Complete Genome Analysis Of Banana Streak GF Virus And Its Study On ORFⅠand ORFⅡ Encoding Proteins

Banana(Musa spp.)is one of the important economic crops in the south of China,and also an important food crop in the world.With the development of the banana plantating,viral diseases have become one of the limiting factors for banana yield reduction,such as Banana streak virus(BSV),Cucumber mosaic virus(CMV)and Banana bunchy top virus(BBTV).Among them,Banana streak virus(BSV)belongs to a member of the genus Badnavirus.At present,there are only 9 BSVs by ICTV classification,and the genome size is about 7500 bp with 3 open reading frames(ORFs).Among them,ORFⅠand ORFⅡ encodes two small proteins of 20.8 k Da and 14.7 k Da in size,but their functions are remaining unknown.In addition,the BSV genomes of different species are diversity and polymorphic.The study found a new BSV strain in China and identified the protein functions encoded by its ORFⅠand ORFⅡ,which will not only provided sequence resources for the study the polymorphism of BSV species,but also laying the foundation for the study of the functions of ORFⅠ and ORFⅡ genes in BSV and Badnavirus.In this study,the complete genome sequence of BSGFV-YN was obtained by segmental PCR amplification,and the structural characteristics of BSGFV-YN and its three ORFs were determined.Subsequently,the Yeast two-hybrid technology and the Bimolecule fluorescence complementary technology were used to explore the interactions between ORF Ⅰ and ORF Ⅱ.Meanwhile,ORFⅠor ORFⅡor both were transformed into Arabidopsis plants by agrobacterium-mediated transformation method and the transgenic plants containing ORFⅠor ORFⅡand both were obtained.The effect of heterologous expression of ORFⅠor ORFⅡon Arabidopsis plants was further clarified.The specific results are as follows:(1)Cloning and sequence analysis of BSGFV-YN complete genomeBanana leaves showing streak symptoms were collected from Yunnan,China in2009.The banana belongs to the Cavendish Musa AAA group.Using its total DNA as the template,seven specific pair primers were designed according to the known sequence of BSGFV isolate(NC＿007002.1),and each fragment of BSGFV-YN complete genome was amplified by PCR,and cloned into the vector(p MD-18T)and sequenced.The seven overlapping DNA fragments were assembled via head-to-tail method by Bio Edit software,and a circular double-stranded DNA of 7325 bp with 42%GC was finally obtained.The complete genome sequence of BSGFV-YN was deposited in Gen Bank under accession number MN296502.Complete genome sequence analysis showed that BSGFV-YN was 98.14% sequence similarity with BSGFV-Goldfinger,while it was 49.10-57.09% sequence similarity with other BSV species.The complete genome and ORFⅢ polyprotein of BSGFV-YN and 37 other Badnaviruses were used to construct the phylogenetic trees.The results showed that BSGFV-YN is highest homology with BSGFV-Goldfinger and belonged to the same large branch with the other BSV strains.Although they are highly homologous,their hosts are obviously different.The former banana host is Cavendish Musa AAA group while the latter banana host is Goldfinger Musa AAAB group.Further analysis of the noncoding regions of these two virus strains showed that BSGFV-YN had an extra specific sequence longer than BSGFV-Goldfinger between the intergenic region of ORFⅢ and ORFⅠ.Sequence analysis indicated that two kinds of repeated sequence were found in the intergenic region of ORFⅢ and ORFⅠ.The evidence indicate that the repeated sequence of the intergenic region may provide an important clue for the BSGFV infecting different banana hosts,but further experimental evidences are needed.(2)ORFⅠand ORFⅡinteraction verificationThe activation domain recombinant vector(p GBKT7-ORFⅠ)and the bait domain recombinant vector(p GADT7-ORFⅡ)were constructed based on the Yeast two-hybrid system,and the self-activation and toxicity tests were carried out.The results showed that the ORFⅠwas not toxic to yeast cells and no self-activating activity.The p GBKT7-ORFⅠand p GADT7-ORFⅡ were transformed into the yeast cells(Y2HGold),and positive clones of p GBKT7-ORFⅠand p GADT7-ORFⅡwere obtained by screening with SD-Leu-Trp-His medium containing X-α-Gal,and the result indicated that ORFⅠinteracted with ORFⅡ.In order to further verify the interaction between ORFⅠand ORFⅡ,the interaction between ORFⅠand ORFⅡwas verified by Bimolecular fluorescent complementary technology.The vector(YN1-ORFⅠand YC1-ORFⅡ)were constructed and transferred into the chemically competent cell(GV1301)respectively,and co-injected into the tobacco leaves for transient expression.After 3days,the plant leaves were observed under Fluorescence confocal microscopy.The green fluorescence was observed in the cytoplasm of tobacco cells,indicating that ORFⅠinteracted with ORFⅡin tobacco cells.(3)The subcellular localization of ORFⅠand ORFⅡThe vector(GV1300-ORF Ⅰ and GV1300-ORF Ⅱ)were constructed and transferred into the chemically competent cell(GV1301)respectively,and injected into the tobacco leaves for transient expression.After 3 days,the plant leaves were observed by Fluorescence confocal microscopy.The result showed that both of ORFⅠand ORFⅡ were located in the cytoplasm of tobacco cells.The result further demonstrates that ORFⅠinteracts with ORFⅡand plays biological functions in the cytoplasm of plant cell.(4)The effect of ORFⅠor ORFⅡexpression on Arabidopsis plantsThe recombinant plant expression vectors(GV1300-ORFⅠand GV1300-ORFⅡ)were transferred into the chemically competent cells(GV1301),and further transformed into the Arabidopsis plants.After antibiotic screening,the transgenic Arabidopsis plants were obtained.Phenotypic observation and statistical analysis showed that the Arabidopsis plants containing the ORFⅠgene did not show the difference with the wild phenotype,while the Arabidopsis plants containing the ORFⅡor ORFⅠ/ORFⅡ showed leaves rosette.The study enriched the genome sequences of BSV from different banana hosts,and provided viral sequence resources for the polymorphism study and the evolutionary relationship analysis between BSV and host selection.In addition,the protein functions of BSV ORFⅠand ORFⅡwere preliminary carried out.The interactions between ORFⅠand ORFⅡin the cytoplasm of plant cell and the effect of their heterologous expression on Arabidopsis plants phenotype were clarified.