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作物学报 ›› 2023, Vol. 49 ›› Issue (10): 2665-2676.doi: 10.3724/SP.J.1006.2023.34020

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗谷胱甘肽硫转移酶ScGSTF1与P3N-PIPO互作应答甘蔗花叶病毒侵染的研究

杨宗桃(), 焦文迪, 张海, 张克闽, 程光远, 罗廷绪, 曾康, 周营栓, 徐景升()   

  1. 福建农林大学农业农村部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗工程技术研究中心, 福建福州 350002
  • 收稿日期:2023-01-30 接受日期:2023-04-17 出版日期:2023-10-12 网络出版日期:2023-04-27
  • 通讯作者: 徐景升, E-mail: xujingsheng@126.com
  • 作者简介:E-mail: YZT@fafu.edu.cn
  • 基金资助:
    国家自然科学基金项目(31971991);福建农林大学科技创新基金项目(CXZX2019132G);广西甘蔗生物学重点实验室开放课题(GXKLSCB-202003)

Interaction of sugarcane glutathione S-transferase ScGSTF1 with P3N-PIPO in response to SCMV infection

YANG Zong-Tao(), JIAO Wen-Di, ZHANG Hai, ZHANG Ke-Ming, CHENG Guang-Yuan, LUO Ting-Xu, ZENG Kang, ZHOU Ying-Shuan, XU Jing-Sheng()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University / National Engineering Research Center for Sugarcane, Fuzhou 350002, Fujian, China
  • Received:2023-01-30 Accepted:2023-04-17 Published:2023-10-12 Published online:2023-04-27
  • Contact: E-mail: xujingsheng@126.com
  • Supported by:
    National Natural Science Foundation of China(31971991);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2019132G);Open Project of Guangxi Key Laboratory of Sugarcane Biology(GXKLSCB-202003)

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摘要：

谷胱甘肽S-转移酶(glutathione S-transferase, GST, EC2.5.1.18)广泛分布于具有细胞结构的生物中。在植物中, GST参与调控生长发育、异生物质解毒及逆境应答。本课题组以甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)编码蛋白P3N-PIPO为诱饵, 从甘蔗(Saccharum spp. hybrid)叶片cDNA酵母文库中筛选到1个GST基因, 并从甘蔗原始栽培种Badila中克隆了该基因, 其开放读码框(open reading frame, ORF)长度为645 bp, 编码214个氨基酸。序列比对及进化树分析表明, 该基因编码蛋白属于GST家族的Phi (F)类型(GSTF), 因此命名为ScGSTF1。进一步的生物信息学分析表明, ScGSTF1不存在跨膜区域, 为稳定的亲水性脂溶蛋白; GST蛋白在单子叶和双子叶植物中及C3和C4植物中存在明显的分化。酵母双杂交(yeast two-hybrid, Y2H)和双分子荧光互补(bimolecular fluorescence complementation, BiFC)试验表明, ScGSTF1与SCMV-P3N-PIPO蛋白互作。亚细胞定位试验表明ScGSTF1定位于内质网。实时荧光定量PCR分析表明, ScGSTF1在甘蔗各个组织中显著差异表达, 在第3节间中的表达量最高, 在心叶及根中的表达量最低; SCMV侵染显著性影响ScGSTF1基因的表达水平, 侵染早期上调表达, 随后下调, 但仍保持显著高于对照的水平。

关键词: 甘蔗, 甘蔗花叶病毒, 谷胱甘肽硫转移酶, P3N-PIPO, 蛋白互作

Abstract:

The Glutathione S-transferases (GSTs) are widely distributed in cellular organisms and participate in the regulation of growth and development, detoxification of xenobiotics, and response to stress in plants. In the present study, we screened a sugarcane (Saccharum spp. hybrid) cDNA library and obtained a GST gene from sugarcane original cultivar Badila using Sugarcane mosaic virus (SCMV)-encoded protein P3N-PIPO as bait. The open reading frame (ORF) of the cloned GST gene was 645 bp in length, encoding a protein with 214 amino acids. Sequence alignment and phylogenetic tree showed that this GST belonged to the Phi (F) class (GSTF) designated as ScGSTF1 in GST family. Further bioinformatics revealed that ScGSTF1 protein was a stable hydrophilic lipoprotein without transmembrane domain. Phylogenetic tree demonstrated that ScGSTF1 was divergent between monocotyledons and dicotyledonous, as well as in C3 and C4 plants. Yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays confirmed the interaction of ScGSTF1 with SCMV-P3N-PIPO. Subcellular localization assays indicated that ScGSTF1 was localized in the endoplasmic reticulum. The qRT-PCR analysis showed that ScGSTF1 gene was significantly differently expressed in different tissues of sugarcane plants with the highest expression level in the 3rd internode and the lowest expression level in leaf roll or roots. SCMV infection affected the relative expression level of ScGSTF1 gene, which was up-regulated at the early stage of infection and down-regulated later, but still maintained a significantly higher level than the control.

