不结球白菜BcCHC1基因与TuMV互作机理的初步研究
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1.南京农业大学园艺学院;2.南京农业大学,中国科学院华南植物园 广东省应用植物学重点实验室/中国科学院华南农业植物分 子分析与遗传改良重点实验室

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江苏省自然科学基金资助项目(BK20200560),江苏省农业科技自主创新资金[CX(19)3038]


A preliminary study on the expression analysis of the BcCHC1 gene in non-heading Chinese cabbage
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    摘要:

    【目的】为了提高不结球白菜对TuMV的抗性,该研究初步探究了不结球白菜BcCHC1基因与TuMV的互作机制。【方法】从不接球白菜中鉴定clathrin网格蛋白重链CHC基因家族成员,并克隆了1个CHC1,对其进行亚细胞定位、从候选基因中利用双分子荧光互补检测筛选出CI与6K2,VIGS诱导BcCHC1沉默后植株枯死,BiFC试验验证BcCHC1与TuMV蛋白之间存在的互作关系。【结果】(1)成功克隆得到不结球白菜BcCHC1基因,其编码序列长度为5 124 bp,共编码1 708个氨基酸。(2)在TuMV侵染不结球白菜30 d后,通过实时荧光定量PCR结果显示,接种TuMV株系中BcCHC1的相对表达量显著下降。(3)亚细胞定位发现BcCHC1定位在烟草表皮细胞的细胞膜与细胞核。(4)BcCHC1基因沉默株系观察发现,在接种TuMV前,BcCHC1沉默植株就已经死亡。(5)通过BiFC实验验证分析,发现BcCHC1可以与CI、6K2互作,与CI互作的位置主要在细胞核,与6K2互作的位置主要在细胞膜上。【结论】研究推测BcCHC1与TuMV的CI和6K2相互作用,通过影响网格蛋白依赖性内吞途径以及病毒复制等方式调控TuMV侵染不结球白菜的过程,但BcCHC1具体如何调控TuMV侵染不结球白菜的作用机理还需进一步研究。

    Abstract:

    【Objective】 In order to improve TuMV resistance of non-forming Chinese cabbage, the interaction mechanism between BcCHC1 gene and TuMV was investigated. 【Methods】 In this study, a member of the clathrin heavy chain CHC gene family was identified from cabbage and a CHC1 was cloned, and subcellular localization of the CHC1 was performed. CI and 6K2 were screened from the candidate genes by bicolecular fluorescence complementary detection. VIGS induced BcCHC1 silencing and the plants died. BiFC test confirmed the interaction between BcCHC1 and TuMV proteins. 【Results】 The results showed that: (1) BcCHC1 gene was cloned successfully, and its coding sequence length was 5 124 bp, encoding 1 708 amino acids. (2) After 30 days of TuMV infection, the results of real-time fluorescence quantitative PCR showed that the relative expression of BcCHC1 in TuMV inoculated lines decreased significantly. (3) Subcellular localization found that BcCHC1 was located in the cell membrane and nucleus of tobacco epidermal cells. (4) The observation of BcCHC1 gene silenced lines showed that BcCHC1 silenced plants had died before TuMV inoculation. (5) Through BiFC experimental verification analysis, it was found that BcCHC1 could interact with CI and 6K2, and the interaction position with CI was mainly in the nucleus, while the interaction position with 6K2 was mainly in the cell membrane. 【Conclusion】 The study speculated that BcCHC1 interacts with TuMV''s CI and 6K2 to regulate TuMV infection of non-knotting Chinese cabbage by influencing clastrin-dependent endocytosis pathway and viral replication, etc. However, the specific mechanism of BcCHC1 regulating TuMV infection of non-knotting Chinese cabbage needs further study.

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刘芳,吕善武,袁梦果,等.不结球白菜BcCHC1基因与TuMV互作机理的初步研究[J].西北植物学报,2024,44(8):1229-1238

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  • 收稿日期:2023-07-02
  • 最后修改日期:2024-01-11
  • 录用日期:2024-01-24
  • 在线发布日期: 2024-07-15
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