棉花磷胁迫响应基因GhWRKY6克隆及功能分析

Cloning and Functional Analysis of a Phosphorus Stress Response Gene, Gh WRKY6,in Cotton(Gossypium hirsutum L.)

【目的】提高棉花在低磷胁迫下的抗性,挖掘棉花磷胁迫相关功能基因。【方法】用生物信息学方法分析Gh WRKY6基因的结构特征和进化关系;构建基因超表达载体转化拟南芥,分析转基因拟南芥在低磷胁迫下的抗性;利用荧光定量PCR(Real-time quantitative polymerase chain reaction, qRT-PCR)分析拟南芥中磷信号途径Marker基因表达量的变化。【结果】以陆地棉cDNA为模板克隆得到Gh WRKY6基因,生物信息学分析表明该基因序列含有一个WRKY保守结构域,是WRKY家族转录因子。在低磷胁迫下,转基因拟南芥与野生型相比,种子萌发速度、主根长度、侧根数目、生物量均低于野生型,而在正常生长条件下两者并无差别。qRT-PCR分析表明GhWRKY6能够影响关键的磷转运蛋白基因的表达。【结论】GhWRKY6作为一个负调控因子参与到植物低磷胁迫响应信号途径中。

[Objective] To improve the stress resistance of cotton(Gossypium hirsutum L.) under low-phosphorus(Pi) stress, the roles of related functional genes were investigated.[Methods] The structural characteristics of GhWRKY6 were analyzed using bioinformatics methods. To assess low Pi-stress resistance, an overexpression vector was constructed and transformed into Arabidopsis thaliana. The expression levels of Pi stress-related genes in A. thaliana were analyzed using real-time quantitative polymerase chain reaction.[Results] The GhWRKY6 gene was cloned from the cDNA of Upland cotton, and a bioinformatics analysis indicated that the gene contains a WRKY conserved domain and is a WRKY family transcription factor. Under low-Pi stress,the transgenic lines showed delayed germination, shorter primary root lengths, fewer lateral roots, and lower biomasses compared with wild-type, but there were no differences between wild-type and over-expression lines under normal growth conditions. The real-time quantitative polymerase chain reaction analysis indicated that GhWRKY6 can affect the expression of key phosphate transporter genes.[Conclusion] GhWRKY6 acts as a negative regulator in plant low-Pi response signaling pathways.