Structure Characteristics Of Complete Genome Of Strawberry Vein Banding Virus Chinese Isolate And Function Identification Of Its Gene ORF VI

The complete genome sequence of Strawberry vein banding virus (SVBV)Chinese isolate has been determined in our research. The exact position of each ORFof SVBV American isolate located at the genome was determined according to thecharacteristics of sequences of each ORFs of SVBV American isolates. Moreover,sequence of six ORFs of SVBV Chinese isolate were cloned according to thecomplete sequence of SVBV Chinese isolate, and the necessary sequence resourceswere provided for the following research of function genes. Pathogenic determinantORF VI of SVBV Chinese isolate was transformed into Arabidopsis thaliana toobserve the influence of ORF VI to symptom phenotype of A. thaliana.1. Determination and sequence analysis of complete genome of SVBV ChineseisolateTotal DNA was extracted from strawberry leaves infected with SVBV Chineseisolate by CTAB method. Sequence of complete genome of SVBV Chinese isolatewas amplified by designing specific primers on basis of complete sequence of genomeof SVBV American isolate, and PCR product was cloned and sequenced. Completesequence containing7838nts of genome of SVBV Chinese isolate was determined bysequence splicing. Comparing nucleotide sequence of SVBV Chinese isolate with thatof SVBV American isolate and other members of Caulimovirus. It indicated thatSVBV Chinese isolate shared the highest similarity (86.14%) with SVBV Americanisolate. Furthermore, the exact position of each ORF of SVBV American isolatelocated at the genome was determined according to the characteristics of sequences ofeach ORFs of SVBV American isolates. Comparing nucleotide sequence of each ORFof SVBV Chinese isolate with that of each corresponding ORF of other members ofCaulimovirus, and phylogenetic trees of ORFs based on alignments of nucleotidesequences of ORFs of Caulimovirus were constructed.2. Cloning of ORFs of SVBV Chinese isolateOn the basis of spliced sequence of SVBV Chinese isolate, specific primers ofeach ORF were designed to amplify the integrity sequence of ORFs of SVBV Chineseisolate. PCR products were cloned and sequenced. Comparing and checking sequenceof each ORF with the splicing complete sequence of SVBV Chinese isolate, and theclone of each ORF whose sequenc was correct were preserved.3. Construction of plant expression vector of ORF VI and A. thaliana plants weretransformed via Agrobacterium tumefaciens Recombinated plant expression vector pCAM P6was constructed by inserting ORFVI of SVBV Chinese isolate into plant expression vector pCAM2300. pCAM P6wasintroduced into A. thaliana plants via Agrobacterium, and transgenic A. thalianaplants of generation T0were screened via antibiotic resistance. It was found that ORFVI transgenic A. thaliana plants didn’t show any obviously symptom phenotypecomparing with wild type plants of A. thaliana by symptom observating.