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松材线虫细胞色素cyp-13A11基因RNA干扰载体的构建及其功能分析

盛东萍 张晓阳 冯梦婷 叶建仁 邱秀文

盛东萍, 张晓阳, 冯梦婷, 叶建仁, 邱秀文. 松材线虫细胞色素cyp-13A11基因RNA干扰载体的构建及其功能分析[J]. 北京林业大学学报, 2021, 43(1): 96-102. doi: 10.12171/j.1000-1522.20200025
引用本文: 盛东萍, 张晓阳, 冯梦婷, 叶建仁, 邱秀文. 松材线虫细胞色素cyp-13A11基因RNA干扰载体的构建及其功能分析[J]. 北京林业大学学报, 2021, 43(1): 96-102. doi: 10.12171/j.1000-1522.20200025
Sheng Dongping, Zhang Xiaoyang, Feng Mengting, Ye Jianren, Qiu Xiuwen. Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(1): 96-102. doi: 10.12171/j.1000-1522.20200025
Citation: Sheng Dongping, Zhang Xiaoyang, Feng Mengting, Ye Jianren, Qiu Xiuwen. Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(1): 96-102. doi: 10.12171/j.1000-1522.20200025

松材线虫细胞色素cyp-13A11基因RNA干扰载体的构建及其功能分析

doi: 10.12171/j.1000-1522.20200025
基金项目: 国家自然科学基金项目(31660205),江西省林业厅林业科技创新项目(201711)
详细信息
    作者简介:

    盛东萍。主要研究方向:森林病理学研究。Email:1973661421@qq.com 地址:332005 江西省九江市前进东路551号

    责任作者:

    邱秀文,博士,副教授。主要研究方向:森林病理学研究。Email:qiuxiuwen3@163.com 地址:同上

Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus

  • 摘要:   目的  构建松材线虫cyp-13A11基因RNA干扰载体,研究cyp-13A11基因的功能,为松材线虫病的生物防治提供理论依据。  方法  本文通过设计特异性引物,扩增松材线虫cyp-13A11基因片段,构建至pEASY-T1干扰载体上并转入Trans1-T1大肠杆菌菌株。采用浸泡法对松材线虫进行RNA干扰,通过qRT-PCR检测cyp-13A11基因的表达,将干扰后的线虫分别接种至长满灰葡萄孢菌丝的培养皿中和黑松苗上,测定RNA干扰后线虫的取食、繁殖、致病力和干扰效率等指标。  结果  成功构建了带有pEASY-T1干扰载体的Trans1-T1菌株并且能够合成cyp-13A11 dsRNA,通过RNA干扰抑制了松材线虫cyp-13A11基因的表达。松材线虫RNA干扰后接种至灰葡萄孢第3天,cyp-13A11 dsRNA处理松材线虫的取食面积较小,ddH2O处理松材线虫取食面积达到了整体的2/3;接种第6天,cyp-13A11 dsRNA处理松材线虫的取食面积为整体的1/3,而ddH2O处理松材线虫已将灰葡萄孢菌丝取食殆尽。ddH2O处理松材线虫的繁殖数量比cyp-13A11 dsRNA处理高4.28倍。松材线虫接种至黑松苗第10天,ddH2O处理黑松发病率为22.2%,而cyp-13A11 dsRNA处理黑松发病率为5.6%;接种至20天,ddH2O处理的黑松发病率为44.4%,cyp-13A11 dsRNA处理的黑松发病率为33.3%;直至接种30天,ddH2O和cyp-13A11 dsRNA处理的黑松全部枯萎,发病率达到100%。  结论  成功构建了松材线虫cyp-13A11基因RNA干扰载体,cyp-13A11基因表达沉默后影响了松材线虫的取食,降低了松材线虫的繁殖能力和致病力。

     

  • 图  1  松材线虫cyp-13A11基因的克隆

    Figure  1.  Cloning of cyp-13A11 gene from Bursaphelenchus xylophilus

    图  2  Trans1-T1表达菌株PCR验证

    Figure  2.  PCR verification of Trans1-T1 expression strain

    图  3  cyp-13A11基因dsRNA合成结果

    Figure  3.  Double-stranded RNA synthesis of cyp-13A11 gene

    图  4  RNA干扰后cyp-13A11基因表达量

    不同字母表示不同处理间差异显著性(P < 0.01)。下同。Different letters indicate significant differences between treatments (P < 0.01). The same below.

    Figure  4.  Expression level of cyp-13A11 gene after RNAi treatment

    图  5  松材线虫在灰葡萄孢上的取食情况

    A. 接种3 d;B. 接种6 d。对照组:ddH2O浸泡松材线虫;RNAi处理组:cyp-13A11 dsRNA浸泡松材线虫。下同。A,inoculated for three days; B,inoculated for six days. Control group,Bursaphelenchus xylophilus soaked in ddH2O; RNAi treatment group,Bursaphelenchus xylophilus soaked in cyp-13A11 dsRNA. The same below.

    Figure  5.  Feeding condition of Bursaphelenchus xylophilus on Botrytis cinerea

    图  6  RNA干扰对松材线虫繁殖的影响

    Figure  6.  Effects of RNA interference on the reproduction of Bursaphelenchus xylophilus

    图  7  不同接种处理第20天黑松发病情况

    Figure  7.  Symptoms of Pinus thunbergii seedlings after 20 days inoculation

    表  1  不同接种处理黑松的发病情况

    Table  1.   Disease status of Pinus thunbergii under different treatments

    接种时间
    Inoculating
    time/d
    黑松发病率 Wilting rate of Pinus thunbergii/%
    对照组1
    Control group 1
    RNAi处理组
    RNAi treatment group
    对照组2
    Control group 2
    1005.622.2
    20033.344.4
    300100.0100.0
    注:对照组1:cyp-13A11dsRNA接种至黑松苗;RNAi:cyp-13A11 dsRNA干扰后的线虫悬液接种至黑松苗;对照组2:ddH2O浸泡后的线虫悬液接种至黑松苗。Notes: control group 1, cyp-13A11 dsRNA is inoculated into Pinus thunbergii; RNAi, the suspension of nematodes after interference with cyp-13A11 dsRNA is inoculated into Pinus thunbergii; control group 2, the ddH2O soaked nematode suspension is inoculated into Pinus thunbergii.
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出版历程
  • 收稿日期:  2020-01-20
  • 修回日期:  2020-04-09
  • 网络出版日期:  2020-12-24
  • 刊出日期:  2021-02-05

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