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

doi:10.3969/j.issn.1004-1524.2022.06.10

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1193-1204.DOI: 10.3969/j.issn.1004-1524.2022.06.10

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

洪森荣¹^,²^,³^,⁴(), 向琼钰¹, 谢颖¹, 熊晨露¹, 徐晨慧¹, 徐璐珂¹, 陈荣华⁵, 蔡红⁵

1.上饶师范学院生命科学学院,江西上饶 334001
2.上饶市药食同源植物资源保护与利用重点实验室,江西上饶 334001
3.上饶市三叶青保育与利用技术创新中心,江西上饶 334001
4.上饶农业技术创新研究院,江西上饶 334001
5.上饶市红日农业开发有限公司,江西上饶 334700

收稿日期:2021-02-21 出版日期:2022-06-25 发布日期:2022-06-30
作者简介:洪森荣（1974—）,男,江西永新人,硕士,教授,主要从事植物生物技术研究工作。E-mail： hongsenrong@163.com
基金资助:
国家自然科学基金(31960079);江西省科技厅重点研发计划一般项目(20192BBGL70050);江西省科技厅重点研发计划一般项目(20202BBG73010);江西省教育厅科学技术研究项目(GJJ201704);上饶市科技局重点研发计划一般项目(2020C002);上饶市科技局平台载体建设项目(2020J001);上饶市科技局平台载体建设项目(2019I017)

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

HONG Senrong¹^,²^,³^,⁴(), XIANG Qiongyu¹, XIE Ying¹, XIONG Chenlu¹, XU Chenhui¹, XU Luke¹, CHEN Ronghua⁵, CAI Hong⁵

1. College of Life Sciences, Shangrao Normal University, Shangrao 334001, Jiangxi, China
2. Shangrao Key Laboratory of Protection and Utilization of Medicinal and Edible Homologous Plant Resources, Shangrao 334001, Jiangxi, China
3. Shangrao Tetrastigma hemsleyanum Diels et Gilg Conservation and Utilization Technology Innovation Center, Shangrao 334001, Jiangxi, China
4. Shangrao Agricultural Technology Innovation Research Institute, Shangrao 334001, Jiangxi, China
5. Shangrao Red Sun Agricultural Development Co., Ltd., Shangrao 334700, Jiangxi, China

Received:2021-02-21 Online:2022-06-25 Published:2022-06-30

摘要/Abstract

摘要：

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33 173.36 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。

关键词: 怀玉山三叶青, 烟草病毒增殖蛋白1, 基因克隆, 亚细胞定位, 表达分析

Abstract:

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 33 173.36 u and isoelectric point of 9.16, which was hydrophobic protein. The secondary structure was composed of α-helix (43.73%), β-lamella (21.69%), 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 2 had the highest expression in leaves and Huaiyu 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.

Key words: Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan, tobamovirus multiplication protein 1, gene cloning, subcellular localization, expression analysis

中图分类号:

S687.3

洪森荣, 向琼钰, 谢颖, 熊晨露, 徐晨慧, 徐璐珂, 陈荣华, 蔡红. 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[J]. 浙江农业学报, 2022, 34(6): 1193-1204.

HONG Senrong, XIANG Qiongyu, XIE Ying, XIONG Chenlu, XU Chenhui, XU Luke, CHEN Ronghua, CAI Hong. Gene cloning, subcellular localization and tissue expression analysis of tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1193-1204.

图/表 15

图1 怀玉山三叶青烟草病毒增殖蛋白1基因PCR扩增

Fig.1 PCR amplification of tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图2 怀玉山三叶青烟草病毒增殖蛋白1基因碱基组成

Fig.2 Base composition of tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图3 怀玉山三叶青烟草病毒增殖蛋白1基因各碱基的比例

Fig. 3 Proportion of each base of tobamovirus multiplication protein 1 gene of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图4 怀玉山三叶青烟草病毒增殖蛋白1氨基酸序列

Fig. 4 Amino acid sequence of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图5 怀玉山三叶青烟草病毒增殖蛋白1亲疏水值分布

Fig. 5 Distribution of hydrophilic and hydrophobic water values of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图6 怀玉山三叶青烟草病毒增殖蛋白1二级结构

Fig. 6 Secondary structure of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图7 怀玉山三叶青烟草病毒增殖蛋白1二级结构各部分的比例蓝色代表 α-螺旋;红色代表β -片层;紫色代表不规则卷曲。

Fig. 7 Proportion of the secondary structure of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan Blue represents alpha helix; Red represents extended strand; Purple represents random coil.

图8 怀玉山三叶青烟草病毒增殖蛋白1三级结构

Fig. 8 Tertiary structure of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图9 怀玉山三叶青烟草病毒增殖蛋白1亚细胞定位 E.R.,内质网;E.R._plas,内质网_质膜;extr,细胞外;cyto,细胞质;mito,线粒体;plas,质膜。

Fig.9 Subcellular localization of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan E. R., Endoplasmic reticulum; E.R_ plas, Endoplasmic reticulum_plasma membrane; extr, Extracellular; cyto, Cytoplasm; mito,Mitochondria; plas, Plasma membrane.

图10 怀玉山三叶青烟草病毒增殖蛋白1系统进化分析

Fig. 10 Phylogenetic analysis of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图11 怀玉山三叶青烟草病毒增殖蛋白1基因氨基酸序列的同源性比较

Fig. 11 Homology comparison of amino acid sequences of tobamovirus multiplication protein 1 of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan

图12 烟草病毒增殖蛋白1基因克隆电泳图（左）和菌落PCR电泳图（右）

Fig. 12 Clonal electrophoresis of tobacco virus proliferating protein 1 gene (left) and colony PCR electrophoresis (right)

图13 载体101-TP1-GFP的6号克隆测序结果比对第一行是参考序列,第二至三行是构建的GFP载体6号克隆用两端测序引物的测序结果。

Fig. 13 Comparison of sequencing results of clone 6 of vector 101-TP1-GFP The first line is the reference sequence, and the second to third lines are the sequencing results of the constructed GFP vector clone 6 using two end sequencing primers.

图14 TP1-GFP基因（上）和GFP对照（下）亚细胞定位照片

Fig. 14 Subcellular localization of TP1-GFP gene (top) and GFP control (bottom)

图15 怀玉山三叶青2个栽培种不同器官中烟草病毒增殖蛋白1基因相对表达量分析柱上无相同字母的表示差异显著（P<0.05）。

Fig. 15 Relative expression analysis of tobamovirus multiplication protein 1 in different organs of two cultivars of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan Bars marked without the same leters indicated significant difference at P<0.05.

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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

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