Silencing Trehalase And Chitin Synthase Gene Of Cnaphalocrocis Medinalis Using RNA Interference

Rice is major food crops for people in the world.The yield of rice is related to people's life.The rice leaf folder,Cnaphalocrocis medinalis?Lepidoptere:Pyralidea?,commonly known as bracts worm etc,is one of the most seriously pest of rice.Chitin is a major component of insect epidermis and midgut and other important organs,and is essential for the growth and reputation of insect.Trehalase?Tre?and chitin synthase?CHS?are the first and last enzymes in the process of chitin synthesis,respectively.They play the important roles in the synthesis of insect chitin and ideal targets for pest control.In recent years,With the clarification of RNA interference?RNAi?technology that has been widely applied to the research on insect gene function and pest control.In this study,C.medinalis was acted as the research object;trehalase and chitin synthase genes were acted as the target;double-stranded RNA?dsRNA?was synthesized in vitro.The inhibition efficiency of dsRNA on target genes of rice leaf roller was detected by injecting RNAi technology,and then the dsRNA of trehalase and chitin synthase was expressed in rice by using transgenic RNAi technology.The rice leaf roller became stunting;less body size;malformation;and mortality after feeding the transgenic rice,to get the goal to control C.medinali.Which laying ay the foundation for the green control of C.medinalis using plant-mediated RNA interference technology.1.Scilencing of CmTre and CmCHS through RNAiThe subject sequence was deviseed based on the open reading frame?ORF?of trehalase gene?CmTre?and chitin synthase gene?CmCHS?.The double-stranded RNA of the subject site from CmTre?dsCmTre2?and CmCHS?dsCmCHS?were in vitro combined and then injected into the 3^rd instar larvae.RNAi effect were determined by phenotypic and mortality changes.CmTre and CmCHS expression levels were detected by real-time quantitative PCR?RT-qPCR?.The results showed that ingestion of dsCmTre and dsCmCHS led to larval less feeding,stunting,less body size,malformation,and mortality.However,the larvea were no significant change in the body type of the control group injected with dsGFP.RT-qPCR results showed that after5 d of dsRNA injection,the expression of the four gene,CmTre1,CmTre2,CmCHSA and CmCHSB was decreased by48.54%,51.03%,59.07%and 58.80%,respectiveiy in dsCmTre and dsCmCHS group compared with the control group injected with dsGFP.The above results indacated that the growth of C.medinalis were blocked and the ecdysis was difficult after injection of dsCmTre and dsCmCHS.The expression of four gene CmTre and CmCHS was significantly inhibited,which had obvious lethal effect.2.Structure and appraisal of recombinant vectorTaking the target sequence verified by RNAi as the target fragment.The positive and negative complementary sequences were inserted into both sides of the intron of rice phospholipase D gene?PLD?,which were synthesized and cloned into vector PUC57.The RNAi recombinant plant expression vector p1301-Tre-CHS was constructed by ligating to the expression vector pCAMBIA1301.The recombinant RNAi expression vector was transferred into Agrobacterium tumefaciens LBA4404after PCR,restriction and sequencing appraisal etc.constructed genetically engineered bacteria.3.Genetic transformation of rice and detection of RNA effect in transgenic plantsA.tumefaciens containing RNAi expression vector was used to infect the callus of Shuhui 527 rice,and 10 strains of transgenic rice were obtained by co-culture,resistance screening,differentiation and rooting.The transgenic rice was fed with the rice leaf roller larvae,and its phenotypic changes,mortality changes,and RT-qPCR were used to detect whether the transgenic rice could mediate the RNAi effect in insects.The results showed that after the test insects were ingested transgenic rice,the body size was smaller than normal,and there was a significant lethal phenotype.Some of them could not be normalized,or they could grow to sputum,but the sputum deformed or less than normal sputum.After fed transgenic rice for 5 days,the death rate of test insects in the experimental group was 46.67%,and the corrected death rate was 45.45%.Compared with the control group,the mortality increased by 44.45%.RT-qPCR results showed that,compared with the control group,the expression of CmTre1,CmTre2,CmCHSA and CmCHSB gene in rice leaf roller fed on transgenic was decreased by48.06%,49.84%,54.22%and 54.63%,respectiveiy.The above results showed that rice transfected with dsCmTre and dsCmCHS could effectively mediate RNAi effect in insect and can better resist C.medinalis.