木纳格葡萄谷胱甘肽-S-转移酶VvGST1基因克隆与序列分析

doi:10.6048/j.issn.1001-4330.2023.08.013

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (8): 1922-1930.DOI: 10.6048/j.issn.1001-4330.2023.08.013

• 园艺特产·农产品加工工程 • 上一篇下一篇

木纳格葡萄谷胱甘肽-S-转移酶VvGST1基因克隆与序列分析

王曼¹(), 张政², 伊丽达娜·迪力夏提¹, 吴斌²()

1.新疆农业大学食品科学与药学学院,乌鲁木齐 830052
2.新疆农业科学院农产品贮藏加工研究所/新疆农产品加工与保鲜重点实验室,乌鲁木齐 830091

收稿日期:2022-12-01 出版日期:2023-08-20 发布日期:2023-08-14
通信作者: 吴斌(1973-),男,新疆塔城人,研究员,博士(后),硕士生/博士生导师,研究方向为农产品贮藏与加工,(E-mail)42042615@qq.com
作者简介:王曼(1995-),女,新疆温泉人,硕士研究生,研究方向为农产品贮藏与加工,(E-mail)1803348774@qq.com
基金资助:
国家自然科学基金项目(U2003213);国家科学自然基金项目(31860460);新疆维吾尔自治区区域协同创新专项(2021E01005)

Cloning and bioinformatics analysis of VvGST1 from Munage tab.grapes

WANG Man¹(), ZHANG Zheng², Yilidana Dilixiati¹, WU Bin²()

1. College of Food and Pharmaceutical Sciences, Xinjiang Agricultural University, Urumqi 830052, China
2. Institute of Agro-products Storage and Processing, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Processing and Preservation of Agro- products, Urumqi 830091,China

Received:2022-12-01 Online:2023-08-20 Published:2023-08-14
Correspondence author: WU Bin(1973-), male, native place: Tacheng, Xinjiang.Professor, research field: Storage and processing of agricultural products, (E-mail)42042615@qq.com.
Supported by:
National Natural Science Foundation of China(U2003213);National Natural Science Foundation of China(31860460);Xinjiang Uygur Autonomous Region Regional Collaborative Innovation Project(2021E01005)

摘要/Abstract

摘要：

【目的】克隆木纳格葡萄果实中谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)基因VvGST1全长并研究其序列特征,为该基因在葡萄果实抗病作用和功能的研究奠定基础。【方法】根据已有的基因序列,设计3'和5'端RACE引物,利用RT-PCR和RACE技术克隆VvGST1基因cDNA全长。【结果】该基因序列全长719 bp,均为开放阅读框(ORF),共编码221个氨基酸。该基因编码蛋白相对分子质量为25.55 kDa;理论等电点pI值为6.32;分子式为C₁₁₇₈H₁₇₉₉N₂₉₃O₃₂₁S₁₁;不稳定指数为39.22;该蛋白质是稳定的亲水性蛋白,不含跨膜结构域,没有预测到信号肽,可能存在于细胞基质中,是典型的基质蛋白。VvGST1氨基酸序列与棉花、桃、西梅、樱桃、李子、板栗等植物聚为一类,其中与棉花属亲缘关系最近。【结论】获得了木纳格葡萄谷胱甘肽-S-转移酶VvGST1基因全长编码序列,探明了该序列的结构特征。

关键词: 葡萄; 克隆; 谷胱甘肽-S-转移酶; VvGST1基因; 序列分析

Abstract:

【Objective】 To clone and study the sequence characteristics of the glutathione-S-transferase, (GST) gene from the Vitis vinifera L.cv ‘Munage’ table grapes in the hope of laying a foundation for the disease resistance and function of this gene in grape fruit.【Methods】 Full-length VvGST1 gene cDNA was isolated from grape fruit according to the existed ESTs sequence,and 3' and 5' end of the RACE primers were designed by using RT-PCR and RACE.【Results】 A variety of bioinformatics analysis methods were used to predict and analyze the gene, and the results showed that the ORF (Open Reading Frame) length was 719 bp, which encoded a 221 amino acid polypeptide.The VvGST1 gene was forecasted and the relative molecular mass of encoding the protein was 25.55 kDa, the theoretical isoelectric point was 6.32, the atomic composition was C₁₁₇₈H₁₇₉₉N₂₉₃O₃₂₁S₁₁.Its instability coefficient was 39.22, showing it was astable hydrophilic protein without signal peptide or transmembrane structure.It might exist in the cell matrix and was a typical matrix protein.Through analyzing the evolutionary tree, the amino acid sequence of VvGST1 was clustered with cotton, peach, prunus, cherry, plum, chestnut and other plants, among which it was closely related to cotton.【Conclusion】 The full-length coding region of VvGST1 gene in Vitis vinifera L.cv ‘Munage’ table grapes is successfully obtained,and the structural characteristics of the sequence are confirmed.

