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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

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

      摘要
    • 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。

       

      Abstract:
        Objective  In order to reveal the function of cyp-13A11 gene in Bursaphelenchus xylophilus, the RNA interference vector was constructed and the function of cyp-13A11 gene of Bursaphelenchus xylophilus was analyzed to provide theoretical basis for biological control of pine wilt diseases.
        Method  Specific primers were designed to amplify the cyp-13A11 gene fragment of Bursaphelenchus xylophilus. The products of amplification were constructed into pEASY-T1 interference vector, which was further transferred into Trans1-T1 escherichia coli strain. RNA interference efficiency was determined by qRT-PCR. The feeding reproduction and pathogenicity of pine wood nematodes were investigated after the nematodes soaked in cyp-13A11 dsRNA solution were inoculated on Botrytis cinerea and pine trees, respectively.
        Result  Trans1-T1 strain with pEASY-T1 interference vector was successfully constructed and cyp-13A11 dsRNA was synthesized. RNA interference inhibited the expression of cyp-13A11 gene in Bursaphelenchus xylophilus. In addition, the feeding area was less in cyp-13A11 dsRNA treatment than that in ddH2O treatment, as two thirds of the Botrytis cinerea were fed by nematodes in ddH2O treatment on day 3. After 6 days, one third of Botrytis cinerea was fed by nematodes in cyp-13A11 dsRNA treatment, while almost all of the Botrytis cinerea were exhausted by nematodes in ddH2O treatment. The reproduction number of nematodes in ddH2O treatment was 4.28 times higher than that in cyp-13A11 dsRNA treatment. Furthermore, the wilting rates in both ddH2O and cyp-13A11 dsRNA treatments were 22.2% and 5.6% after Bursaphelenchus xylophilus being inoculated on pine trees for 10 days, respectively. The wilting rates of pine trees in both ddH2O and cyp-13A11 dsRNA treatments were 44.4% and 33.3% after 20 days, respectively. Obviously, the wilting rates were 100% in both treatments after 30 days.
        Conclusion  The RNA interference vector of cyp-13A11 gene in pine wood nematode was successfully constructed. Silencing of cyp-13A11 gene exhibited an important effect on feeding of Bursaphelenchus xylophilus, as well as reduced the reproduction ability and pathogenicity of Bursaphelenchus xylophilus.

       

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