目的 克隆铁皮石斛DoWRKY5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DoWRKY5基因转录因子cDNA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。qRT-PCR分析表明,DoWRKY5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 DoWRKY5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 In order to isolate and analysize the bioinformatics and expression pattern of DoWRKY5 gene from Dendrobium officinale. Methods A WRKY gene was first obtained by transcriptome sequencing and reverse transcription-polymerase chain reaction (RT-PCR) from D. officinale and analyzed by bioinformatics tools. The tissue expression pattern and the low temperature stress, abscisic acid (ABA) stress, and sucrose stress responses were analyzed by qRT-PCR. Results The cDNA sequence of DoWRKY5 gene was isolated, which was 1 336 bp in length, with an open reading frame (ORF) of 834 bp and an encoded polypeptide of 277 amino acid. The amino acid sequence contained a conserved WRKY domains and a zinc finger structures (C₂H₂), belonging to GroupⅡof WRKY family. Expression analysis by qRT-PCR showed that DoWRKY5 was expressed in the roots, stems, and leaves of D. officinale, and the most abundant in leaves. The amount of DoWRKY5 expression were significantly increased under low temperature of 4 ℃ and different time. Moreover, the expression of DoWRKY5 could be induced by ABA and source. Conclusion DoWRKY5 may be an important transcription factor to response cold stress and other abiotic stresses in D. officinale, which provides a foundation for further study of cold tolerance mechanism and cold-resistant breeding of D. officinale.

浙江省重大科技专项(2012C12912-9);浙江省林学重中之重一级学科研究生创新项目(201530)