香蕉条斑病毒ORF Ⅰ基因克隆及编码蛋白和同源物的生物信息学分析

Cloning of Banana streak virus ORF Ⅰ and Bioinformatic analysis of its protein and homologues

【目的】探明杆状DNA病毒属病毒的ORFⅠ基因功能。【方法】本研究从香蕉条斑病毒云南分离株(BSV-YN)中克隆了BSV ORFⅠ基因,通过核苷酸和氨基酸序列分析和同源关系分析,确定了BSV-YN的分类地位;对其编码蛋白及同源物的理化性质、氨基酸序列组成、亲疏水性、跨膜区域、信号肽、亚细胞定位、磷酸化位点、同源结构域、蛋白二级和三级结构等进行预测和分析。【结果】BSV ORFⅠ基因片段大小为528 bp,编码175个氨基酸,通过核苷酸和氨基酸序列分析和同源关系分析,确定了BSV-YN属于BSGFV种。生物信息学分析表明,BSV-YN ORFⅠ及同源蛋白都是不稳定的、亲水性蛋白,以丝氨酸和苏氨酸磷酸化为主。这些蛋白二级结构主要由α-螺旋和无规则卷曲组成,无信号肽或跨膜区域。亚细胞定位预测表明,BSV-YN ORFⅠ及同源蛋白主要在细胞核分布,推测它们在病毒感染的宿主细胞核中发挥重要作用。【结论】推测BSV-YN ORFⅠ在病毒感染的宿主细胞核中参与病毒复制、转录等相关过程。本研究为进一步开展该蛋白的功能研究提供科学线索。

【Objective】According to the report of the Tenth International Committee on Taxonomy of Viruses(ICTV),genus Badnavirus has 46 species of viruses,including 9 distinctive species of Banana streak virus(BSV),which are Banana streak GF virus(BSGFV),Banana streak UA virus(BSUAV),Banana streak IM virus(BSIMV),Banana streak MY virus(BSMYV),Banana streak OL virus(BSOLV),Banana streak UI virus(BSUIV),Banana streak UL virus(BSULV),Banana streak UM virus(BSUML)and Banana streak VN virus(BSVNV).Badna viruses are widely distributed in tropical and temperate regions of the world and cause severe diseases in several important monocot and dicot crops,such as banana,yam,cocoa,citrus,black pepper and taro.Their genome size ranges from 7.2 to9.2 kb with three to seven open reading frames(ORFs).However,the functions of many predicted ORFs including BSV ORFⅠgene and its homologues have been kept unknown.【Methods】Banana leaves with black stripe,chlorotic stripe,spindle and eye-shaped symptoms of banana streak disease(BSD)in Yunnan Province,China were collected in the year of 2008.The pathogen was tentatively named Banana strike virus Yunnan isolate(BSV-YN).The total DNA was extracted by using CTAB method and it was used as a DNA template for PCR amplification.The ORFⅠgene of the BSV-YN was cloned and the classification relationship of BSV-YN in badna viruses was determined based on the ORFⅠsequences analysis.The sequence similarity of ORFⅠgene and its homologues genes were analyzed by NCBI blastn and BioEdit software,while the homology relationship of BSV-YN ORFⅠand its homologues proteins were conducted by MEGA6.0 with Maximum Likelihood method.In order to further investigate the characterization and function of ORFⅠand its homologues proteins,physicochemical properties,amino acid sequence composition,hydrophobicity,transmembrane structure,signal peptide,subcellular localization,phosphorylation site,structural functional domain,secondary and tertiary structures were predicted based on the online databases and softwares on bioinformatics websites.【Results】The BSV-YN ORFⅠgene was 528 bp,encoding 175 amino acids with unknow function.NCBI blastn and BioEdit software analysis showed that BSV-YN ORFⅠgene was 99.0%sequences similarity with BSGFV ORFⅠgene(Genbank accession No.KJ013507.1),while its protein showed 100%identity with BSGFV ORFⅠ(Genbank accession No.AHM92949.1).Phylogenetic tree analysis indicated that BSV-YN ORFⅠhad the highest homology with the BSGFV ORFⅠ,followed by the BSUAV ORFⅠand Commelina yellow mottle virus(ComYMV)ORFⅠ.Therefore,BSV-YN belonged to the BSGFV species.ORFⅠs of BSV-YN,BSGFV,BSUAV and BSIMV had 175 to 177 amino acid residues with their relative molecular weights of 20.80 kDa.Furthermore,the isoelectric points(pIs)among 6.98 to8.45,about 30 negative and 30 positive amino acid residues,the hydrophobicity among-0.741 to 0.606,and protein instability coefficients among 45.85 to 63.27 were also predicted.These results indicated that BSV-YN,BSGFV,BSUAV and BSIMV ORFⅠs were unstable,hydrophilic or water-soluble proteins.BSV-YN ORFⅠhad fourteen potential phosphorylation sites,including nine serine sites(Amino acid sites at 3,14,18,32,33,34,88,103 and 126 in the peptide chain),four threonine sites(Amino acid sites at 45,72,148,152 in the peptide chain)and one tyrosine(Amino acid site at 27 in the peptide chain).The results predicted that the serines and threonines of BSV-YN ORFⅠwere mainly phosphorylation sites.The secondary structure of BSV-YN ORFⅠand its homologous proteins consisted mostly of alpha helix(73.00%to 88.57%),followed by random coil(10%),extended strand(1.70%to 9.00%)and extended strand(less than 2%).Further analysis showed that the tertiary structure BSV-YN ORFⅠhad high similarity with BSGFV,BSIMV,SCBV and KTSV ORFⅠs,which were mainly consisted of alpha helix and random coil as well.Bioinformatic analysis indicated that BSV-YN ORFⅠand its homologous proteins did not have signal peptide region or transmembrane domain region.Subcellular localization prediction indicated that these proteins are mainly distributed in the cell nucleus.Therefore,BSV-YN ORFⅠand its homologous proteins were presumably involved in the viral infection process in the host cell nucleus.【Conclusion】The gene ORF of BSV-YN ORFⅠwas 528 bp long,encoding 175 amino acids with unknow function.Sequences similarity and homology relationship analyses of the BSV-YN ORFⅠand homologues genes indicated BSV-YN belonged to the BSGFV species.Bioinformatic analysis showed that BSV-YN ORFⅠand homologous proteins were unstable,hydrophilic proteins,with serine and threonine phosphorylation.The secondary structure was mainly composed ofα-helix and random coil in BSV-YN ORFⅠand homologous proteins,but no signal peptide or transmembrane domain of these proteins.Subcellular localization prediction indicated that these proteins were mainly distributed in the cell nucleus,presumably involved in the viral infection process.The proteins might play some key roles in the virus-infected cell nucleus,such as viral replication and transcription.This study would provides scientific clues for further research on the function of this protein.