Construction Of Infectious CDNA Clones Of Citrus Yellow Vein Clearing And Virus Citrus Leaf Blotch Virus

Every year,citrus virus diseases cause serious economic losses in citrus industry worldwide.Citrus yellow clearing vein virus on citrus was been reported recently.Some typical leaf symptoms include yellow vein clearing,chlorosis,leaf rewinding and shrinking were observed on CYVCV-infected lemon and sour oranges.Even it will appear leaves off,veins necrosis,resulting in tree weakness,fruit production decline,serious or even crops.In recent years,the occurrence of the CYVCV in citrus producing areas in China has been increasing year by year,and has become an important issue affecting our country's citrus production.Citrus leaf blotch virus(CLBV),a representative member of the putative genus Citrivirus,family Flexiviridae.The virus can infect most citrus varieties.In 2016,China first discovered the virus on sweet cherries and kiwifruit and subsequent reports on lemon.Currently,there are few studies on the molecular and biological characteristics of CYVCV and CLBV,and the infectious clone of plant virus is an important tool to study the function of virus gene.However,when constructing a large RNA viral full-length cDNA clone in E.coli,it often showed the instability of the genome structure(mutation and deletion).Therefore,this study first respectively for CYVCV and CLBV isolates were amplified full-length cDNA was cloned by TAR,Then through the yeast transformation based on homologous recombination(transformation-associated recombination TAR)technology were constructed by cloning full-length infectious CYVCV and CLBV,which laid the foundation for the further study of molecular biology,two kinds of virus pathogenic mechanism and carrier application.Main contributions of this work are as follows:1.Rapid construction of virus-invasive cloneThe binary vectors DK1317-2 backbone and the fragment pYES1L-2117 containing the yeast-related replication origin were ligated with In-Fusion HD Cloning Kit.In this way a ternary shuttle vector pCY is obtained which can be replicated and expressed in Escherichia coli,yeast and Agrobacterium.Then the recombinant cloning system was validated by PVX infection clone construction.Then Potato Virus X(PVX)was used to verify the system,and the PVX infected clone was obtained within 2 weeks.2.Construction of full-length infectious clones of CYVCVThis study established one-step RT-PCR amplification system of CYVCV genome.The cloned full-length cDNA of the citrus yellow vein clearing vitus Sichuan Province Anyue isolate was successfully cloned into the ternary vector pCY by TAR technology.The sequencing results showed that the CYVCV-AY genome is 7529 bp and contains 6 open reading frames.It is consistent with the CYVCV isolates that have been reported so far.Select one of them submitted to Genbank,named as CYVCV-AY(accession number: MG878869).Randomly select the 4 sequences obtained were compared.The results showed that the 4 sequences of CYVCV were highly similar to the whole genome sequence.They were all over 99%.Phylogenetic tree showed that the 4 isolates were clustered with theCYVCV isolates from citrus.The full-length infectivity cDNA clone of CYVCV was first obtained at domestic and foreign through TAR clone.The results of symptom observation and RT-PCR detection showed that the obtained pCY-CYVCV was infective.Agrobacterium-mediated inoculation experiments showed that the inoculation method and the selected host had a great influence on the infection efficiency.The total length of cDNA obtained by pCY-CYVCV was invasive by citrus with Agrobacterium tumefaciens,and its infection efficiency was relatively high.3.Construction of full-length infectious clones of CLBVThe invasive clone HBYD of the CLBV Chinese isolate was obtained for the first time using the established cloning rapid clone system of citrus virus(submitted to Genbank,accession number: MG572236).The sequence analysis showed that CLBV-HBYD was 8747 nt in length,containing 5' monomethylated cap structur and 3?poly-(A)tail.,and encoding 3 open reading frames(Open reading frame,ORF).The ORF1,ORF2 and ORF3 is 5889,1089,and 1092 nt respectively.Genome-wide sequence alignment shows : The identity of nucleotide sequences between CLBV-HBYD and 9 CLBV isolates was 79%~98%,and the identity of EU857540.1 from citrus was up to 98%,and the identity of JN983454.1,JN983455.1,JN983456.1 and JN900477.1 from kiwifruit all are 79%.In phylogenetic tree constructed using MEGA6 software,CLBV-HBYD citrus sources were clustered into a cluster with other isolates from citrus sources.Based on the invasive clones,the pCLBV expression vector was constructed by inserting the subgenomic promoter and open reading frame of the GFP gene.