南方根结线虫促分裂原活化蛋白激酶(MAPK)基因的RNAi效应分析

Analysis of silencing MAPK by RNA interference of root-knot nematode

促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在线虫的生长发育等方面具有重要作用,本研究探索利用RNAi技术沉默南方根结线虫mapk-1基因来抑制线虫的生长发育。根据mapk-1基因的cDNA序列设计引物,通过体外转录合成约550bp的dsRNA对南方根结线虫的卵进行RNAi以沉默mapk-1基因。试验结果表明,将南方根结线虫的卵块浸泡在含有2mg/mL dsRNA的M9缓冲溶液中,24h后卵块孵化出的2龄幼虫数量明显多于对照组(无dsRNA),但孵化出的幼虫死亡率高达90%,而对照组的死亡率低于5%,说明mapk-1基因的沉默抑制了卵块的孵化和线虫的生长,同时将孵化的幼虫接种番茄,14d后番茄根部无根结产生,35d后无卵块产生;而浸泡72h后卵块孵化出的2龄幼虫几乎全部死亡,并且孵化的线虫数量明显少于对照。提取导入dsRNA的卵块的RNA进行半定量PCR分析,结果表明mapk-1基因的mRNA被降解。

Mitogen-activated protein kinase（MAPK）plays an important role in development of nematode.By using RNA interference technique,controlling was studied the expression of mapk-1.Primers were designed based on the cDNA sequence of mapk-1 and a dsRNA about 550 bp was synthesized by vitro transcription to soak the egg masses of Meloidogyne incognita.The results showed that the amount of J2 in egg masses treated with 2 mg/mL dsRNA for 24 hours was significantly greater than that in control group,but the mortality rate in treated group was reached 90%,however,the value in control group was not more than 5%.In addition,the tomato roots had no knot at 14 days postinfection（dpi）and no egg mass at 35 dpi when inoculated the J2.When egg masses were soaked in 2 mg/mL dsRNA for 72 hours,the morality rate of J2 was almost 100% and the amount of J2 was markedly less than control group.RT-PCR analysis showed lower transcript abundance for the targeted mRNAs than control.