陆地棉GhWRKY33的克隆及抗旱功能分析

Cloning and Drought Resistance Analysis of GhWRKY33 in Upland Cotton

【目的】通过克隆陆地棉GhWRKY33并研究其抗旱功能,为棉花抗旱机制解析及分子育种奠定基础。【方法】利用同源克隆的方法从陆地棉中棉所10号中克隆GhWRKY33的开放读码框(open reading frame,ORF),并进行生物信息学分析,分析该基因的二级结构、亲疏水性,预测磷酸化位点和启动子区域的顺式作用元件,在NCBI中通过BLASTP检索同源性高的蛋白序列进行序列比对并构建系统发育树;构建35S::GhWRKY33-GFP融合表达载体,通过农杆菌介导注射烟草叶片,观察荧光信号;利用qRT-PCR检测基因的组织表达特异性,以及干旱、ABA、JA、ET处理下的基因表达模式;构建GhWRKY33过表达载体并转化拟南芥,利用20%PEG6000对野生型和T3代转基因拟南芥进行干旱处理,观察处理后野生型和转基因拟南芥的表型,并测定脯氨酸和丙二醛含量等生理生化指标,分析目的基因与干旱响应基因AtP5CS、AtRD29A、AtCOR15A的表达水平。【结果】从陆地棉品种中棉所10号克隆获得GhWRKY33的ORF全长为1533 bp,编码一个含510个氨基酸残基的蛋白,含有2个WRKY保守结构域和2个C2H2型锌指结构,属于第Ⅰ类WRKY转录因子家族;二级结构预测其编码的蛋白主要由无规则卷曲构成,含有26个苏氨酸磷酸化位点,推测可能与磷酸化有密切的关系,亲疏水性预测表明该蛋白属于亲水性蛋白;系统发育树分析显示该蛋白与GrWRKY33同源性最高。亚细胞定位将GhWRKY33定位于细胞核。qRT-PCR显示该基因具有明显的组织表达特异性,在棉花顶芽的表达量最高;干旱、ABA、JA、ET处理后,基因的表达量明显上升。干旱胁迫后,与野生型相比,转基因拟南芥的抗旱性水平明显提高,植株萎蔫程度较轻,其脯氨酸含量显著升高,丙二醛含量显著降低,且目的基因与干旱响应基因AtP5CS、AtRD29A、AtCOR15A的表达水平均明显提高,表明PEG诱导了该基因的表达,进而调控了干旱响应基因表达上调,使转基因拟南芥表现出对干旱胁迫的抗性。【结论】GhWRKY33响应干旱胁迫,过表达后能明显提高转基因拟南芥的抗旱性。

【Objective】In this study,Gh WRKY33 gene in upland cotton was cloned,and its function was analyzed in the drought resistance,which will lay a foundation for the mechanism dissection of drought resistance and molecular breeding in cotton.【Method】The ORF of Gh WRKY33 was cloned from Upland cotton CCRI10 by homologous cloning method.Bioinformatics analysis was carried out to analyze the secondary structure and hydrophobicity,and to predict the phosphorylation sites and cis-acting elements in the promoter region.In the NCBI website,the protein sequences with high homology were searched by BLASTP for sequence alignment,and the phylogenetic tree was constructed.The expression vector of 35 S::GhWRKY33-GFP fusion protein was constructed and injected into tobacco leaves mediated by Agrobacterium tumefaciens to observe the fluorescence signal.qRT-PCR was conducted to detect the tissue-specific expression of the gene and the expression pattern under drought,ABA,JA and ET treatments.Plant overexpression vector was constructed and transformed into Arabidopsis thaliana.T3 transgenic lines were performed to observe the phenotype after drought treatment and to determine the physiological and biochemical indexes such as the content of proline and malondialdehyde.The expression levels of drought response genes AtP5 CS,AtRD29 A and AtCOR15 A were identified in the wild type and transgenic lines before and after PEG treatment.【Result】The open reading frame(ORF)of Gh WRKY33 gene was 1533 bp,which encoded 510 amino acid residues.GhWRKY33 protein included two WRKY conserved domains and two C2 H2 zinc finger structures,and belonged to the family of type I WRKY transcription factors.Secondary structure prediction showed that GhWRKY33 was mainly composed of random curl and contained 26 threonine phosphorylation sites,which was closely related to phosphorylation.The hydrophobicity prediction showed that the protein belonged to a hydrophilic protein.Phylogenetic tree analysis displayed that the gene had the highest homology with GrWRKY33.The GhWRKY33 protein was located in the nucleus.qRT-PCR showed that the expression of the gene possessed tissue specificity and was the highest in the apical bud tissue,and significantly increased after drought,JA and ET treatments.Compared to the wild type,the transgenic lines displayed stronger drought resistance,and the wilting degree of the plants was weaker.Under drought treatment,the content of proline was significantly higher than that of wild type,while the content of MDA was lower than that of wild type.The expression levels of Gh WRKY33 and drought response genes AtP5 CS,AtRD29 A and At COR15 A were significantly increased.It was speculated that PEG could induce the expression of GhWRKY33,and then regulated the up-regulation expression of the drought response genes,thus,transgenic Arabidopsis thaliana showed higher resistance to drought stress.【Conclusion】Gh WRKY33 responded to drought stress and improved the drought resistance of transgenic Arabidopsis thaliana.