Constraction Of Infectious CDNA Clones Of Youcai Mosaic Virus And Interaction With TuMV And PVX

Youcai mosaic virus(YoMV),also known as oilseed rape mosaic virus(ORMV),is a member of subgroup Ⅲ of Tobamovirus.The virus is widely distributed in rapeseed(Brassica napus L.)growing areas,and mainly damages cruciferous plants through mechanical transmission.In 2018,we detected YoMV on Brassica juncea var.gemmifera and Brassica juncea var.tumida for the first time in the investigation of cruciferous vegetable diseases in Chongqing.There is no further study on the biological characteristics of YoMV isolates from spore mustard.In order to better understand and prevent virus,the infection clone of YoMV was constructed successfully with DNA homologous recombination technology,which provides materials for the interaction between the virus and the host.It laid the foundation for further research on the host range of YoMV and its interaction with other viruses.The main findings are as follows:1)Development of a full-genome cDNA clone of YoMV.In this study,the genomes of YoMV spore mustard isolates were amplified by RT-PCR,and the infectious cDNA clone pCaYoof YoMV was successfully constructed using seamless cloning technology.After Agrobacterium inoculated,pCaYohas infective activity and pathogenicity through symptom observation,RT-PCR identification and electron microscope observation.2)Development of two mutant of YoMV containing GFP.On the one hand,the fusion expression strategy is adopted,the GFP is inserted before the stop codon of the YoMV CP,and the pCaYo-GFP mutant is obtained by seamless cloning construction.On the other hand,using the strategy of adding subgene promoters,replacing YoMV CP with GFP and introducing the CP gene of TMV-GFP,the YoMV-GFP mutant was successfully constructed through yeast homologous recombination technology.The Agrobacterium was inoculated into Nicotiana benthamiana,and the symptoms were observed,RT-PCR identification and electron microscope observation.The results showed that GFP was expressed in the inoculated leaves and systematically infect N.benthamiana,but cannot express GFP in the upper leaves.As the virus replicates,the GFP gene is cut and lost by the virus,but the remaining amino acid residues reduce the pathogenicity of pCaYo-GFP.YoMV-GFP can systematically infected N.benthamiana and express GFP,but its fluorescence intensity is weaker than TMV-GFP,and its pathogenicity is weaker than pCaYo.It only causes N.benthamiana to curl without yellowing and necrosis.Inoculating YoMV-GFP by mechanical friction on N.benthamiana can still infect and express GFP systemically.3)YoMV host range identification.Inoculate YoMV by friction in Cruciferae,Solanaceae and Chenopodiaceae.The results show that YoMV cause necrosis on Chenopodium quinoa、Chenopodium amaranticolor、Nicotiana glutinosa.YoMV can infect and cause the upper leaves of crops such as Brassica juncea var.gemmifera Lee Lin.,Brassica juncea var.tumida Tsen Lee Lin.,and Shanghaiqing to curl;it can infect Cruciferae such as Oilseed rape and Chinese cabbage,but will not produce symptoms of viral diseases.YoMV can infect Yunyan 87,Yunyan116,Yunyan 8704,NC292,and only Yunyan 116 can produce mosaic symptoms on the upper leaves.YoMV can cause HR in tobacco such as KRK26,NC102,Chuanyan No.1 and MSY48.4)The interaction between YoMV and TuMV or PVX.Mixed compound infection of YoMV,PVX and TuMV,the results showed that TuMV and YoMV have an antagonistic relationship,compound infection will alleviate the symptoms of N.benthamiana;YoMV and PVX are synergetic relationship,compound infection aggravate the disease symptoms of N.benthamiana,and PVX can promote the proliferation of YoMV in the early stage of infection.