Construction Of Jasmine Virus C Infectious CDNA Clone&CP Gene Cloning And Sequence Analysis Of Jasmine Virus C Isolates

Jasmine virus C(JaVC)is a new member of Carlavirus,which commonly coexists with Jasmine virus T(JaVT)and Jasmine virus H(JaVH)on jasmine.The virus was first reported in Taiwan,and the presence of JaVC may be associated with the yellowing mosaic of jasmine leaves.In 2017,we discovered that the jasmine leaves in Fujian also showed signs of yellow mosaic,so we tested and identified the JaVC.Then we obtained the whole genome sequence of JaVC-FJ and constructed its full-length cDNA clone.In order to clarify the distribution and variations of the JaVC virus in other jasmine producing area in China,the 3' end of JaVC was cloned from the jasmines leaves collected from nine provinces,including Sichuan,Yunnan,Guangxi,Hunan,Guangdong,Fujian,Jiangsu,Shandong and Jilin.Sequences analysis shows variations in different provinces and provides the basis for molecular detection and gene function research of JaVC.In addition,virus propagation,virus coating and the genetic diversity are closely related to the Coat protein(CP).Therefore,the gene encoding the CP protein of JaVC was cloned and expressed in prokaryotic cells to prepare polyclonal antibodies for further studys.The main research contents are as follows:1.Construction of JaVC infectious cDNA clonesPrimers were designed according to the JaVC sequence with GenBank accession number NC030926 and 3 cDNA fragments of JaVC Fujian isolates were obtained through RT-PCR amplification.The full-length cDNA clone pXT-JaVC was constructed with the above 3 fragments using Gibson seamless cloning technique and transformed into Agrobacterium tumefaciens GV3101.Samples were taken after 5 days infiltration of Nicotiana benthamiana.Total RNA of inoculated and systemic leaves of Nicotiana benthamiana was extracted,and a specific fragment of JaVC gene was detected by RT-PCR.It was found that JaVC could be detected in both inoculated and systemic leaves of N.benthamiana.Then we constructed a pXT-JaVC plasmid vector with red fluorescent protein mCherry tagged to CP and the specific red fluorescence in N.benthamiana cells was observed by confocal laser scanning electron microscopy.Therefore the full-length cDNA clone pXT-JaVC was infectious.2.Cloning and sequence analysis of JaVC CP genes nine provincesRT-PCR was used to amplify the 3' end of JaVC from from jasmine leaves collected from Sichuan,Yunnan,Guangxi,Hunan,Guangdong,Fujian,Jiangsu,Shandong and Jilin.The fragments were cloned into pTOPO vector which were then subjected to sequencing.The 3' end encodes the coat protein and 11K protein of JaVC.We then compared these 9 CPs and the previous reported CP of Taiwan.Compared to Taiwan isolate,the nucleotide sequence similarities of 9 CPs were 82.27%?91.36%,and the amino acid sequence similarities were 92.23%?96.82%.The nucleotide sequence of JaVC Taiwan isolate has the highest similarity with that of Guangdong.The phylogenetic analysis showed that Taiwan,Guangdong,Sichuan,Fujian,Jilin and Yunnan isolates grouped together,Shandong,Guangxi,Hunan and Jiangsu grouped together.No obvious pattern was found and the question of whether the host's regional differences affect the classification of JaVC still needs further research.3.Expression of CP genes and preparation of polyclonal antibodiesThe JaVC CP gene was amplified by PCR and inserted into the prokaryotic expression vector pET-28a(+).The recombinant plasmid was named pET-28a-CP.After transformed into Escherichia.coli Rosetta,the target protein was induced by IPTG and purified by Ni-NTA Agarose affinity chromatography.The polyclonal antibody was prepared by immunizing New Zealand white rabbits with the purified 33 kDa protein.