Silencing Tre2 Gene Of Cnaphalocrocis Medinalis Using Transgenic RNA Interference

Rice is one of the most important food crops and the staple food for more than half of people in the world.However,insect pest is one of the main reasons causing reduction of rice yield.There are more than 350 known pests in Rice in our country,including feeding fresh leaves class,boring class,sucking the juice class,feeding fresh root class.The rice leaf folder is more serious hazards than others.The rice leaf folder,Cnaphalocrocis medinalis(Lepidoptera: Pyralidae),commonly known as leaf roller,bracts worm etc,is one of the most seriously pest of rice.Trehalase is a key enzyme in insects' trehalose metabolism and plays an important role in development and energy regulation of insects and primary energy storage material for insect flight.It also could be an ideal target for pest control.At present,there are two kinds of trehalase gene,belonging to the soluble trehalase gene(Tre1)and membrane-bound trehalase gene(Tre2).RNA interference(RNAi)refers to a phenomenon in which RNA molecules inhibit gene expression which typically by causing the degradation of the homologous mRNA molecules,and the gene silencing process induced by double-stranded RNA(dsRNA),which is the highly evolutionarily conserved mechanism of gene regulation.The main purpose of this study is to obtain transgenic rice that expresses dsRNA of the rice leaf folder membrane-bound trehalase gene(CmTre2)by transgenic RNAi technology;after the rice leaf folder feeds on these rice leaves,CmTre2 will be silenced.And injection in vitro RNAi technology from C.medinalis,so as to achieve the purpose to control C.medinali,which laying the foundation for biological control of C.medinalis.This research will be laying the foundation for biological control of C.medinalis.The main results are as follows: 1.Interference the expression of CmTre gene by injection of dsRNAThe target sequence specific to CmTre2 gene and two kinds of CmTre were designed for RNAi.Designed two RNAi target sites — CmTre?* and CmTre*.Two double-stranded RNAs(dsRNAs)were synthesized in vitro and were separately introduced into the 3rd instar larvae through micro-injection.After injection of dsRNA,the larvae developed loss of appetite,growth retardation,and malformation;some even died.RNAi effects were detected by phenotype observation and real-time quantitative PCR(RT-qPCR).RT-qPCR showed that at the 96 th h after injection of CmTre?-dsRNA and CmTre-dsRNA,the mRNA expression of CmTre decreased by 48.33% and 71%,respectively,in comparison with the control.After the 7th d injection of PBS,CmTre?-dsRNA and CmTre-dsRNA,the larval mortality rates were 23.33%,63.33% and 83.33%,respectively,increased by 11.66%,51.66% and 71.66%,compared to the control.Moreover,the corrected mortality rate of each group is 13.18%?58.50% and 82.26%,respectively(P<0.05).2.Construction and identification of recombinant RNAi expression vectorEqually,the same sequence specific to CmTre?* and CmTre*,synthesized with BamH I and Spe I restriction enzyme cutting sites at both sides,respectively.After double enzyme digestion,these fragment were inserted into vector p1301 to construct recombinant expression vector p1301-CmTre ? * and p1301-CmTre*.The results showed that the recombinant vector had been successfully constructed by PCR,double enzyme digestion and sequencing identification.Then p1301-Cm Tre?* and p1301-CmTre* were to transformed into Agrobacterium tumefaciens LBA4404 by liquid nitrogen freezing and thawing method to construct genetic engineering bacteria.3.Acquirement of the transgenic riceThe calli of rice Zhonghua 11 were infected by A.tumefaciens LBA4404 which contained p1301-Cm Tre?* and p1301-CmTre* plasmid,respectively.Then the calli were screened by higromycin,differentiation of the resistant calli and plant regeneration,17 transgenic rice plants were obtained.And 7 rice plants were found to be positive ones containing CmTre?* and 9 rice plants were found to be positive ones containing CmTre* through RT-PCR detection.4.Detection of RNAi effectsUsed the C.medinalis feeding on normal rice as controls,observed each C.medinalis feeding on transgenic rice found that its life activity decreased significantly,growth and development lagged,presented the molt blocked and the worm varied small,deformity even death,etc.Using real time quantitative PCR detected of ramets CmTre mRNA expression levels in C.medinalis feeding on different transgenic rice and counted its deaths.The results showed that the average expression level of CmTre in C.medinalis feeding on CmTre?* transgenic rice decreased by 29% and feeding on CmTre* transgenic rice decreased by 55%,compared to the control groups.And the mortality was also increased 46.67% and 66.67%,respectively.The results indicated that the transgenic rice plants containing the interference sequence of CmTre gene which could strongly silence CmTre in C.medinalis that fed on these transgenic rice,Moreover,the corrected mortality rate of each group is 51.85% and 74.67%(P<0.05).In addition to the study of insect resistance of two transgenic rice in the laboratory,the experiment of insect resistance of two transgenic rice was carried out in outside.The results show that C.medinalis feeding amount of leaf of two kinds of transgenic rice were significantly reduced,showed leafroll rate of rice CmTreII gene and white leaf rate were 13.40% and 12.47%,the roll leaf rate of leaves of CmTre transgenic rice and white leaf rate were 12.61% and 11.84%,respectively,compared with the control the roll leaf rate of leaf decreased by 11.32% and 12.11%;white leaf rate decreased by 10.10% and 10.73%;The results show that the RNA interference effect of two kinds of transgenic rice are quite obvious.It can play a very significant role in silence of trehalase gene of C.medinalis,which fed the leaves of CmTreII and CmTre transgenic rice and leaf of transgenic rice showed obvious antifeedant to C.medinalis.