目的 对广丰千金薯Dioscorea polystachya Turczaninow.cv.Guangfeng Qianjin烟草花叶病毒进行鉴定、原核蛋白表达和序列分析，为广丰千金薯脱毒苗的培育和鉴定提供理论依据。方法 通过lncRNA测序鉴定广丰千金薯烟草花叶病毒蛋白基因，利用大肠杆菌重组技术表达广丰千金薯烟草花叶病毒蛋白，并采用生物信息学方法进行序列分析。结果 广丰千金薯烟草花叶病毒蛋白基因包括复制酶1（ORF1）、复制酶2（ORF2）、RNA聚合酶、运动蛋白（movement protein，MP）、带电蛋白和外壳蛋白（coat protein，CP）；广丰千金薯烟草花叶病毒CP、MP、RNA聚合酶、带电蛋白、ORF1和ORF2基因cDNA总长度分别为480、807、1425、123、4851、3351 bp，其蛋白分别由159、268、474、40、1613和1114个氨基酸组成；广丰千金薯烟草花叶病毒蛋白除带电蛋白为疏水性蛋白质外，其余如ORF1、ORF2、RNA聚合酶、运动蛋白和外壳蛋白均为亲水性蛋白质；广丰千金薯烟草花叶病毒带电蛋白二级结构由β-片层和无规则卷曲构成，CP、MP、RNA聚合酶、ORF1和ORF2的二级结构均由α螺旋、β-片层、无规则卷曲构成；广丰千金薯烟草花叶病毒CP、MP、RNA聚合酶、带电蛋白、ORF1和ORF2三级结构均为单体；广丰千金薯烟草花叶病毒CP、MP、RNA聚合酶、带电蛋白、ORF1和ORF2的亚细胞定位分别为内质网、细胞核、细胞质、细胞核、线粒体、叶绿体；广丰千金薯烟草花叶病毒CP、MP、RNA聚合酶、带电蛋白、ORF1和ORF2与烟草花叶病毒的亲缘关系较近，同源性分别为99.79%、99.38%、99.23%、100%、99.38%、99.49%；广丰千金薯烟草花叶病毒复制酶、RNA聚合酶、MP和CP可实现大肠杆菌重组表达，表明广丰千金薯烟草花叶病毒的lncRNA测序结果准确。结论 广丰千金薯烟草花叶病毒与烟草花叶病毒同源性极高，氨基酸序列及核酸序列与烟草花叶病毒其他植物分离物相似度高，在进化上高度保守。

Objective To provide theoretical basis for the cultivation and identification of virus-free seedlings of Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin, its tobacco mosaic virus was identified, detected its protein expression and analyzed its sequence. Methods The protein gene of tobacco mosaic virus of Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin was identified by lncRNA sequencing, and its protein of tobacco mosaic virus was expressed by E. coli recombinant technology, and its gene sequence was analyzed by bioinformatics method. Results Tobacco mosaic virus proteins in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin included includes replicase (ORF1), replicase (ORF2), RNA polymerase, motion protein, charged protein and coat protein; The total length of cDNA of CP, MP, RNA polymerase, charged protein, replicase (ORF1) and replicase (ORF2) genes of tobacco mosaic virus in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin was 480, 807, 1425, 123, 4851 and 3351 bp respectively, and their protein were composed of 159, 268, 474, 40, 1613 and 1114 amino acids respectively; Proteins of tobacco mosaic virus in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin, except that the charged protein was hydrophobic protein, others such as replicase (ORF1), replicase (ORF2), RNA polymerase, motion protein and coat protein were hydrophilic proteins; The secondary structure of the charged protein of tobacco mosaic virus in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin was composed of extended strand and random coil, and its secondary structures of CP, MP, RNA polymerase, replicase (ORF1) and replicase (ORF2) were composed of alpha helix, extended strand and random coil; The tertiary structures of CP, MP, RNA polymerase, charged protein, replicase (ORF1) and replicase (ORF2) of tobacco mosaic virus protein in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin were monomers; The subcellular localization of CP, MP, RNA polymerase, charged protein, replicase (ORF1) and replicase (ORF2) of tobacco mosaic virus in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin were endoplasmic reticulum, nucleus, cytoplasm, nucleus, mitochondria and chloroplast respectively; CP, MP, RNA polymerase, charged protein, replicase (ORF1) and replicase (ORF2) of tobacco mosaic virus in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin are closely related to tobacco mosaic virus, and the homologies are 99.79%, 99.38%, 99.23%, 100%, 99.38% and 99.49% respectively.; The replicase, RNA polymerase, motion protein, and coat protein of tobacco mosaic virus protein genes in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin could achieve recombinant expression of E. coli, indicating that its lncrna sequencing results were accurate. Conclusion Tobacco mosaic virus protein genes in Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin had high homology with tobacco mosaic virus, and its amino acid sequence and nucleic acid sequence were highly similar to other plant isolates of tobacco mosaic virus, which was highly conserved in evolution.

R286.12

国家自然科学基金资助项目（32060092）；国家自然科学基金资助项目（31960079）；2022年上饶市科技专项项目（饶科发[2023]5号社发类）（2022A008）；江西省科技厅重点研发计划一般项目（20202BBG73010，20192BBGL70050）；江西省现代农业产业技术体系建设专项（JXARS-13-赣东站）；江西省教育厅科学技术研究项目（GJJ211729）；上饶市科技局平台载体建设项目（2019I017，2020J001）