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

作　　者：盛东萍 张晓阳 冯梦婷[1,2] 叶建仁 邱秀文 Sheng Dongping;Zhang Xiaoyang;Feng Mengting;Ye Jianren;Qiu Xiuwen(Jiangxi Yangtze River Economic Zone Research Institute of Jiujiang University,Jiujiang 332005,Jiangxi,China;College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,Jiangxi,China;Jiangsu Key Laboratory for Prevention and Management of Invasive Specie,College of Forest,Nanjing Forestry University,Nanjing 210037,Jiangsu,China)

机构地区：[1]九江学院江西长江经济带研究院,江西九江332005 [2]江西农业大学生物科学与工程学院,江西南昌330045 [3]江苏省有害生物入侵预防与控制重点实验室,南京林业大学林学院,江苏南京210037

出　　处：《北京林业大学学报》2021年第1期96-102,共7页Journal of Beijing Forestry University

基　　金：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)。

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。[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 furthertransferred into Trans1-T1 escherichia coli strain. RNA interference efficiency was determined by qRTPCR.The feeding reproduction and pathogenicity of pine wood nematodes were investigated after thenematodes 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 constructedand cyp-13A11 dsRNA was synthesized. RNA interference inhibited the expression of cyp-13A11 gene inBursaphelenchus xylophilus. In addition, the feeding area was less in cyp-13A11 dsRNA treatment than thatin ddH2O treatment, as two thirds of the Botrytis cinerea were fed by nematodes in ddH2O treatment on day3. After 6 days, one third of Botrytis cinerea was fed by nematodes in cyp-13A11 dsRNA treatment, whilealmost all of the Botrytis cinerea were exhausted by nematodes in ddH2O treatment. The reproductionnumber 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% afterBursaphelenchus xylophilus being inoculated on pine trees for 10 days, respectively. The wilting rates ofpine 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] TheRNA interference vector of cyp-13A11 gene in pine wood nematode was successfully constructed. Silencingof cyp-13A11 gene exhibited an important eff

关 键 词：松材线虫 cyp-13A11基因 载体构建 RNA干扰 功能分析 

分 类 号：S763.7[农业科学—森林保护学] S433.1[农业科学—林学]