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目的克隆铁皮石斛Do WRKY5基因,并对生物信息学和表达模式进行分析。方法通过转录组测序和RT-PCR方法相结合首次从铁皮石斛中克隆到一个WRKY基因,利用生物信息学工具对其进行分析,并利用荧光定量PCR分析其在铁皮石斛中的组织表达模式及在低温胁迫、脱落酸(ABA)胁迫和蔗糖渗透胁迫下的表达模式。结果获得长1 336 bp的Do WRKY5基因转录因子c DNA全长序列,开放阅读框为834 bp,编码277个氨基酸残基,含有一个WRKY基因保守结构域和一个C2H2锌指结构域,属于WRKY基因家族的第II类成员。q RT-PCR分析表明,Do WRKY5基因在铁皮石斛根、茎、叶中均有表达,但在叶中的表达量最高。在不同低温和4℃不同时间诱导处理后,该基因表达量均显著升高,并且该基因在ABA胁迫和蔗糖渗透胁迫下均可被诱导表达。结论 Do WRKY5基因在铁皮石斛应答低温胁迫等多种非生物胁迫中可能起重要的调控作用,为进一步研究铁皮石斛的抗寒机制和抗寒性品种的选育提供基础。
Objective To clone Do WRKY5 gene from Dendrobium candidum and analyze its bioinformatics and expression pattern. Methods A WRKY gene was cloned from Dendrobium candidum by transcriptome sequencing and RT-PCR. Bioinformatics tools were used to analyze the expression of WRKY. The expression patterns of WRKY in Dendrobium officinale were analyzed by fluorescence quantitative PCR. Low temperature stress, abscisic acid (ABA) stress and sucrose osmotic stress. Results The full-length cDNA sequence of Do WRKY5 gene with a length of 1 336 bp was obtained. The open reading frame of the Do WRKY5 gene was 834 bp encoding a polypeptide of 277 amino acids. It contained a WRKY gene conserved domain and a C2H2 zinc finger domain, belonging to the WRKY gene Family of Class II members. q RT-PCR analysis showed that Do WRKY5 gene was expressed in roots, stems and leaves of Dendrobium candidum, but its expression was the highest in leaves. The expression of this gene was significantly increased after induction at different temperatures and at different times of 4 ℃, and the gene was induced to express under ABA stress and sucrose osmotic stress. Conclusion Do WRKY5 gene may play an important regulatory role in response to low abiotic stress such as low temperature stress on Dendrobium candidum, which will provide a basis for further research on the mechanism of cold resistance of Dendrobium candidum and breeding of cold-tolerant cultivars.