Study On Rice Stripe Virus Resistance By Silencing The Related Proteins Gene Of Virus Infection In Laodelphax Striatellus

Rice is one of the most important food crops in China and even in the world scope.Laodelphax Striatellus is a kind of piercing,sucking mouthpart insect,which is a member of homoptera Delphacidae.It can jeopardize rice by piercing and sucking the phloem sap.Rice stripe virus(RSV)and rice black streaked dwarf virus(RBSDV)are transmitted by it in a persistent,circulative-propagative manner.It has a destructive impact on rice growth and development,causing a great losse in rice production.At present,we use RNAi technology to control plant viruses and most of the time we use single strategy,the traditional chemical control methods caused serious environmental pollution.To prevent and control the spread of the virus,it is imperative to find a new effective anti-virus strategy.In this study,we used Laodelphax Striatellus and rice stripe virus as research materials,and verified the related proteins genes’ function of virus transmission in Laodelphax Striatellus by the technology of RNAi,through silencing the expression of virus gene and virus transmission related proteins genes,the contents of virus has decreased significantly in insect and rice.We also have cultivated transgenic rice which has a higher resistance quality.Based on this study,we focus on researching the prevention and control effects of virus in different RNA interference strategies,to explore effective strategy to counter virus.The main experiment results and conclusions are as follows:(1)Cloning and functional verification of virus transmission related proteins genes in Laodelphax StriatellusThe total RNA was extracted from the Laodelphax Striatellus,and cDNA was synthesized with reverse transcription.We use PCR technology to amplify the target genes with the corresponding primers.After gel extraction,the genes was linked with cloning vector to construct recombinant plasmid and then transform the competent cell E.coli DH5α.After sequence the positive bacterial colonies,it showed that the full-length is 999 bp of GAPDH3,encoding 332 amino acids(Gen Bank accession number KT724283.1),the full-length is 948 bp of RACK,encoding 315 amino acids(GenBank accession number is KT724285.1).The dsRNA of these two genes were synthesized by in vitro reverse transcription kit,and then add it into the artificial diet(the final concentration is 500ng/μL)which will fed to the Laodelphax Striatellus of 2 instar with RSV.The results of qRT-PCR showed that dsRNA reduced the expression of two genes in the Laodelphax Striatellus significantly,and the interfering effect was observered from the first day to the eighth day.These results indicate that GAPDH3 and RACK genes play an important role in the spread of RSV virus in Laodelphax Striatellus.(2)Research of prevention and control virus with prokaryotic expression dsRNA of virus transmission related proteins genes and virus geneConstruction prokaryotic expression vector of the target gene and virus gene single hairpin and the two chimeric gene double hairpin,and then transform Escherichia coli M-JM109 LacY,IPTG induce expression of dsRNA.We extract dsRNA and add in to artificial diet(final concentration is 500ng/μL)to fed 2 years old Laodelphax Striatellus with RSV.qRT-PCR results showed that the dsRNA which was induced by different prokaryotic expression vectors all can decrease the expression level of the corresponding genes significantly,and the effect was observered from the first day to the eighth day.And it can significantly reduce the content of virus in the Laodelphax Striatellus.The interfering effect of dsRNA expression which induced by chimeric hairpin prokaryotic expression vector is better than that of dsRNA expression which induced by single hairpin prokaryotic expression vector.On the basis of these results,we sprayed the wild-type rice which was induced by different chimeric hairpin prokaryotic expression vector and dissolved in TE buffer(final concentration 500ng/μL),then inoculated the rice with 2 year old Laodelphax Striatellus with RSV.We removed the Laodelphax Striatellus in the sixth day and the eighth day respectively.The results of qRT-PCR showed that different kind of dsRNA all can reduce the content of virus in rice.(3)The antiviral research of transgenic rice which express dsRNA of virus gene and virus transmission related proteins genesWe have constructed the chimeric double hairpin plant expression vector of GAPDH3,RACK gene and RSV SP gene,and then transform Agrobacterium tumefaciens EHA105.We use Agrobacterium tumefaciens EHA105 to infiltrate the rice of L10.Thus we obtained transgenic rice plants with different vectors.The analysis results of transgenic plants Southern blot showed that different exogenous genes can integrated into the rice genome with different copy number.The analysis results of northern blot showed that the exogenous genes can be transcribed into mRNA normally,then mRNA can produced into siRNA.We inoculate five leaf stage of T1 transgenic rice seedlings with the Laodelphax Striatellus instar with RSV,and the wild L10 was used as control.We remove the Laodelphax Striatellus after three days.After three weeks,we found the resistance ratio of RSV in transgenic rice including SP+GAPDH3 is 33.33% while SP+RACK is 50%.Previous study had shown that the resistance ratio of RSV in transgenic rice including RSV SP is 29.41%.Studies have shown that chimeric gene double hairpin has a higher resistance to RSV.