番木瓜WRKY转录因子CpWRKY11的克隆和表达  

Cloning and expression analysis of transcription factor CpWRKY11 in papaya(Carica papaya L.)

作　　者：杨敏 李庆萌 周陈平 邝瑞彬[1] 杨护[1] 黄炳雄[1] 魏岳荣[1] YANG Min;LI Qingmeng;ZHOU Chenping;KUANG Ruibin;YANG Hu;HUANG Bingxiong;WEI Yuerong(Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs,Guangdong Provincial Key Laboratory of Tropical and Subtropical Fruit Tree Research,Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences,Guangzhou,Guangdong 510640,China)

机构地区：[1]广东省农业科学院果树研究所,农业农村部南亚热带果树生物学与遗传资源利用重点实验室/广东省热带亚热带果树研究重点实验室,广东广州510640

出　　处：《西北农林科技大学学报（自然科学版）》2023年第5期119-130,138,共13页Journal of Northwest A&F University(Natural Science Edition)

基　　金：广东省基础与应用基础研究基金项目(2022A1515010697,2021A1515010739);广东省农业科学院果树研究所培育项目(21113);广东省科技专项资金项目(2021A05192)。

摘　　要：【目的】克隆番木瓜炭疽菌诱导的WRKY转录因子CpWRKY11,研究其在生物胁迫、外源激素处理及非生物胁迫条件下的表达特征,为CpWRKY11基因在后续番木瓜分子育种中的应用提供理论支持。【方法】从自主选育的炭疽病抗性品种中克隆WRKY转录因子CpWRKY11,采用SMART、ExPaSy、NetPhos和DNAMAN软件对该基因及其编码的蛋白序列进行生物信息学分析,利用MEGA-X软件进行系统进化树构建和分析;构建亚细胞定位载体CpWRKY11-GFP,通过转化水稻原生质体,确定CpWRKY11的亚细胞定位情况;利用酵母单杂交试验验证CpWRKY11是否具有转录激活活性;采用实时荧光定量PCR(qRT-PCR)分析CpWRKY11在番木瓜不同组织(根、茎、叶、花和果实)及生物胁迫(短孢炭疽菌和番木瓜畸形花叶病毒侵染)、外源激素(水杨酸(SA)、茉莉酸甲酯(MeJA)、乙烯利(ETH))和非生物胁迫(高盐、干旱和机械损伤)处理下的表达情况。【结果】CpWRKY11基因编码序列全长1719 bp,编码572个氨基酸;CpWRKY11含有60个磷酸化位点,理论分子量为62.31 ku,理论等电点为6.75,不稳定指数为51.17,亲水性为-0.792,是一个亲水不稳定蛋白。CpWRKY11有2个典型的WRKY结构域,同源氨基酸比对及进化树分析结果显示,CpWRKY11与拟南芥AtWRKY20的同源性最高,属于WRKY家族Ⅰ类。亚细胞定位结果显示,CpWRKY11只定位在细胞核上。酵母转录激活验证试验结果显示,CpWRKY11蛋白不具有转录激活活性。CpWRKY11在番木瓜中为组成型表达,在茎中表达量最高,其次为根、果实和花,在叶中的表达量最低。CpWRKY11受短孢炭疽菌和PLDMV的诱导上调表达,在短孢炭疽菌诱导48 h内持续上调,与0 h对照相比其表达量均显著上调,且在反应后期(24和48 h)表达量更高。在PLDMV处理条件下,除接种第3天外,CpWRKY11也均呈现出明显的上调趋势。外源激素处理结果显示,CpWRKY11受SA、ETH和MeJA的诱导上调表达,尤其是在MeJA处【Objective】The transcription factor CpWRKY11 of papaya induced by Colletotrichum brevisporum was cloned and its expression characteristics in biotic stress,exogenous hormone treatments and abiotic stress were studied to provide support for subsequent application of CpWRKY11 in molecular breeding of papaya.【Method】CpWRKY11 was cloned from the anthracnose tolerance cultivar of papaya.Bioinformatics analysis on its gene sequence and encoded protein sequence was performed using SMART,ExPaSy,NetPhos and DNAMAN softwares,and the phylogenetic tree analysis was constructed using MEGA-X software.The subcellular localization vector CpWRKY11-GFP was constructed to detect the subcellular localization of CpWRKY11.Yeast hybridization assay was used to verify whether CpWRKY11 possessed transcriptional activation activity.The expression of CpWRKY11 in different tissues and its dynamic expression under biological stress(Colletotrichum brevisporum and papaya leaf distortion mosaic virus),exogenous hormones(salicylic acid,methyl jasmonate and ethephon)and abiotic stress(high salt,drought and mechanical injury)were analyzed by quantitative real-time PCR(qRT-PCR).【Result】The full length coding sequence of CpWRKY11 was 1719 bp,encoding 572 amino acids and containing 60 phosphorylation sites.The theoretical isoelectric point,theoretical molecular weight,instability index and hydrophilicity of CpWRKY11 protein were 6.75,62.31 ku,51.17 and-0.792,respectively,showing that it was an unstable hydrophilic protein.CpWRKY11 contained two typical WRKY domains.Multiple sequence alignment and phylogenetic tree analysis showed that CpWRKY11 had the highest homology with AtWRKY20,belonging to classⅠWRKY family.Subcellular localization revealed that CpWRKY11 was localized in the nucleus.Yeast transcriptional activation verification experiments indicated that CpWRKY11 protein had no transcriptional activation activity.The qRT-PCR analysis revealed that CpWRKY11 was constitutively expressed in papaya,with the highest expression level in

关 键 词：番木瓜 炭疽病 CpWRKY11 生物胁迫 

分 类 号：S667.9[农业科学—果树学] S436.679[农业科学—园艺学]