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

doi:10.3969/j.issn.1004-1524.2022.06.10

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (6): 1193-1204.DOI: 10.3969/j.issn.1004-1524.2022.06.10

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S687.3

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.

Figures/Tables 15

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

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

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

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

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

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

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.

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

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.

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

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

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

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.

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

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.

References 13

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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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