Vector Constructions Based On Citrus Mandarivirus And Rhabdovirus

VIGS utilizes the posttranscriptional gene silencing(PTGS)machinery of plants to restrain viral infections systemically and is used to downregulate the plant’s endogenous genes.However,the citrus virus-based VIGS vector has not been widely used in citrus genetic engineering because of its own limitations.Citrus yellow vein clearing virus(CYVCV)and citrus yellow mottle-associated virus(CiYMaV)belong to the subgenus Mandarivirus,which can infect many citrus varieties and cause mild or none symptoms in non-sensitive hosts.Therefore,they are suitable to be developed as VIGS vectors.As the first rhabdovirus identified in citrus,citrus-associated rhabdovirus(CiaRV)also has great potential to be developed into a vector.Therefore,this study carried out research on the construction of VIGS vector,identification of subgenomic promoter and construction of rhabdovirus minireplicon(MR).The main results and conclusions are as follows:1.Based on CiYMaV and CYVCV infectious c DNA clone vectors,VIGS vectors pCiYCP,pCiYUTR,pCYVCP,and pCYVUTR were constructed by inserting endonuclease sites before the start codon of CP or between CRP and 3’ UTR,respectively.Fragments of the citrus endogenous genes,Sulfur(Su)Phytoene Desaturase(PDS),was cloned and inserted into the constructed VIGS vector.The recombinant plasmids pCiYCP-PDS,pCiYCP-Su,pCiYUTR-PDS,and pCiYUTR-su based on CiYMaV and pCYVCP-PDS,pCYVCP-Su,pCYVUTR-PDS,and pCYVUTR-Su based on CYVCV were obtained.The above vectors with P19 of tomato rosette stunt virus(TBSV)were transformed into Agrobacterium tumefaciens and infiltrated citrus seedlings in vacuum.Combined with symptoms,gene silencing was evaluated by molecular biology methods such as dot blot,RT-PCR and RT-qPCR.The results showed that only pCiYCP,pCiYCP-PDS,and pCiYCP-Su could infect citrus plants and produced gene silencing in citrus for 200 days.the silencing efficiency was more than 70% after 80 days of inoculation.Therefore,it is considered that pCiYCP can be used as a VIGS vector in citrus,but it still needs to be further optimized.However,the VIGS vector based on CYVCV cannot be constructed by directly inserting the clone site.2.In order to solve the problem of non-infectious activity of CYVCV-based VIGS vector,a series of deletion mutant vectors were constructed to identify the subgenomic promoter of CYVCV CP.It is speculated that the core promoter region is located at nucleotide 39 to 71 upstream of the CP transcription starting site,and the downstream activity enhancement region is located at amino nucleotide 120-135 of CP ORF.The results laid a foundation for the construction of CYVCV-based vectors that contain repetitive CP subgenomic promoters.3.The minireplicon of CiaRV was constructed.Using homologous recombination or transformation-associated recombination(TAR),binary expression vectors pGD-N,pGD-P,pGD-L,and minireplicon vector pCiaRV-MR that contain CiaRV cis-acting element and reporter genes,were constructed.A.tumefaciens harboring these vectors with three silencing suppressors were co-injected into Nicotiana benthamiana leaves.The green fluorescence was observed by the fluorescence confocal microscope 5 days post inoculation.The study also proved that the replication and transcription of CiaRV must involve the core proteins N,P and L.The successful construction of minireplicon has created conditions for the infectious c DNA cloning and vector construction of CiaRV.