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水稻齿叶矮缩病毒(Rice ragged stunt virus,RRSV)由介体褐飞虱(Nilaparvata lugens)以持久增殖型方式传播。RRSV编码的非结构蛋白6(nonstructural protein 6,Pns6)是病毒原质的组分,但其在介体昆虫传毒中的功能未知。本研究利用ds RNA诱导的RNA干扰(RNA interference,RNAi)技术研究Pns6蛋白在病毒侵染介体昆虫中的功能。通过膜饲喂法将源于Pns6基因的ds RNA(ds Pns6)以及源于绿色荧光蛋白(green fluorescent protein,GFP)基因的ds RNA(ds GFP)分别导入2龄褐飞虱体内,并以饲喂10%蔗糖的处理为对照,之后于病株上获毒2 d。应用免疫荧光标记技术检测发现,昆虫获毒9 d后,ds GFP和10%蔗糖处理组褐飞虱的带毒率分别为27%和30%,而ds Pns 6处理组褐飞虱的带毒率仅为10%;同时q RT-PCR检测发现,Pns6蛋白的表达量受到抑制后,病毒编码的结构蛋白P8和P9的表达量也显著下降(P<0.05)。此外,ds GFP和10%蔗糖处理组褐飞虱的传毒率分别为20%和22%,干扰Pns6蛋白的表达后褐飞虱的传毒率仅为6%。研究结果表明,Pns6是RRSV在褐飞虱体内增殖的重要蛋白,抑制Pns6的表达显著降低褐飞虱的带毒率并抑制病毒的增殖,同时削弱褐飞虱的传毒能力,明确了Pns6可作为理想的靶标用于阻断RRSV在褐飞虱体内的增殖。
Rice ragged stunt virus (RRSV) is propagated in a prolonged proliferative manner by the Nilaparvata lugens. Nonstructural protein 6 (Pns6), encoded by RRSV, is a component of the viral progenitor but its function in mediating insect insects is unknown. In this study, dsRNA-induced RNA interference (RNAi) was used to study the function of Pns6 in virus-infected mediator insects. The dsRNA derived from the Pns6 gene (ds Pns6) and the dsRNA derived from the green fluorescent protein (dsfFP) gene (ds GFP) were introduced into N. lugens by membrane feeder method and fed in a 10 % Sucrose treatment as a control, then poisoned the diseased plants 2 d. The results of immunofluorescence labeling showed that the rates of virulence of BPH in ds GFP and 10% sucrose treated groups were 27% and 30%, respectively, after 9 days of virulent toxicity, while those in ds Pns 6 treatment group were only 10 %. At the same time, the expression of Ps6 and P9 was also significantly decreased by q RT-PCR (P <0.05). In addition, the transmission rates of N. lugens in ds GFP and 10% sucrose treatments were 20% and 22%, respectively, and only 6% of them were interfered with the expression of Pns6 protein. The results showed that Pns6 is an important protein that RRSV can proliferate in N. lugens. Inhibiting the expression of Pns6 significantly reduces the virulence of N. lugens and inhibits the proliferation of N. lugens and weakens the ability of N. lugens to transmit. Pns6 can be used as an ideal target Blocking RRSV Proliferation in Nilaparvata lugens.