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作物学报 ›› 2025, Vol. 51 ›› Issue (7): 1736-1746.doi: 10.3724/SP.J.1006.2025.44141

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘薯谷胱甘肽S-转移酶基因IbGSTU7的克隆及功能分析

尹雨萌1**(), 王雁楠1**(), 康志河1, 乔守晨1, 卞倩倩1, 李亚蔚1, 曹郭郑1, 赵国瑞1, 徐丹丹2, 杨育峰1,*()   

  1. 1河南省农业科学院粮食作物研究所, 河南郑州 450002
    2通许县农业科学研究所, 河南开封 475400
  • 收稿日期:2024-08-30 接受日期:2025-04-27 出版日期:2025-07-12 网络出版日期:2025-05-07
  • 通讯作者: *杨育峰, E-mail: yyfyyf5@163.com
  • 作者简介:尹雨萌, E-mail: yinyumeng0521@163.com;
    王雁楠, E-mail: alman001@hnagri.org.cn
    **同等贡献
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-10);河南省现代农业产业技术体系建设专项(HARS-22-04-G1);河南省农业科学院优秀青年科技基金项目(2024YQ17);河南省农业科学院自主创新项目(2024ZC014);河南省中央引导地方科技发展资金项目(Z20231811177)

Cloning and functional analysis of glutathione S-transferase gene IbGSTU7 in sweetpotato

YIN Yu-Meng1**(), WANG Yan-Nan1**(), KANG Zhi-He1, QIAO Shou-Chen1, BIAN Qian-Qian1, LI Ya-Wei1, CAO Guo-Zheng1, ZHAO Guo-Rui1, XU Dan-Dan2, YANG Yu-Feng1,*()   

  1. 1Cereal Crop Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
    2Tongxu Academy of Agricultural Sciences, Kaifeng 475400, Henan, China
  • Received:2024-08-30 Accepted:2025-04-27 Published:2025-07-12 Published online:2025-05-07
  • Contact: *E-mail: yyfyyf5@163.com
  • About author:**Contributed equally to this work
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-10);Special Fund for Henan Agriculture Research System(HARS-22-04-G1);Outstanding Youth Science and Technology Fund of Henan Academy of Agricultural Sciences(2024YQ17);Autonomous Innovation Project of Henan Academy of Agricultural Sciences(2024ZC014);Central Government Guides Local Funds for Scientific and Technological Development of Henan Province(Z20231811177)

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摘要：

本研究通过分析前期的薯皮色变异转录组数据, 克隆得到IbGSTU7基因, 该基因开放阅读框(ORF)全长660 bp, 编码219个氨基酸。蛋白序列分析表明, IbGSTU7为酸性、亲水性、稳定性蛋白, 且与甘薯近缘野生种Ipomoea triloba的亲缘关系最近, 定位于细胞质中。通过实时荧光定量PCR (qRT-PCR)对IbGSTU7基因的表达特征进行分析发现, IbGSTU7在甘薯主要组织中均有表达, 且在薯皮中的表达量最高。此外, 干旱、盐胁迫以及外源脱落酸(ABA)均可诱导IbGSTU7基因上调表达。通过农杆菌转化法对IbGSTU7在甘薯中进行过表达发现, 过表达植株的薯皮花青苷含量以及花青苷合成途径相关基因IbPAL与IbUFGT的表达量均显著提高。同时, PEG模拟干旱胁迫下过表达植株表现出更强的抗旱性, 鲜重和根长显著高于野生型对照, 过氧化氢(H2O2)含量显著降低, 活性氧(ROS)清除相关基因IbMDHAR和IbPOD显著上调表达。本研究为进一步探索IbGSTU7基因在甘薯花青苷积累和干旱胁迫应答中的功能提供了新的理论支撑。

关键词: 甘薯, IbGSTU7, 过表达, 花青苷, 抗旱性

Abstract:

In this study, the IbGSTU7 gene was cloned based on our previously obtained transcriptome data from a sweetpotato skin color mutant. The open reading frame (ORF) of IbGSTU7 is 660 bp in length and encodes a protein comprising 219 amino acids. Protein sequence analysis revealed that IbGSTU7 is an acidic, hydrophilic, and structurally stable protein, showing the closest phylogenetic relationship to its homolog in Ipomoea triloba, a wild relative of sweetpotato. Subcellular localization analysis indicated that IbGSTU7 is localized in the cytoplasm. Quantitative RT-PCR analysis demonstrated that IbGSTU7 is expressed across major sweetpotato tissues, with the highest transcript level observed in the skin of storage roots. Furthermore, its expression was inducible by drought, salt, and exogenous abscisic acid (ABA) treatments. The IbGSTU7 gene was subsequently overexpressed in sweetpotato using an Agrobacterium tumefaciens-mediated transformation method. In overexpression (OE) lines, anthocyanin content in the skin of storage roots and the expression levels of anthocyanin biosynthesis-related genes IbPAL and IbUFGT were significantly increased. Additionally, the OE plants exhibited enhanced tolerance to PEG-simulated drought stress, as evidenced by significant increases in fresh weight and root length compared to wild-type (WT) plants. Under drought conditions, OE lines showed a marked reduction in hydrogen peroxide (H2O2) levels, accompanied by significant upregulation of reactive oxygen species (ROS) scavenging-related genes IbMDHAR and IbPOD. These findings provide novel insights into the role of IbGSTU7 in regulating anthocyanin accumulation and improving drought stress tolerance in sweetpotato.

