拟禾本科根结线虫FMRF酰胺类多肽基因的克隆及RNAi分析

Meloidogyne graminicola was a kind of harm concealment, high reproduction rate, strong parasitic and, was one of the main pathogen of rice root knot nematode diseases. In recent years, M. graminicola spread rapidly on rice in south China and, was threated to rice production. FMRFamide-related peptides (flp) gene mediated neural signal transmission, it plays an important role for nematodes with feeding, moving and parasiting. This study based on the nematode genomics, comparison function genome analysis means to screen flp gene homologous genes.The full length cDNA sequence of flp gene was obtained by RT-PCR and RACE from M. graminicola. Flp gene was localized by in stiu hybridization and, analysis of its function. Through the in vitro synthesis of dsRNA and , using M. graminicola J2 to carry out inierference effects in vitro validation . At the same time, using QPCR method to analysis of mRNA. Choose the best effect of RNAi flp genes for much of RNAi effect validation. Obtain M. graminicola flp genes and analysis their functions can provide new theoretical basis for rice root knot nematode disease prevention and control .

拟禾本科根结线虫（Meloidogyne graminicola）是一种为害隐蔽、繁殖速率高、致病力强的植物寄生线虫，是诱发水稻根结线虫病的主要病原之一。近年来，该线虫在我国南方水稻产区快速蔓延，严重威胁水稻安全生产。FMRF酰胺类多肽（flp）基因介导的神经信号传递对线虫的运动、取食和寄生起着重要作用。本研究以线虫基因组学为基础，运用比较功能基因组分析手段筛选flp同源基因，结合RT-PCR和RACE方法，获得拟禾本科根结线虫flp基因的cDNA全长序列，利用原位杂交的方法对flp基因进行定位，深入分析其功能；通过体外合成dsRNA，对拟禾本科根结线虫二龄幼虫进行离体干扰试验，同时，采用QPCR方法分析病原线虫mRNA表达量；选择RNAi效果良好的flp基因进行多基因RNAi效应验证。拟禾本科根结线虫flp基因的获得及其功能的解析，可为水稻根结线虫病防控提供新的理论依据。

本研究以M. graminicola为对象，对田间采集的水稻根结线虫进行了鉴定，克隆出M. graminicola的4个flp基因，并采用原位杂交定位及RNAi技术对其中Mg-flp-1、Mg-flp-14、Mg-flp-18基因进行了功能分析。. 结合形态学鉴定、分子生物学鉴定及同源性分析结果，将两地采集的水稻根结线虫病病原线虫1MSY和10MDZ鉴定为拟禾本科根结线虫（M. graminicola）。.通过RT-PCR克隆了M. graminicola的4个flp基因Mg-flp-1（GenBank登录号MK886770）、Mg-flp-13（GenBank登录号MK886771）、Mg-flp-14、Mg-flp-18的片段，其片段大小分别为493 bp、269 bp、673 bp、721 bp。运用RACE技术扩增了Mg-flp-14基因1,047 bp全长（GenBank登录号MK855119）及Mg-flp-18基因3′端序列931 bp（GenBank登录号MK886769），并对4个基因进行了分析。.采用原位杂交技术对M. graminicola的Mg-flp-1、Mg-flp-13、Mg-flp-14和Mg-flp-18四个基因进行了定位分析。结果显示Mg-flp-1、Mg-flp-13两个基因在线虫环咽神经环均有表达，Mg-flp-1基因在线虫头部有表达，Mg-flp-13基因在线虫的口针基部球附近有表达；Mg-flp-14基因在线虫口针、环咽神经环及排泄孔周围的神经环部位均显著表达；Mg-flp-18基因在线虫排泄孔周围的神经环处显著表达。采用RNAi技术检测了Mg-flp-1、Mg-flp-14和Mg-flp-18基因的沉默效果。体外浸泡M. graminicola J2和卵块；Mg-flp-1 dsRNA、Mg-flp-14 dsRNA和Mg-flp-18 dsRNA分别处理线虫J2后，采用半定量技术检测M. graminicola J2体内Mg-flp-1、Mg-flp-14和Mg-flp-18基因的沉默效果发现，Mg-flp-1和Mg-flp-14 dsRNA无沉默效果，Mg-flp-18 dsRNA的沉默效果较低，发现卵的孵化率均在97%左右，没有差异。多基因干扰均无沉默效果。J2受干扰后侵染水稻与对照组没有明显的差别。