怀玉山三叶青烟草病毒增殖蛋白1基因克隆、亚细胞定位和组织表达分析

Gene cloning,subcellular localization and tissue expression analysis of tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

通过怀玉山三叶青试管苗转录组数据库筛选到怀玉山三叶青烟草病毒增殖蛋白1基因的核心片段,利用反转录PCR(RT-PCR)技术克隆怀玉山三叶青烟草病毒增殖蛋白1基因,并采用生物信息学方法和实时荧光定量PCR进行序列分析和器官表达分析。结果表明,怀玉山三叶青烟草病毒增殖蛋白1基因cDNA总长度为888 bp,G+C含量为51.58%;怀玉山三叶青烟草病毒增殖蛋白1由295个氨基酸组成,分子量33173.36 u,等电点9.16,为疏水性蛋白;二级结构由α-螺旋(43.73%)、β-片层(21.69%)、无规则卷曲(34.58%)构成;三级结构为单体;怀玉山三叶青烟草病毒增殖蛋白1主要存在于内质网、内质网-质膜、细胞外、细胞质、线粒体和质膜中;怀玉山三叶青烟草病毒增殖蛋白1在进化上与Aegilops tauschii subsp.tauschill(节节麦)、Triticum turgidum subsp.durum(硬粒小麦)、Hordeum vulgare(大麦)的亲缘关系较近,尤其是与Aegilops tauschii subsp.tauschill(节节麦)烟草病毒增殖蛋白1在进化上具有最高的亲缘关系。通过烟草叶片亚细胞定位分析表明,烟草病毒增殖蛋白1定位于细胞质(可能包括细胞膜)和细胞核膜中。实时荧光定量PCR(qRT-PCR)结果显示,烟草病毒增殖蛋白1基因在怀玉山三叶青2个栽培种中的表达存在器官特异性,怀玉2号在叶中表达量最高,怀玉1号在茎中表达量最高。怀玉山三叶青烟草病毒增殖蛋白1具有典型烟草病毒增殖蛋白1的结构特征,氨基酸序列及核酸序列与同源物种相似度高,在进化上高度保守,对进一步揭示该酶生物学功能具有重要意义。

In this paper,the core fragment of tobamovirus multiplication protein 1 gene was screened from the transcriptome database of plantlets of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan.The tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan was cloned by reverse transcription-PCR(RT-PCR)technique,sequenced by bioinformatics method and analyzed in organ expression by real-time quantitative PCR(qRT-PCR).The results showed that the total length of tobamovirus multiplication protein 1 gene cDNA of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan was 888 bp and the content of G+C was 51.58%.The tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan was made up of 295 amino acids with molecular weight of 33173.36 u and isoelectric point of 9.16,which was hydrophobic protein.The secondary structure was composed ofα-helix(43.73%),β-lamella(21.69%)and irregular curl(34.58%).The tertiary structure is monomer.The tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan mainly existed in the endoplasmic reticulum,endoplasmic reticulum-plasma membrane,extracellular,cytoplasmic,mitochondrial and plasma membrane.The evolution of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan was closely related to Aegilops tauschii subsp.tauschill,Triticum turgidum subsp.durum and Hordeum vulgare,especially showed the highest phylogenetic relationship with tobamovirus multiplication protein 1 of Aegilops tauschii subsp.tauschill.Subcellular localization analysis of tobacco leaves showed that tobamovirus multiplication protein 1 was located in cytoplasm(possibly including cell membrane)and nuclear membrane.The results of qRT-PCR showed that the expression of tobamovirus multiplication protein 1 gene was organ specific in the two cultivars of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan.Huaiyu No.2 had the highest expression in leaves and Huaiyu No.1 had the highest expression in stems.The tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan has the structural characteristics of typical tobamovirus multiplication protein 1.The amino acid sequence and nucleic acid sequence are highly similar to the homologous species and highly conserved in evolution,which is of great significance to further reveal the biological function of tobamovirus multiplication protein 1.