Key words: sweetpotato, IbGSTU7, overexpression, anthocyanin, drought resistance

表1

本研究所用的引物信息"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 引物目的
Primer purpose
IbORF-F ATGGCAGAAGTGAAGGTTTT ORF扩增引物
IbORF-R TTACATGAATACCTTGGTGTAGTAG ORF amplification
GSTU7-p1300-F CGAGCTCATGGCAGAAGTGAAGGTTTTAG 亚细胞定位
GSTU7-p1300-R CGGATCCCATGAATACCTTGGTGTAGTAGGG Subcellular localization
GSTU7-p1302-F GACTAGTATGGCAGAAGTGAAGGTTTT 过表达
GSTU7-p1302-R GGGTGACCTTACATGAATACCTTGGTGTAGTAG Overexpression
1302-35S-F GAAGATAGTGGAAAAGGAAGGTG 转基因阳性鉴定
GSTU7-324-R CCTTTGGTAAATACTGCTTTCTCC Transgenic positive identification
QGSTU7-F AGTGAAGGTTTTAGGGTCGTGG 实时荧光定量PCR
QGSTU7-R GACAGGGTTGGATTTGAGAAGC Real-time quantitative PCR
Actin-F AGCAGCATGAAGATTAAGGTTGTAGCAC
Actin-R TGGAAAATTAGAAGCACTTCCTGTGAAC
IbMDHAR-F CTACTCCCGTGCCTTTGATT
IbMDHAR-R CTCCAAGAATGCACCAACAA
IbPOD-F TTCACGACTGCTTCGTTGA
IbPOD-R TTCTCAACCGCGGTCTTAA
IbCHI-F CCGATCATAGAAGCCGCCAT
IbCHI-R CTCCGGCATTGAACCCTCTT
IbPAL-F CCCTGCAGTGCTAACTACCC
IbPAL-R GAATAGCCGGGTTCCCACTC
IbUFGT-F CAAACGGAAACGGGTTGGAC
IbUFGT-R CGGTGGGTTCTAGCTTCTGG

图1

IbGSTU7基因的生物信息学分析 A: IbGSTU7蛋白的三级结构分析; B: IbGSTU7蛋白和其他物种中相关蛋白的同源进化树分析。"

图2

IbGSTU7蛋白的亚细胞定位 标尺为30.0 μm。"

图3

IbGSTU7基因在苏薯8号中的表达分析 A: IbGSTU7基因在苏薯8号不同组织中的表达。B: IbGSTU7基因对不同处理的响应。S: 茎; L: 叶片; PR: 柴根; SSR: 薯皮; FSR: 薯肉。*、**分别表示在0.05和0.01水平差异显著。"

图4

过表达IbGSTU7基因栗子香植株的获得 A: 栗子香胚性愈伤组织; B: Hyg抗性愈伤组织的形成; C: 体细胞胚的形成; D: 拟转基因植株的再生; E: 长大的拟转基因植株; F: 完整的拟转基因植株; G: 试管苗扩繁; H: 温室驯化; I: 大田种植。标尺为2 cm。"

图5

过表达转基因甘薯植株中的IbGSTU7基因的qRT-PCR分析 *、**分别表示在0.05和0.01水平差异显著。"

图6

过表达IbGSTU7基因促进薯皮中的花青苷积累 A: WT和IbGSTU7过表达株系在田间生长4个月后的薯皮和薯肉颜色, 标尺为5 cm; B~C: WT和IbGSTU7过表达株系薯皮的类胡萝卜素(B)和花青苷含量(C); D: WT和IbGSTU7过表达株系薯肉的花青苷含量; E~G: WT和IbGSTU7过表达株系中IbCHI (E)、IbPAL (F)和IbUFGT (G)基因的表达量。*、**分别表示在0.05和0.01水平差异显著。"

图7

过表达IbGSTU7基因增强了甘薯的抗旱性 A~F: 在含20% PEG-6000的MS培养基上培养4周的WT和IbGSTU7过表达株系的表型(A)、鲜重(B)、根长(C)、IbMDHAR基因表达量(D)、IbPOD基因表达量(E)和H2O2含量(F)。标尺为5 cm。*、**分别表示在0.05和0.01水平差异显著。"

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