Key words: sugarcane, Sugarcane mosaic virus, glutathione S-transferase, P3N-PIPO, protein interaction

表1

本研究使用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 策略
Strategy
pPR3-ScGSTF1-F GCAGAGTGGCCATTACGATGGCCGGCGGTGGCGGCGA 酵母双杂交载体构建
pPR3-ScGSTF1-R ATTCTCGAGAGGCCGAGCTGATGGCTTCATCATGG Vector generation for Y2H
221-ScGSTF1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGGCGGTGGCGGCGA 双分子荧光互补载体构建
221-ScGSTF1-R GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCTGATGGCTTCATCATGG Vector generation for BiFC
ScGSTF1-qF TGGACGAGGCGAACGAGACC 定量PCR
ScGSTF1-qR AGCGGCGGTGAGGCAGAC Real-time-qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因[69]
GAPDH-R TCCTCAG GGTTCCTGATGCC Real-time-qPCR[69]
Actin-F CCTGAAGATCACCCTGTGCT 内参基因[70]
Actin-R GCAGTCTCCAGCTCCTGTTC Reference gene[70]
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测[71]
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV[71]

图1

ScGST与拟南芥和水稻的GST蛋白的系统进化树分析 ScGST以红色字体的突出显示; 粉色进化枝代表Tau (GSTU)类, 紫色进化枝代表dehydroascorbate reductase (DHAR)类, 蓝色进化枝代表Lambda (GSTL)类, 红色进化枝代表Zeta (GSTZ)类, 绿色进化枝代表tetrachlorohydroquinone dehalogenase (TCHQD)类, 棕色进化枝代表Theta (GSTT)类, 青色进化枝代表γ-subunit of translation elongation factor (EF1G)类, 黑色进化枝代表Phi (GSTF)类。"

图2

ScGSTF1与其他物种GSTF蛋白的系统进化树分析 甘蔗: ScGSTF1 (OQ333021); 高粱: SbGSTF1 (XP_002458541.1); 玉米: ZmGSTF2 (ACG25283.1); 柳叶稷: PvGSTF1 (XP_039849196.1); 狗尾草: SvGSTF1 (XP034596925.1); 二穗短柄草: BdGSTF1 (XP_010232378.1); 大麦: HvGSTF1 (XP_044977142.1); 小麦: TaGSTF1 (XP_044350191.1); 烟草NbGSTF1 (XP_016472830.1); 马铃薯: StGSTF1 (KAH0657425.1); 拟南芥: AtGSTF13 (AT3G62760); 大豆: GmGSTF21 (NP_001291446.1); 葡萄: VvGSTF13 (RVW54521.1); 桃树: PpGSTF13 (XP_007209587.1); 毛果杨: PtGSTF13 (XP_006386755.1)。红色框、绿色框和黄色框分别代表亚群I-1、亚群I-2和群II。"

图3

甘蔗ScGSTF1与其他植物GSTF的二级结构 甘蔗: ScGSTF1 (OQ333021); 拟南芥: AtGSTF13 (At3g62760); 杨树: PtGSTF1 (Potri.002G015100.1); 水稻: OsGSTF1 (XP_015642391.1)。灰色箭头为β-折叠, 蓝色螺旋为α-螺旋。"

图4

ScGSTF1-YFP在本氏烟表皮细胞中的定位 ScGSTF1-YFP亚细胞定位。标尺为25 μm。"

图5

Y2H检测ScGSTF1与SCMV-P3N-PIPO的互作 pNubG-Fe65和pTSU2-APP组合作为阳性对照, pNubG-Fe65和pPR3-N组合作为阴性对照。DDO+X-Gal: 添加了5-溴-4-氯-3-吲哚-β-D-半乳糖苷的缺少亮氨酸(Leu)和色氨酸(Trp)的酵母合成限定基本培养基; QDO+X-Gal: 添加了X-Gal的缺少亮氨酸(Leu)、色氨酸(Trp)、组氨酸(His)和腺嘌呤(Ade)的酵母合成限定基本培养基。"

图6

BiFC检测ScGSTF1与SCMV-P3N-PIPO的互作 A: cYFP融合于P3N-PIPO的N末端, nYFP融合于ScGSTF1的C末端。B: cYFP融合于ScGSTF1的N末端, nYFP融合于SCMV-P3N-PIPO的C末端。将P3N-PIPO-YC和ScGSTF1-YN (A)、ScGSTF1-YC和P3N-PIPO-YN (B)分别共注射到本氏烟叶片中进行瞬时表达, 48 h后激光共聚焦观察。比例尺为25 μm。"

图7

ScGSTF1基因的组织特异性表达分析 LR: 心叶; +1 L: +1叶; +7 L: +7叶; +3 I: 第3节间; +8 I: 第8节间; R: 根。误差线为每组处理的标准误差(n = 3)。柱上不同的小写字母表示在P < 0.05时显著性差异。"

图8

ScGSTF1基因应答SCMV侵染的表达模式 误差线为每组处理的标准误差(n = 3)。柱上不同的小写字母表示在P < 0.05时显著性的差异。"

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