmWRKY gene.

Key words: Castanea mollissima BL, CmWRKY, Gene cloning, Sequence analysis, Prokaryotic expression

中图分类号: 

• Q785

• Q786

• S664.03