广藿香(Pogostemon cablin)PatWRKY40和PatWRKY53的基因克隆与表达分析

Gene Cloning and Expression Analysis of PatWRKY40 and PatWRKY53 in Pogostemon cablin

WRKY转录因子在转录调控、次生代谢调控和生长发育中起关键作用。为了了解WRKY转录因子在广藿香中的调控模式,本研究从广藿香转录组数据中,筛选获得WRKY家族中的PatWRKY40和PatWRKY53基因,利用PCR技术克隆得到基因的全长编码区。对PatWRKY40和PatWRKY53基因进行生物信息学分析。结果表明:PatWRKY40和PatWRKY53基因全长分别为1074 bp和1150 bp,编码213个和340个氨基残基,蛋白分子量为33.31 kD和37.79 kD,都属于亲水蛋白,预测其均定位于细胞核。此外,PatWRKY40和PatWRKY53分别属于Ⅱ类和Ⅲ类WRKY转录因子,并具有不同的蛋白结构,系统发育树分析这2个基因在不同分枝上。利用RT-PCR对PatWRKY40和PatWRKY53的表达模式进行分析,发现PatWRKY40具有组织表达差异(叶>花>茎),PatWRKY40和PatWRKY53在MeJA诱导6 h后表达量被显著上调了1.6倍和3.2倍。表明PatWRKY40和PatWRKY53明显受到JA诱导,可能介导JA信号通路参与广藿香的发育及代谢调控。本研究首次克隆广藿香的WRKY类转录因子并解析其表达模式,为后续PatWRKY40和PatWRKY53的功能鉴定及对其调控网络研究提供理论参考。

WRKY transcription factors play a key role in transcriptional regulation,secondary metabolic regulation,and plant development.In order to understand the regulatory pattern of WRKY transcription factors in Pogostemon cablin,the PatWRKY40 and PatWRKY53 genes in the WRKY family was screened from the transcriptome data of the Pogostemon cablin,and the full-length coding region of the gene was cloned by PCR.Using bioinformatics analysis,PatWRKY40 and PatWRKY53 genes are 1074 bp and 1150 bp in length,encoding 213 and 340 amino residues,with molecular weights of 33.31 kD and 37.79 kD,all of which are hydrophilic proteins,all of which are predicted to be located in the nucleus.In addition,PatWRKY40 and PatWRKY53 respectively belong to classⅡand classⅢWRKY transcription factors,and with different protein structure,phylogenetic tree analysis on the two genes in different branches.The expression patterns of PatWRKY40 and PatWRKY53 were analyzed by RT-PCR.It was found that PatWRKY40 had organ-specific expression(leaf>flower>stem),and the expression levels of PatWRKY40 and PatWRKY53 were significantly up-regulated by 1.6-fold and 3.2-fold after 6 h of MeJA treatment.PatWRKY40 and PatWRKY53 were obviously induced by JA,which may mediate JA signaling pathway to participate in the development and metabolism regulation of patchouli.In this study,WRKY transcription factor genes and their expression patterns were firstly cloned and analyzed from the Pogostemon cablin,which will lay a foundation for the follow-up functional characterization of PatWRKY40 and PatWRKY53 and the regulatory network.