A Preliminary Study Of Papaya Broad Spectrum Resistance Against PRSV By Using CRISPR/FnCas9|Cas9 Technology

Through the sampling survey on the incidence of papaya in Hainan Island,this study further confirmed that papaya ring spot virus(papaya ringspot virus,PRS V)is still a major disease that seriously endangering the production of papaya in Hainan,which seriously restricts the development of papaya industry and brings huge economic losses to farmers.The effect of conventional field management or cultivation measures on virus control is poor or high cost.The most direct and effective measures for the prevention and control of PRS V is to plant antiviral varieties.The main antiviral varieties of commercialized plant are genetically modified lines at present.But through our investigation,we find that the existing transgenic lines have weakened the resistance of some PRSV lines,and even showed no resistance.In recent years,CRISPR technology has provided new against virus strategies.By cooperating with the Dan Voytas Laboratory of University of Minnesota in the United States,we established a system of constructing CRISPR gene editing vector by golden gate method in our laboratory and constructed two sets of gene editing vectors.(1)modified the CRISPR/Cas9 system of editing DNA double chain,CRISPR/FnCas9 can recognize and cleave RNA.In this study,the conservative region of different strains and different genes of the virms was analyzed,and several different gRNA were designed and connected to the plant transformation expression vector.The different strains of virus could be recognized and its genome RNA was clave to achieve the purpose of broad-spectrum resistance papaya against PRSV.(2)CRISPR/Cas9 gene editing system is used to mutate the gene of eIF4E protein that plays a key role in the replication of PRSV VPg protein in papaya.It is expected to block the interaction with the virus without affecting the other functions of papaya,so that the virus can not be replicated in the papaya.Thus,the plant can obtain genetic resistance to the virus.The CRISPR/FnCas9 gene editing vector,pCRISPR-FP-FnCas9 was verified by transient expression on part of the leaves of papaya,showing a preliminary control effect of the virus.The CRISPR/Cas9 gene editing plant transformation vector pRLG 103-PeIF4E-ET,which editing eIF4E gene in papaya genome,was also verified by sequencing.The two sets of vectors are being transformed into papaya.After transformation,the callus has entered the stage of resistance screening.