| 刘开，余炳伟，李丹丹，陈娜，曹必好.茄子 SmWRKY65 基因克隆及其对青枯病的抗性分析[J].广东农业科学,2021,48(3):42-52 |
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茄子 SmWRKY65 基因克隆及其对青枯病的抗性分析 |
| Cloning of SmWRKY65 Gene and Its Resistance to Bacterial Wilt in Eggplant |
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| DOI：10.16768/j.issn.1004-874X.2021.03.006 |
| 中文关键词: 茄子 青枯病 WRKY 转录因子 实时荧光定量 PCR VIGS |
| 英文关键词: eggplant bacterial wilt WRKY transcription factor real-time fluorescence quantitative PCR virus induced gene silencing（VIGS） |
| 基金项目:国家重点研发计划项目（2017YFD0101904） |
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| 中文摘要: |
| 【目的】通过克隆 SmWRKY65（Solanum melongena L. WRKY65）基因并分析其生物信息学与功能，以期探究 SmWRKY65 对茄子青枯病的响应情况。【方法】以茄子抗病植株（E-31）为试验材料、叶片 cDNA 为模板，通过 PCR 技术获得 SmWRKY65 基因，并利用 ExPASy ProtParam tool 对其进行生物信息学分析。运用实时荧光定量 PCR（quantitative real-time PCR，qRT-PCR）的方法对其不同组织部位与抗病、感病材料（E-32）中的响应情况进行分析；通过双酶切法构建亚细胞定位载体；运用病毒诱导的基因沉默（virus induced gene silencing, VIGS）技术构建 pTRV2-SmWRKY65 载体并侵染茄子抗病植株。【结果】在茄子中成功克隆了 SmWRKY65 基因，该基因开放阅读框为 834 个核苷酸序列，编码 277 个氨基酸残基，在 71~131 区域具有 1 个含 60 个氨基酸的 WRKY 保守域，定位于细胞核中。时空表达分析发现，SmWRKY65 在茄子根组织中的表达量最高，叶中次之，茎中最低；qRT-PCR 分析表明，SmWRKY65 在茄子抗病和感病材料中均响应青枯病菌的诱导上调表达；VIGS 结果表明，与清水和 pTRV2 空载对照相比，接种青枯病菌后，pTRV2-SmWRKY65 沉默植株的叶片表现出明显的萎蔫症状，结合 qRT-PCR 分析发现，pTRV2-SmWRKY65 表达量较对照组明显下降。【结论】SmWRKY65
在茄子抗病材料中的表达水平明显高于感病材料，且 pTRV2-SmWRKY65 植株与对照相比表现为易感症状，说明SmWRKY65 沉默后茄子抗青枯病的能力下降，表明 SmWRKY65 可能涉及茄子青枯病抗性相关过程的调控。 |
| 英文摘要: |
| 【Objective】The response of SmWRKY65（Solanum melongena L. WRKY65）to bacterial wilt of eggplant was explored by cloning SmWRKY65 gene and analyzing its bio-information and function.【Method】The eggplant disease-resistant plants（E-31）and cDNA from its leaves were used as tested materials and template, respectively. The SmWRKY65 was obtained by PCR technology and bioinformatics analysis was constructed by ExPASy ProtParam tool. The expression analysis of different tissue sites and disease-resistant and susceptible materials（E-32）was performed by quantitative real-time PCR（qRT-PCR）method. Subcellular localization vector was constructed by double digestion method. The pTRV2-SmWRKY65 vector was constructed by virus induced gene silencing（VIGS）technology and the disease-resistant eggplant plants were infected.【Result】The SmWRKY65 gene was successfully cloned in eggplant. The gene contains 834 bp nucleotide sequences, codes 277 amino acid residues in its open reading frame, and concludes a WRKY conserved domain containing 60 amino acid in the regions of 71-131. The analysis of subcellular localization showed that SmWRKY65-GFP fusion protein was found in the cell nucleus. The expression analysis of different tissues revealed that SmWRKY65 had highest expression in eggplant roots, followed by leaves and stems. The qRT-PCR analysis testified that SmWRKY65 was up-regulated in response to the induction of bacterial wilt in eggplant resistant and susceptible plants. The VIGS results showed that the leaves of pTRV2-SmWRKY65 silent plants was observed clear wilting symptoms when compared
with water and pTRV2 control vector. By qRT-PCR profiling, it was observed that pTRV2-SmWRKY65 expression was obviously decreased when compared with control groups.【Conclusion】The expression level in disease-resistant eggplant materials was significantly higher than that in susceptible materials, and the pTRV2-SmWRKY65 plants showed more symptoms of susceptibility when compared with control groups, indicating that the silence of SmWRKY65 reduced the resistance to bacterial wilt of eggplants and SmWRKY65 was possibly related to the process regulation of eggplant resistance to bacterial wilt. |
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