摘要
【目的】探讨利用RNA干扰技术切断灰飞虱体内水稻条纹病毒(Rice stripe virus,RSV)繁殖的可行性,研究RSV病毒不同基因间的互作关系。【方法】利用喂食的方法测定体外合成的dsRNA对灰飞虱体内RSV基因表达的抑制作用。根据RSV基因序列设计并合成3种dsRNA(dsRdRp、dsNSvc4和dsCP),分别喂食携带RSV病毒的2龄灰飞虱,96 h后利用荧光定量PCR技术同时检测灰飞虱体内RSV病毒不同基因RdRp、NSvc4和CP的表达量。【结果】经过特异性dsRNA喂食处理的灰飞虱体内RSV病毒靶基因NSvc4、CP的表达量大幅下调,下调幅度分别为93.2%和94.9%;靶基因RdRp表达量下调幅度为45.6%。dsRNA喂食处理对其它非靶标RSV基因的表达均具有一定的下调作用,表明RSV病毒不同基因RdRp、NSvc4和CP间可能存在互作关系。【结论】本研究为利用转dsRNA工程微生物或转基因水稻进行RSV病毒的防治提供了重要依据。
【Objective】 The objective of this study is to use RNA interference（RNAi） technique to explore the feasibility of inhibiting the reproduction of Rice stripe virus（RSV） and to probe the interactions among different RSV genes in vitro.【Method】 The inhibition function of exogenous dsRNAs to the expression of RSV genes in Laodelphax striatellus（Fallén） were investigated through feeding synthesized dsRNA in vitro.According to the sequences of RSV,three types of dsRNAs（dsRdRp,dsNSvc4,dsCP） were designed and synthesized and then used to feed 2nd instar L.striatellus that infested with RSV.The dsRNA were synthesized by using the T7 RiboMax Express RNAi System.Then 1 μg？μL-1 dsRNA was added to the artificial diet for feeding.The expression level of each RSV gene in insects was examined by quantitative real-time PCR（qRT-PCR） at 96th hour after dsRNA treatment.【Result】 The results of qRT-PCR indicated that the expression of two RSV genes,NSvc4 and CP,decreased by 93.2% and 94.9%,respectively.Although the decreasing amplitude of RdRp gene was significantly lower,it still decreased by up to 45.6%.Further studies showed that the expressions of other non-target RSV genes decreased when feeding specific dsRNA,which indicated that interaction might exist among different RSV genes.【Conclusion】 This study offered an important basis for controlling RSV through dsRNA microbe engineering or transgenic rice technology.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第6期1158-1165,共8页
Scientia Agricultura Sinica
基金
国家“973”计划项目(2010CB126200)
国家自然科学基金项目(31272042)
