不结球白菜响应ABA和低温基因WRKY18的克隆及表达分析

Cloning and expression analysis of WRKY18 gene in non-heading Chinese cabbage under ABA and low temperature treatments

[目的]WRKY转录因子是植物特有的一类转录因子,广泛参与植物的生长发育、生物及非生物胁迫响应。克隆和鉴定不结球白菜共同响应ABA和低温的WRKY基因,对研究不结球白菜抗逆分子机制和改良其抗逆性具有重要意义。[方法]采用电子克隆的方法在NCBI数据库中进行不结球白菜WRKY18基因全长拼接,然后在不结球白菜‘苏州青’中,克隆到1个与低温及ABA响应相关的WRKY基因,通过生物信息学手段和实时定量RT-PCR(qRT-PCR)技术分析了该基因的序列结构特征及其在低温和ABA处理下的表达变化。[结果]该基因全长为1080bp,含有978bp的开放阅读框,编码326个氨基酸,与油菜及拟南芥的WRKY18直系同源,氨基酸序列相似性分别为87%和73%,命名为BcWRKY18。序列分析表明,该编码蛋白为核蛋白,不含跨膜区,无信号钛,具有WRKYGQK基序(motif)约60个氨基酸残基的WRKY保守结构域,为WRKY转录因子家族成员。系统聚类分析指出了BcWRKY18蛋白的保守性及其在开花植物中的进化关系;序列结构和同源建模分析指出了BcWRKY18蛋白的保守结构域、蛋白质的二级及三级结构分布模型;qRT-PCR检测了BcWRKY18基因在ABA和低温胁迫下的表达,表明低温和ABA都能诱导BcWRKY18基因表达,其表达模式都存在相似的过程,且BcWRKY18基因对ABA的响应比对低温的响应更快、更明显。[结论]从不结球白菜中克隆获得1个新的WRKY类转录因子基因BcWRKY18,基因表达分析发现BcWRKY18可能存在对ABA和低温的共响应及自身反馈调控。

[Objectives]WRKY transcription factors are one kind of plant-specific transcription factors,involved in plant growth and development,biotic and abiotic stress responses.We cloned and characterized WRKY genes to explore the resistance molecular mechanisms of non-heading Chinese cabbage（Brassica campestris ssp.chinensis Makino） WRKY genes（Bc WRKYs） responding to ABA and low temperature.[Methods]The full-length c DNA of WRKY 18 was cloned from Brassica campestris cultivar‘Suzhouqing＇by silicon cloning method.Bioinformatics approach was utilized for analyzing nucleotide and acid sequence of WRKY 18.Quantitative RT-PCR（qRT-PCR） was employed to investigate the expression patterns at low temperature and ABA treatment.[Results]BcWRKY 18 c DNA was 1 080bp,including an ORF（open reading frame） of 978bp,and encoded a protein of 326 amino acids.The deduced amino acid sequence was orthologous with Brasscia napus and Arabidopsis thaliana WRKY 18（87% and 73%,respectively）.Sequence analysis showed that the predicted protein is a nuclear protein and has no transmembrane domain and signal peptide.BcWRKY 18 has a typical conserve domain of WRKY transcription factors family with WRKY domain.It is a member of WRKY transcription factors family.Phylogenetic analysis indicated its evolutionary relationship in flowering plants.Analysis of sequence structure and homology modeling revealed conserved domains,secondary and 3D structure of Bc WRKY 18.qRT-PCR analysis showed that both low temperature and ABA induce the expression of Bc WRKY 18.Moreover,ABA and low temperature treatment have the similarexpression pattern.The expression level of Bc WRKY 18 was faster and more obvious at ABA treatment.[Conclusions]The BcWRKY 18 gene,which encodes WRKY transcription factor,was cloned from non-heading Chinese cabbage.The Bc WRKY 18 gene may co-respond to ABA and low temperature and possess itself feedback regulation.