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WRKY转录因子是植物中发现的新型转录调控因子,在调控植物生长发育和代谢等生理过程中发挥重要作用。该研究采用RT-PCR和RACE技术从铁皮石斛Dendrobium officinale中克隆得到一个新的WRKY转录因子基因cDNA全长,命名为DoW RKY1,提交GenBank获得登录号KF716131。生物信息学分析表明,该基因全长cDNA为1 704 bp,其中开放阅读框(ORF)1629bp,编码1条由542个氨基酸组成的多肽,具有2个WRKY保守结构域,属于WRKY家族第Ⅰ类转录因子。酵母单杂交表明,DoW RKY1可以激活下游报告基因(His3,Ade2)的表达,具有转录激活功能,属于转录激活子。半定量RTPCR显示,DoWRKY1在根、茎、叶和类原球茎中均有表达。qRT-PCR分析表明,DoW RKY1响应茉莉酸甲酯(MeJ A)和壳聚糖(chitosan)诱导,分别在2h和1h,表达量达到最高峰。研究结果为进一步研究其在铁皮石斛次生代谢过程的调控功能奠定基础。
WRKY transcription factors are novel transcriptional regulators discovered in plants and play an important role in the regulation of physiological processes such as plant growth and development and metabolism. In this study, a new full-length cDNA of WRKY transcription factor was cloned from Dendrobium officinale by RT-PCR and RACE, named DoW RKY1 and submitted to GenBank for accession number KF716131. Bioinformatics analysis showed that the full-length cDNA of this gene was 1 704 bp in length, including an open reading frame (ORF) of 1629 bp encoding a polypeptide consisting of 542 amino acids with two WRKY conserved domains belonging to class I of the WRKY family Transcription factor. Yeast single hybridization shows that DoW RKY1 can activate the expression of downstream reporter gene (His3, Ade2), has the function of transcriptional activation and belongs to the transcriptional activator. Semi-quantitative RTPCR showed that DoWRKY1 was expressed in roots, stems, leaves and protocorm-like bodies. qRT-PCR analysis showed that DoW RKY1 peaked at 2 h and 1 h, respectively, in response to induction by MeJ A and chitosan. The results laid the foundation for further study on its regulatory function in secondary metabolism of D. officinale.