Key words: grape; cloning; glutathione-S-transferase; VvGST1 gene; sequence analysis

中图分类号:

S663.1

王曼, 张政, 伊丽达娜·迪力夏提, 吴斌. 木纳格葡萄谷胱甘肽-S-转移酶VvGST1基因克隆与序列分析[J]. 新疆农业科学, 2023, 60(8): 1922-1930.

WANG Man, ZHANG Zheng, Yilidana Dilixiati, WU Bin. Cloning and bioinformatics analysis of VvGST1 from Munage tab.grapes[J]. Xinjiang Agricultural Sciences, 2023, 60(8): 1922-1930.

图/表 10

图1 葡萄VvGST1基因PCR扩增电泳图、测序峰图和对比

Fig.1 PCR amplification of electrophoresis chart sequencing peak diagram and comparison resultsof VvGST1 gene in grape

图2 VvGST1与其他物种氨基酸序列多重对比

Fig.2 Multiple sequence alignment of amino acids of the VvGST1 proteins isolated to other species

图3 VvGST1与其它植物GST氨基酸序列系统进化树

Fig.3 Phylogentic relationship of amino acid sequences between VvGST1 and other GST proteins

图4 VvGST1基因编码蛋白质的氨基酸组成

Fig.4 Amino acid composition of protein encoded and hydrophobicity by the VvGST1 gene

图5 VvGST1蛋白的亲疏水性

Fig.5 Hydrophilicity analysis of VvGST1 protein

图6 VvGST1蛋白信号肽预测

Fig.6 Predictive analysis of VvGST1 signal peptide

图7 VvGST1蛋白跨膜结构域预测注:膜外(outside)、膜内(inside)和跨膜区域(transmembrane)

Fig.7 Prediction of VvGST1 protein transmembrane domain

表1 VvGST1亚细胞定位预测

Tab.1 Prediction of VvGST1 subcellular location

亚细胞定位 Subcellular location	分值Score
亚细胞定位 Subcellular location	LocDB	PotLocDB	Neural Nets	Pentamers	Integral
细胞核 Nuclear	0.0	0.0	0.00	0.00	0.05
质膜 Plasma membrane	0.0	0.0	0.96	0.00	0.08
胞外 Extracellular	0.0	0.0	0.96	0.00	0.00
细胞质 Cytoplasmic	10.0	3.0	0.00	2.44	8.64
线粒体 Mitochondrial	0.0	0.0	0.00	2.42	0.08
内质网 Endoplasm. retic	0.0	0.0	0.00	0.80	0.14
过氧化物酶体 Peroxisomal	0.0	0.0	0.96	0.0	0.00
高尔基体 Golgi	0.0	0.0	0.13	0.26	0.00
叶绿体 Chloroplast	0.0	0.0	0.00	0.03	0.67
液泡 Vacuolar	0.0	0.0	0.00	0.00	0.34

