人参中苯甲酸胁迫响应基因WRKY7的克隆与表达分析

Gene cloning and expression analysis of benzoic acid stress responsive transcription factor WRKY7 in Panax ginseng

WRKY转录因子是高等植物中最重要的转录因子基因家族之一,其在植物形态建成、生长发育以及应对生物(病菌、害虫等)、非生物(干旱、盐、冷、热等)胁迫过程中发挥重要作用。该研究根据人参化感自毒物质苯甲酸诱导的人参转录组数据,设计全长扩增引物,利用RT-PCR方法获得1条编码WRKY转录因子的基因,暂命名为WRKY7,并对其进行TA克隆、测序和序列分析。该基因cDNA全长1 216 bp,其开放阅读框(open reading frame,ORF)为1 014 bp,编码337个氨基酸。序列分析及系统进化分析表明,该蛋白属于WRKY家族第Ⅲ类成员,与西洋参种的WRKY6转录因子亲缘关系最近,同源性为87%。实时荧光定量PCR分析发现,苯甲酸胁迫后WRKY7的表达量显著升高,推测该基因可能参与了人参响应苯甲酸胁迫的过程,为进一步研究人参自毒胁迫响应的分子机制奠定了基础。

WRKY transcription factor is one of the most important transcription factor families widely existing in higher plants, which playing critical role in plant morphogenesis, development, biotic （including phytopathogens, pests etc. ） and abiotic （ drought, salt, chilling, high temperature, etc. ） stress. In the present work, primers used to amplify full-length gene encoding WRKY transcription factor were designed based on the transcriptome data of P. ginseng that induced by benzoic acid, one of the most important autotoxins identified from root exudates and rhizosphere soil of P. ginseng. Then, a WRKY gene, temporarily named as WRKY7, was confirmed by RT-RCR. Furthermore, sequencing and sequence analysis of WRKY7 was conducted. Results indicated that, the full length eDNA of WRKY7 was 1 216 bp, the open reading frame （ORF） of which was 1 014 bp, encodes 337 amino acids. Homologous analysis and phylogenetic tree showed that, WRKY7 belonged to the Ⅲ category of WRKY families, which showing 87% similarity to WRKY6 in P. quinquefolius. Real-time PCR results showed that the expression of WRKY7 in P. ginseng induced by benzoic acid was up-regulated markedly than the control, so we speculated that WRKY7 was involved in the response to benzoic acid stress, which will be helpful for further research on the molecular mechanism of ginseng plant response to benzoic acid stress.