| Carica papaya L.is a popular fruit and an important tropical cash crop,it is also a fruit tree susceptible to virus infection.Papaya leaf distortion mosaic virus(PLDMV)is a new virus disease that threatens the cultivation of Papaya.In recent years,the incidence rate has been increasing year by year,and it has become the most serious virus disease after Papaya ringspot virus(PRSV).Just like PRSV,it is still difficult to prevent and control papaya by chemical control in the cultivation process,so it is urgent to find a more safe and effective antiviral strategy.The acq uisition of new antiviral strategies requires the accumulation of work in the pathoge nic mechanism of PLDMV,the "plant vaccine" of the weak PLDMV strain,and the identification of gene function.A series of researches were carried out based on th ese key technologies,to lay a good theoretical and technical foundation for the dev elopment of a safe and broad-spectrum anti-PLDMV strategy.To carry out the above research work will involve the key link of the construc tion of the plant virus expression vector.The construction of the virus expression v ector is the basis of studying plant viruses and also one of the most critical conten ts.However,due to the large genome of many plant viruses and their toxicity in E.coli,the construction of full-length expression vector with a virus has always been a difficulty in the experiment.In this study,,the full-length of the virus was segm ented amplification,followed by direct transformation of agrobacterium after Gibson assembly in vitro.It’s enabled the rapid,efficient and stable construction of viral in fectious clones,which had higher success rates than traditional in vitro transcription or yeast homologous recombination systems and usually took less than 1 week to c omplete.The study constructed a variety of PLDMV virus expression vectors Using the above methods and did the following work:1.PLDMV expression vector with the reporter gene was constructed and the g ene silencing system of papaya was preliminarily analyzed.Small RNA of PLDMV-GFP in Papaya infected was sequencing and analyzed by NGS.The RNA silencing system of papaya and viral infection mechanism were preliminarily studied by the Vsi RNA characterized of abundance,distribution hots pot,type,length and base preference,and the target genes of Vsi RNAs were p redicted and further verified.The study laid an important theoretical foundation for the in-depth study of the pathogenesis of viruses and the formulation of new strate gies for disease resistance,and provided reference and guidance for the breeding of antiviral diseases.2.The point mutation technique was used to construct a "plant vaccine" with weak mutant strains and its cross-protection effect and principle were verified.It is an effective and broad-spectrum green virus prevention and control techno logy to cross protect papaya plants with weak virus strain " plant vaccine ".In this study,based on the principle of cross-protection,two conserved regions KITC and FRNK in the key pathogenic region of PLDMV HC-Pro were mutated by point mu tation technology,and three mutant weak strains of PLDMV-E,PLDMV-I,PLDMV-EI were constructed.And the cross-protection effect of the constructed weak strain was verified by the challenge experiment of the strong strain with a fluorescent la bel.The double-site mutant weak strain PLDMV-EI could produce no symptoms wit hin 60 days of cross protection,it is a mild、effective and environmentally friendly cross-protection weak strain mutant vaccine.We further verified its protection agai nst the PLDMV virus in nature through field experiments.The relative control effec t of the PLDMV-EI mutant strain was 70.94%.The cross protection experiment of PLDMV and the construction of weak mutant strain provides a new method and str ategy for the prevention and control of the papaya virus.3.Gene function identification platform based on PLDMV-VIGS was establishe d and the function of the CYP83B1 gene was determined.A weak strain that is mild to the host and produces no obvious symptoms is a good material for the VIGS gene functional identification vector.In this study,the PLDMV-VIGS vector was constructed by the weak PLDMV-E mutant strain,whichlargely avoided the interference of plant diseases to the silent phenotype.The PLD MV-VIGS vector was constructed to insert indicator genes PDS and Chl H between NIb protein and CP protein,and the effect of different insert fragment length and ambient temperature on the silencing efficiency of VIGS vector was verified.The P LDMV-VIGS papaya gene function identification platform was established.On this platform,the function of the CYP83B1 gene of papaya was identified.Using char acterization,q RT-PCR,and HPLC,the CYP83B1 gene of papaya was successfully i dentified as a key gene in the pathway of BG(Benzyl glucosinoside)production.V irus-induced gene silencing(VIGS)is a tool for gene function research,which ha s the advantages of simple operation,low cost,short research period and no need for genetic transformation.The establishment of PLDMV-VIGS provides important t echnical support for the research on functional genomics of papaya and an effective tool for occupying the commanding heights of functional genomics in the field of biology in China.The completed papaya genome showed that the papaya genome was the smalle st of the discovered flowering plant(372 Mb).With an excellent genetic transfor mation system(the first commercial transgenic fruit in the world)and a short gr owth cycle.Papaya has significant potential as a "model plant." Therefore,the analys is of the papaya gene silencing system has important scientific significance.PLDMV can also infect cucurbit crops,so the construction of weak PLDMV strain and th e establishment of PLDMV-VIGS also laid a foundation for the application in cu curbit crops.Therefore,it is of great scientific significance to carry out experiments on the interaction between plant virus and host to promote the research of disease resistance breeding in fruit tree science. |
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