图8 VvGST1蛋白的二级结构预测注:蓝色:α-螺旋;绿色:β-折叠;黄色:无规则卷曲;红色:延伸链

Fig.8 Prediction of the secondary structure of VvGST1 protein

图9 VvGST1蛋白的三级结构预测

Fig.9 The tertiary structure prediction of VvGST1 protein

参考文献 23

[1]	Han Q, Chen R, Yang Y, et al. A glutathione S-transferase gene from Lilium regale Wilson confers transgenic tobacco resistance to Fusarium oxysporum[J]. Scientia Horticulturae, 2016, 198: 370-378. DOI URL
[2]	韩键, 白云赫, 朱旭东, 等. 植物谷胱甘肽应答非生物胁迫的分子机制[J]. 分子植物育种, 2020, 18(5): 1672-1680.
	HAN Jian, BAI Yunhe, ZHU Xudong, et al. Molecular Mechanism of Glutathione Response to Abiotic Stress in Plants[J]. Molecular Plant Breeding, 2020, 18(5): 1672-1680.
[3]	吴金华, 张西平, 胡言光, 等. 小麦抗白粉病相关基因GST克隆与表达[J]. 西北植物学报, 2013, 33(1): 34-38.
	WU Jinhua, ZHANG Xiping, HU Yanguang, et al. Cloning and expression of glutathione S-transferase(GST) gene related to powdery mildew of wheat[J]. Acta Botanica Boreali-Occidentalia Sinica, 2013, 33(1): 34-38.
[4]	苏艳杰, 徐元江, 赵芳玉, 等. 藏药喜马拉雅紫茉莉谷胱甘肽-S-转移酶基因的克隆及表达分析[J]. 高原农业, 2021, 5(4): 393-401.
	SU Yanjie, XU Yuanjiang, ZHAO Fangyu, et al. Cloning and Expression Analysis of Glutathione-S-transferase (MhGSTL 3) in Mirabilis himalaica (Edgew.) Heimerl[J]. Journal of Plateau Agriculture, 2021, 5(4): 393-401.
[5]	杨慧萍, 刘雅莉, 娄倩, 等. 葡萄风信子谷胱甘肽S转移酶基因克隆与表达分析[J]. 植物研究, 2016, 36(1): 134-140. DOI
	YANG Huiping, LIU Yali, LOU Qian, et al. Clone and Characterization of A Glutathione-s-transferase Gene in Muscari armeniacum[J]. Bulletin of Botanical Research, 2016, 36(1): 134-140. DOI
[6]	宋文, 单春会, 宁明, 等. 谷胱甘肽-S-转移酶在植物响应冷胁迫方面的研究进展[J]. 食品工业, 2020, 41(7): 239-244.
	SONG Wen, SHAN Chunhui, NING Ming, et al. Advances in Research of Glutathione-S-Transferase in Response to Cold Stress in Plants[J]. The Food Industry, 2020, 41(7): 239-244.
[7]	陈秀华, 王臻昱, 李先平, 等. 谷胱甘肽S-转移酶的研究进展[J]. 东北农业大学学报, 2013, 44(1): 149-153.
	CHEN Xiuhua, WANG Zhenyu, LI Xianping, et al. Research progress on glutathione S-transferases[J]. Journal of Northeast Agricultural University, 2013, 44(1): 149-153.
[8]	马立功, 孟庆林, 张匀华, 等. 向日葵谷胱甘肽-S-转移酶基因的克隆及抗病功能研究[J]. 中国油料作物学报, 2015, 37(5): 635-643.
	MA Ligong, MENG Qinglin, ZHANG Yunhua, et al. Clone and function of a glutathione S-transferases gene from sunflower (Helianthus annuus)[J]. Chinese Journal of Oil Crop Science, 2015, 37(5): 635-643.
[9]	安秀红, 徐锴, 厉恩茂, 等. 苹果抗性相关的谷胱甘肽转移酶基因MdGSTU1的生物信息学和表达分析[J]. 中国农业科学, 2014, 47(24): 4868-4877. DOI
	AN Xiuhong, XU Kai, LI Enmao, et al. Bioinformatics and Expression Analysis of MdGSTU1 GeneEncoding a Resistance-Related Glutathione Transferase from Apple[J]. Scientia Agricultura Sinica, 2014, 47(24): 4868-4877. DOI
[10]	Tiwari M, Kidwai M, Dutta P, et al. A tau class glutathione-S-transferase (OsGSTU5) confers tolerance against arsenic toxicity in rice by accumulating more arsenic in root[J]. Journal of Hazardous Materials, 2022, 426: 128100. DOI URL
[11]	Juhász C, Gullner G. The monoterpenoid (S)-carvone massively up-regulates several classes of glutathione S-transferase genes in tobacco leaf discs[J]. Acta Phytopathologica et Entomologica Hungarica, 2014, 49(2): 163-176. DOI URL
