Cathepsin B基因的沉默对香蕉穿孔线虫繁殖力的影响

RNAi Effect of Cathepsin B Gene on Reproduction of Radopholus similis

【目的】通过研究cathepsin B基因对香蕉穿孔线虫(Radopholus similis)繁殖力的影响,探索cathepsin B基因的功能,为利用该基因防治香蕉穿孔线虫和植物寄生线虫组织蛋白酶的进一步研究提供科学依据。【方法】根据已知香蕉穿孔线虫cathepsin B基因(Rs-cb-1)的序列,从香蕉穿孔线虫克隆cathepsin B基因,以含有目的基因的质粒DNA为模板合成特异的双链RNA(dsRNA),采用dsRNA浸泡的方法对香蕉穿孔线虫进行RNA干扰(RNAi)试验,通过室内接种胡萝卜愈伤组织繁殖线虫的方法,研究cathepsin B基因的沉默效应对香蕉穿孔线虫繁殖力的影响。【结果】用Rs-cb-1dsRNA浸泡12、24、48、72h后香蕉穿孔线虫的平均繁殖倍数分别为165、93、54、53,而未经dsRNA处理的该线虫繁殖倍数均大于420;并且Rs-cb-1dsRNA浸泡的时间不同,对应各处理之间的繁殖倍数差异显著。RT-PCR检测,经Rs-cb-1dsRNA浸泡12h后,目的基因在香蕉穿孔线虫的表达量明显减弱,浸泡24h后其表达量进一步减弱,但还有微量表达,经dsRNA浸泡48、72h后目的基因基本不表达。【结论】Rs-cb-1与香蕉穿孔线虫的繁殖力相关;Rs-cb-1dsRNA的浸泡可以明显抑制cathepsin B基因的表达量,从而影响香蕉穿孔线虫的繁殖力,但浸泡时间不同Rs-cb-1的沉默效率也不同,沉默效率最好的干涉时间是48h。

[Objective] In order to elucidate the function of cathepsin B gene from Radopholus similis and provide a scientific basis for the further research on this cathepsin B gene of plant-parasitic nematodes, and also for developing new control strategies, the RNAi effect of cathepsin B gene on reproduction of R. similis was studied. [ Method] Cathepsin B gene of R. similis （Rs-cb-1） was cloned from R. similis by designed primers and used as the target gene for RNA silencing. The specific double-stranded RNA （dsRNA） was synthesized by using plasmid DNA containing target gene as template. The nematodes of R. similis were soaked with dsRNA of Rs-cb-1 and RNA interference （RNAi） effects were evaluated by examining the reproduction of R. similis on carrot discs and detecting the gene transcription by RT-PCR. [Result] A significant difference in reproduction factors was observed in treatments with different Rs-cb-1 dsRNA soaking time. The average reproduction factor ofR. similis on carrot discs was 165, 93,54, 53 after dsRNA soaked for 12, 24, 48, 72 h, respectively. The reproduction factors of those treatments without Rs-cb-ldsRNA soaking were greater than 420. RT-PCR revealed that the transcription of target gene was significantly degenerated after 12 h Rs-cb-1 dsRNA soaking. After soaked for 24 h, the transcription was decreased more with only a weak band appeared in electrophoresis gel. No transcription of Rs-cb-1 was observed after 48 h and 72 h Rs-cb-1 dsRNA soaking. [Conclusion] The function of Rs-cb-1 is probably related to the reproduction ability of R. similis Rs-cb-1 dsRNA and soaking inhibited the transcription of cathepsin B gene of R. similis, which may further affected the reproduction of R. similis. The RNAi efficiency of Rs-cb-1 was different in Rs-cb-1 dsRNA soaking times, 48 h dsRNA soaking may produce the best silencing effect on target gene.