紫花苜蓿MsWRKY42的分离、鉴定及其对非生物胁迫的响应

Isolation,Identification,and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L.

【目的】WRKY是转录因子大家族,在植物生长发育及逆境胁迫应答中发挥着核心调控作用。通过分析转录因子MsWRKY42在紫花苜蓿抗逆境过程中的作用,为进一步研究WRKY转录因子在紫花苜蓿抗逆分子调控中的作用奠定基础。【方法】通过同源比对方法从紫花苜蓿转录组基础数据库中获得MsWRKY42序列。使用MEGA-X对MsWRKY42蛋白序列及拟南芥序列进行多序列比对,构建系统发育树。通过PlantCARE预测分析MsWRKY42启动子的顺式作用元件。采用实时荧光定量PCR(qRT-PCR)方法分析MsWRKY42在紫花苜蓿不同组织中的表达量及其在NaCl(0.3 mol·L^-1)、PEG(15%)、4℃、40℃、低磷以及ABA(0.1×10^-3 mol·L^-1)处理后的表达量变化。构建pCAMBIA1300-WRKY-GFP融合表达载体,通过农杆菌转化本氏烟草(Nicotiana benthamiana),确定MsWRKY42蛋白的亚细胞定位。利用酵母单杂交系统检测MsWRKY42和启动子区域顺式作用元件W-box的体外结合活性。【结果】该基因包含1个1692 bp的开放阅读框,编码563个氨基酸。多序列比对及系统进化树分析表明,该蛋白属于Ⅱb类WRKY家族成员,含有1个WRKY保守结构域和1个C2H2锌指基序,与拟南芥WRKY家族中的AtWRKY42相似度最高,故将其命名为MsWRKY42。在紫花苜蓿MsWRKY42启动子区域鉴定到多个顺式作用元件,主要包括胁迫响应、激素响应、生长发育等不同生命活动相关的调控作用元件。实时荧光定量PCR结果表明,MsWRKY42在紫花苜蓿各组织中均有不同程度的表达,其中根、叶中的表达量最高,茎、花、荚果中次之,芽中最低。经NaCl、PEG、低温、高温、低磷和ABA处理后的MsWRKY42表达量均有不同程度的上调。亚细胞定位结果显示,MsWRKY42蛋白定位在细胞核上。酵母单杂交系统检测结果显示,MsWRKY42能够与W-box顺式作用元件特异性结合。【结论】MsWRKY42为典型的WRKY转录因子,蛋白质定位在细胞核,能够与W-box顺式作用元件特异性结合;该基因在紫花苜蓿不同组织部位中均有表达,在根和叶中的表达量最高,且表达受NaCl、PEG、低温、高温胁迫和ABA激素的正向诱导。在紫花苜蓿中,该基因可能参与多种非生物胁迫反应。

【Objective】The WRKY gene family in plants encodes a large group of transcription factors(TFs)that play essential roles in diverse stress responses,developmental and physiological processes.It laid a foundation for further research on the role of WRKY transcription factor in alfalfa stress-resistant molecular regulation by analyzing the role of MsWRKY42 transcription factor in alfalfa.【Method】MsWRKY42 sequence was obtained by homologous alignment and sequence characteristics of it were analyzed by online bioinformatics tools.The phylogenetic tree of MsWRKY42 and Arabidopsis WRKY genes was constructed by MEGA-X.The putative cis-elements in promoter region of MsWRKY42 was analyzed by PlantCARE.The real-time quantitative PCR(qRT-PCR)was used to analyze the expression pattern of MsWRKY42 in different organs and its response to abiotic including NaCl(0.3 mol·L^-1),PEG(15%),4℃,40℃,low phosphorus and ABA(0.1×10^-3 mol·L^-1).The fusion expression vector of pCAMBIA1300-WRKY-GFP was constructed and delivered into Nicotiana benthamiana by Agrobacterium mediated method to determine the subcellular localization of WRKY protein.The yeast one-hybrid technique was used to analyze the binding activity of MsWRKY42 and the cis-acting element W-box.【Result】The gene contained a 1692 bp open reading frame encoding 563 amino acids.Multiple sequence alignment and phylogenetic tree analysis results indicated that the protein is a member of the IIb sub-group WRKY family and contains a WRKY conserved domain and a C2H2 zinc finger motif.It has the highest similarity to AtWRKY42 in the Arabidopsis family,therefore,named as MsWRKY42.A variety of cis-acting elements were identified in the promoter region of MsWRKY42,including regulatory elements such as stress response,hormone response,and diurnal regulation.Real-Time PCR analysis showed that MsWRKY42 had the highest expression in roots and leaves.The expression of MsWRKY42 was up-regulated by NaCl,PEG,low temperature,high temperature,low phosphorus and ABA treatments.Subcellular localization indicated that MsWRKY42 was mainly located on the nucleus in plant cells.The results of the binding activity analysis showed that MsWRKY42 was able to specifically bind to W-box.【Conclusion】MsWRKY42 is a typical transcription factor,which can specifically bind to cis-acting elements,and the protein is localized in the nucleus.The gene was expressed in different tissues of alfalfa,and was induced by NaCl,PEG,high temperature,low phosphorus and ABA treatment.It is speculated that the MsWRKY42 gene may play a role in response to multiple stress defense responses as a WRKY transcription factor.