松材线虫cyp-33D3基因的RNA干扰及其功能研究

Study on the Function of cyp-33D3 Gene of Bursaphelenchus xylophilus with RNA Interference Method

[目的]研究松材线虫cytochrome P45033D3(cyp-33D3)基因功能,为揭示松材线虫分子致病机制及其生物防治提供理论依据。[方法]采用双链RNA对cyp-33D3基因的表达进行干扰,测定该基因沉默后对松材线虫取食速率、个体大小、产卵数量、孵化率及致病力的影响。[结果]ddH2O和gfp dsRNA处理的线虫取食速度明显较快,在第5天已经几乎将灰葡萄孢菌丝取食殆尽,而cyp-33D3 dsRNA处理松材线虫取食灰葡萄孢的面积较小;cyp-33D3基因RNA干扰对松材线虫雌、雄成虫的体长无显著影响(P>0.05);与ddH2O和gfp dsRNA处理相比,cyp-33D3dsRNA处理每条雌虫平均产卵数量分别减低了12和11粒,虫卵孵化率分别减低了46%和43%;在接种40 d后,ddH2O处理的黑松苗发病率达到100%,cyp-33D3 dsRNA处理的黑松发病率为43.1%;而gfp dsRNA处理的黑松生长状况良好。[结论]cyp-33D3基因RNA干扰减缓了松材线虫的取食速度,减少了松材线虫的产卵数量,降低了松材线虫的虫卵孵化率和对黑松的致病力;松材线虫cyp-33D3基因RNA干扰对线虫的个体大小无明显影响。

[Objective]To study the function of cytochrome P45033D3(cyp-33D3)gene in Bursaphelenchus xylophilus in order to further reveal the molecular pathogenesis mechanism of B.xylophilus,and provide useful information for its biological control.[Method]Double strand RNA(dsRNA)interference was used to investigate the effect of silencing of cyp-33D3 on feeding speed,individual size,number of eggs,hatching rate and pathogenicity of B.xylophilus.[Result]It was found that the feeding speed of B.xylophilus under ddH2O and gfp dsRNA treatments were faster than that under cyp-33D3dsRNA interference treatment in the first 5 days.Almost all the hyphae of Botrytis cinerea were consumed by B.xylophilus soaked under ddH2O and gfp dsRNA treatments.In contrast,a large portion of the hyphae still existed under cyp-33D3 dsRNA treatment.No significant effect was found on the individual size of B.xylophilus when cyp-33D3 gene was interfered by dsRNA(P>0.05).Compared to ddH2O and gfp dsRNA treatments,the average number of eggs per female decreased 12 and 11 under cyp-33D3 treatment,and the hatching rate of eggs decreased by 46%and 43%,respectively.40 days after the soaked B.xylophilus were inoculated in Pinus thunbergii seedlings,the pathogenicity of B.xylophilus under ddH2O and cyp-33D3 treatments were 100%and 43.1%,respectively,while the P.thunbergii seedlings inoculated with B.xylophilus under gfp dsRNA treatment still grew well.[Conclusion]The RNA interference of cyp-33D3 gene decreases the feeding speed,the number of eggs,hatching rate and pathogenicity of B.xylophilus.However,RNA interference of cyp-33D3 gene exhibits no significant effect on individual size of B.xylophilus.