| 摘要: |
| 【目的】在全基因组水平鉴定番木瓜(Carica papaya L.) WRKY（CpWRKY）转录因子家族基因，并对其在短孢炭疽菌侵染下的表达量进行分析，为CpWRKYs 家族的功能研究和利用奠定基础。【方法】采用生物信息学方法，鉴定和筛选CpWRKYs转录因子家族基因成员，并对其进行系统进化分析、基因结构分析、蛋白保守基序预测和启动子顺式作用元件分析；同时以番木瓜炭疽病耐性品种和敏感性品种为材料，利用短孢炭疽菌侵染采后果实，于0，24，48 h 取样，对其转录组学数据进行分析，筛选2个品种中差异表达的CpWRKY基因，并利用实时荧光定量PCR(RT-qPCR)试验对筛选结果进行验证。【结果】经系统分析从番木瓜中共鉴定出48个CpWRKYs成员，分为Ⅰ、Ⅱ和Ⅲ 3类；其中第Ⅱ类成员最多，可分为Ⅱa、Ⅱb、Ⅱc、Ⅱd和Ⅱe 5个亚类。CpWRKYs家族成员的氨基酸残基数目为97~747 个，等电点为4.83~9.71，为亲水性蛋白，大部分CpWRKYs的结构域高度保守，但有个别蛋白保守域缺失。在 CpWRKYs家族中共预测到10个保守基序，其中包括含有WRKY七肽结构域的基序1、含锌指结构的基序2和同时含有七肽序列和锌指结构的基序3，其中基序3为大多数I类CpWRKY转录因子N端WRKY结构域所特有。CpWRKYs含有1~6个外显子，同一家族或亚家族成员在基因结构上表现出一定的相似性，同时在CpWRKYs启动子中检测到大量与生物胁迫和非生物胁迫相关的元件。对短孢炭疽菌侵染番木瓜的转录组数据进行分析，在番木瓜耐性品种中筛选出了特异表达的CpWRKY22、CpWRKY11、CpWRKY2、CpWRKY33和CpWRKY25，RT-qPCR检测结果显示，这些基因在耐性品种中特异上调表达，在敏感性品种中的表达量无明显变化。【结论】番木瓜WRKY家族共包括48个成员，该家族基因结构和蛋白基序具有组间差异性和组内保守性。CpWRKYs家族成员强烈响应炭疽菌侵染，其中CpWRKY22、CpWRKY11、CpWRKY2、CpWRKY33和CpWRKY25是有潜力的抗炭疽病候选基因。 |
| 关键词: 番木瓜 WRKY家族 炭疽病 短孢炭疽菌 候选基因 |
| DOI： |
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| 基金项目:广东省基础与应用基础研究基金项目（2021A1515010739,2020A1515011166）；广东省农业科学院果树研究所培育项目（21113）；广东省现代农业产业技术体系创新团队建设项目(优稀水果产业)（2021KJ116） |
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| Identification of WRKY transcription factor genes in papaya and response of their expression to Colletotrichum brevisporum infection |
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YANG Min,ZHOU Chenping,YANG Min
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| Abstract: |
| 【Objective】This study aimed to improve future functional studies and application of WRKY genes by identifying the WRKY transcription factor family in papaya and analyzing the changes in expression levels of CpWRKYs after Colletotrichum brevisporum infection.【Method】The CpWRKYs transcription factor family members were identified and screened by bioinformatics methods,and phylogenetic tree construction,gene structure analysis,protein conserved motif prediction and promoter cis acting elements analysis were performed.Then,C. brevisporum was used to infect postharvest fruits of papaya anthracnose tolerant and sensitive cultivars,and samples were collected at different time points (0,24 and 48 h) after infection.Combined with transcriptome data,the differentially expressed CpWRKYs in the two cultivars were screened,and the candidate CpWRKY were verified by RT-qPCR.【Result】A total of 48 CpWRKYs were identified in papaya,which were divided into three subfamilies of Ⅰ,Ⅱ and Ⅲ.Among them,subfamily Ⅱ had the largest number of members,and the CpWRKYs in subfamily Ⅱ were divided into five subgroups of Ⅱ a,Ⅱ b,Ⅱ c,Ⅱ d and Ⅱ e.The number of amino acid residues of CpWRKYs family members was 97―747,the predicted theoretical isoelectric points values of CpWRKYs were 4.83-9.71,and all predicted CpWRKY proteins were hydrophilic.The sequence alignment analysis of CpWRKYs conserved domains showed that most domains of CpWRKYs were highly conserved,but some proteins had the deletion of conserved domains.A total of 10 conserved motifs were predicted in CpWRKYs,including motif 1 containing WRKY heptopeptide domain,motif 2 containing zinc finger structure,and motif 3 containing both heptopeptide sequence and zinc finger structure.The motif 3 was unique to the N terminal WRKY domain of most subfamily Ⅰ CpWRKY transcription factors.The number of exons of CpWRKYs ranged from 1 to 6,and members of the same subfamily or subgroup showed certain similarity.Analysis of cis acting elements showed that a large number of elements related to biotic and abiotic stresses were detected in CpWRKYs promoter.Based on the transcriptome data of papaya postharvest fruits after C. brevisporum inoculation,five specifically expressed CpWRKYs (CpWRKY22,CpWRKY11,CpWRKY2,CpWRKY33 and CpWRKY25) in the tolerance cultivar were screened.The RT-qPCR result showed that the expression levels of these CpWRKYs were specifically up-regulated in the tolerant cultivar,but there were no significant changes in susceptible cultivar.【Conclusion】There were 48 members in WRKY family of papaya and the structures of CpWRKYs and protein motifs of CpWRKY showed diversity among subfamilies and conservation within subfamilies/subgroups.CpWRKY genes strongly responded to C. brevisporum infection and CpWRKY22,CpWRKY11,CpWRKY2,CpWRKY33 and CpWRKY25 were potential candidate genes with resistance to anthracnose in papaya. |
| Key words: papaya WRKY family anthracnose Colletotrichum brevisporum candidate genes |