[12]	Gullner G, Komives T, L Király, et al. Glutathione S-Transferase Enzymes in Plant-Pathogen Interactions[J]. Frontiers in Plant Science, 2018, 9: 1-19. DOI URL
[13]	平怀磊, 王娟, 张怀壁, 等. 滇牡丹DFR基因克隆及生物信息学特征分析[J]. 分子植物育种, 2021: 1-18.
	PING Huailei, WANG Juan, ZHANG Huaibi, et al. Cloning and Bioinformatics Analysis of DFR Gene in Paeonia delavayi[J]. Molecular Plant Breeding, 2021: 1-18.
[14]	De Simone N, Pace B, Grieco F, et al. Botrytis cinerea and table grapes: A review of the main physical, chemical, and bio-based control treatments in post-harvest[J]. Foods, 2020, 9: 1138-1162. DOI URL
[15]	马建辉, 张文利, 高小龙, 等. 山羊草谷胱甘肽S-转移酶基因家族鉴定及表达分析[J]. 作物杂志, 2018(5): 54-62.
	MA Jianhui, ZHANG Wenli, GAO Xiaolong, et al. Identification and Expression Analysis of the Whole Glutathione S-Transferase Genome Family in Aegilops tauschii under Abiotic Stress[J]. Crops, 2018(5): 54-62.
[16]	Wagner U, Edwards R, Dixon D P, et al. Probing the diversity of the Arabidopsis glutathione S-transferase gene family[J]. Plant Molecular Biology, 2002, 49(5): 515-532. DOI PMID
[17]	刘海峰, 王军. 山葡萄谷胱甘肽S-转移酶基因(VAmGST4)克隆及表达分析[J]. 植物生理学报, 2011, 47(12): 1161-1166.
	LIU HaiFeng, WANG Jun. cDNA Cloning and Expression Analysis of Glutathione S-Transferase (VAmGST 4) in Vitis amurensis Rupr.[J]. Plant Physiology Journal, 2011, 47(12): 1161-1166.
[18]	张建平, 贾彩红, 王家保, 等. 采后荔枝果皮中谷胱甘肽转移酶基因的表达与酶活分析[J]. 热带作物学报, 2014, 35(12): 2368-2373.
	ZHANG Jianping, JIA Caihong, WANG Jiabao, et al. Expression Analysis and Determination of Enzyme Activity of the Gultathione Transferase Gene from Postharvest Litchi Pericarp[J]. Chinese Journal of Tropical Crops, 2014, 35(12): 2368-2373.
[19]	李成磊, 蒙华, 张晓伟, 等. 甜荞苯丙氨酸解氨酶基因PAL的克隆及序列分析[J]. 食品科学, 2011, 32(7): 255-257. DOI
	LI Chenglei, MENG Hua, ZHANG Xiaowei, et al. Cloning and Sequence Analysis of Phenylalanine Ammonia-lyase (PAL) Gene from Fagopyrum esculentum[J]. Food Science, 2011, 32(7): 255-257. DOI
[20]	吴忠红, 杜鹃, 谭慧林, 等. 小白杏脂氧合酶基因PaLOX的克隆与序列分析[J]. 生物技术, 2017, 27(1): 1-8.
	WU Zhonghong, DU Juan, TAN Huilin, et al. Cloning and sequence analysis of lipoxygenasegene(LOX) from Xiaobai apricots[J]. Biotechnology, 2017, 27(1): 1-8.
[21]	温琦, 梁塔娜, 张艳欣, 等. 蓖麻GST的基因克隆及生物信息学分析[J]. 分子植物育种, 2020, 18(7): 2161-2166.
	WEN Qi, LIANG Tana, ZHANG Yanxin, et al. Gene Cloning and Bioinformatics Analysis of Castor GST[J]. Molecular Plant Breeding, 2020, 18(7): 2161-2166.
[22]	Ganesh K A, Nam-soo J, Randeep R. A pathogen-induced novel rice (Oryza sativa L.) gene encodes a putative protein homologous to type II glutathione S -transferases[J]. Plant Science, 2002, 163(6): 1153-1160. DOI URL
[23]	Csiszár J, Horváth E, Váry Z, et al. Glutathione transferase supergene family in tomato: Salt stress regulated expression of representative genes from distinct GST classes in plants primed with salicylic acid[J]. Plant Physiology and Biochemistry, 2014, 78: 15-26. DOI PMID

木纳格葡萄谷胱甘肽-S-转移酶VvGST1基因克隆与序列分析

Cloning and bioinformatics analysis of VvGST1 from Munage tab.grapes

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