Genome Sequencing And Construction Of Infectious CDNA Clone Of Tea Virus A

Tea plant is an important economic crop in China,thus the gene function studies of tea plant is of great value for production.The genome of tea plant is highly heterozygous and polyploidy,the traditional method of gene function study is complex and time-consuming,and there is no mature transgenic system for tea plant.In contrast,VIGS(virus induced gene silencing)technology has the characteristics of fast,high efficiency and high throughput,which can be a powerful tool for gene function studies of tea.There are few known tea viruses,and they are not suitable for the construction of VIGS vector after analyzing the genome structure of those viruses.Therefore,the discovery of new tea viruses can provide a possibility for the establishment of tea plant VIGS system and lay an important foundation for the study of tea plant genome function.First,we discovered a new tea virus.High-throughput sequencing was used for collected tea leaves and bioinformatics analysis revealed a virus-like sequence.After amplifying the full length of suspected virus,we found that it has the highest similarity to the members of Capillovirus,and temporarily named it Tea virus A(TVA).Compared with other viruses of this genus,the amino acid sequence of TVA replicase and movement protein was most similar to Apple stem grooving virus(ASGV),32.4% and 36.6% respectively,which far below the standard of establishing new members of this genus,thus TVA is a new member of Capillovirus.Second,we constructed the full-length cDNA infectious clone of TVA.The cDNA fragments of TVA were obtained by RT-PCR,and the full-length cDNA clone of TVA that named pXT-TVA was constructed by seamless cloning technology and reverse PCR.After 5 days of agro-infiltration,the total RNA of the infected leaves and system leaves were extracted.After the genome DNA was removed,the specific fragment of TVA was detected by RT-PCR.The positive results proved that pXT-TVA could infect Nicotiana benthamiana systematically.The strategy of VIGS vector is to create a multiple cloning site in the 3’UTR of TVA,which can be used for the insertion of the target genes.