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作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2472-2484.doi: 10.3724/SP.J.1006.2023.24244

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗VAMP相关蛋白ScPVA12与甘蔗花叶病毒P3N-PIPO的互作研究

玉泉馨1(), 杨宗桃1, 张海1, 程光远1, 周营栓1, 焦文迪1, 曾康1, 罗廷绪1, 黄国强1, 张木清2,*(), 徐景升1,*()   

  1. 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 / 教育部作物遗传育种与综合利用重点实验室, 福建福州 350002
    2广西大学广西甘蔗生物学重点实验室, 广西南宁 530004
  • 收稿日期:2022-10-28 接受日期:2023-02-10 出版日期:2023-09-12 网络出版日期:2023-02-28
  • 通讯作者: *徐景升, E-mail: xujingsheng@126.com; 张木清, E-mail: zmuqing@163.com
  • 作者简介:玉泉馨, E-mail: YuQuanxin_YQX@outlook.com
  • 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 VAMP associated protein ScPVA12 with SCMV P3N-PIPO

YU Quan-Xin1(), YANG Zong-Tao1, ZHANG Hai1, CHENG Guang-Yuan1, ZHOU Ying-Shuan1, JIAO Wen-Di1, ZENG Kang1, LUO Ting-Xu1, HUANG Guo-Qiang1, ZHANG Mu-Qing2,*(), XU Jing-Sheng1,*()   

  1. 1Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / National Engineering Research Center for Sugarcane / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
    2Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, Guangxi, China
  • Received:2022-10-28 Accepted:2023-02-10 Published:2023-09-12 Published online:2023-02-28
  • Supported by:
    National Natural Science Foundation of China(31971991);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2019132G);Open Project Program of Guangxi Key Laboratory of Sugarcane Biology(GXKLSCB-202003)

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摘要：

植物囊泡膜蛋白相关蛋白PVA12 (plant vesicle-associated membrane protein (VAMP)-associated proteins homolog 12)属于VAP27家族蛋白, 在细胞中介导内质网囊泡运输以及膜融合。甘蔗(Saccharum spp. hybrid) PVA12应答甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)侵染尚未见报道。本研究从栽培种新台糖22号(ROC22)中克隆了PVA12基因, 命名为ScPVA12。该基因开放读码框(open reading frame, ORF)的长度为735 bp, 其编码长度为244 aa的蛋白。生物信息学分析表明, ScPVA12是一种不稳定的亲水性脂溶蛋白, C端具有跨膜结构域; 二级结构中无规则卷曲占比最高; 进化树分析表明, 该蛋白在单子叶和双子叶植物中存在明显分化。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, ScPVA12与SCMV-P3N-PIPO蛋白互作。亚细胞定位试验表明ScPVA12定位于内质网。共定位试验表明ScPVA12与SCMV-P3N-PIPO共定位于内质网。实时荧光定量PCR分析发现, ScPVA12基因在甘蔗各组织中均有表达, 在第8节间中的表达量最低, 在正七叶中的表达量最高; SCMV侵染对ScPVA12基因表达影响显著, 在SCMV胁迫下ScPVA12基因先下调表达, 后期恢复正常水平。

关键词: PVA12, 甘蔗花叶病毒, P3N-PIPO, 蛋白互作

Abstract:

PVA12 (plant vesicle-associated membrane protein (VAMP)-associated proteins homolog 12) is a member of the VAP27 family of proteins that mediate endoplasmic reticulum (ER) vesicle transport and membrane fusion in cells. Sugarcane (Saccharum spp. hybrid) PVA12 responding to Sugarcane mosaic virus (SCMV) infection has not been reported. In the present study, the coding gene of PVA12 was cloned from sugarcane cultivar ROC22 and designated as ScPVA12. The open reading frame (ORF) of ScPVA12 was 735 bp in length, which encoded a protein with 244 amino acids. Bioinformatics analysis showed that ScPVA12 was an unstable hydrophilic liposoluble protein with a transmembrane at the C-terminal domain. The ratio of the random coil ranked the highest in the secondary structure. Phylogenetic tree analysis revealed that the ScPVA12 was differentiated in monocotyledons and dicotyledons. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that ScPVA12 interacted with SCMV-P3N-PIPO. Subcellular localization experiments indicated that ScPVA12 was localized in the ER. Co-localization experiments demonstrated that ScPVA12 and SCMV-P3N-PIPO co-localized in the ER. Real-time quantitative PCR analysis showed that ScPVA12 gene was expressed in all sugarcane tissues, with the lowest expression level in the eighth internode and the highest expression level in the 7th leaves. The relative expression level of ScPVA12 gene was significantly affected under the stress of SCMV. ScPVA12 was down-regulated upon SCMV infection and then recovered to the regular level compared with the control.

Key words: PVA12, Sugarcane mosaic virus, 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
ScPVA12-F ATGGCCACCCCCGCCCCTGCCA 克隆
ScPVA12-R TGACTTCATCAGATAGCCCAGGATG Gene cloning
pPPR3-ScPVA12-F ATTAACAAGGCCATTACGGCCATGATGGCCACCCCCGCCCCTGCCA 酵母双杂交载体构建
Construction of yeast two-hybrid vector
pPPR3-ScPVA12-R TTGACTAAGGCCGAGGCGGCCGTTATGACTTCATCAGATAGCCCAG
221-ScPVA12-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGGCCACCCCCGCCCCTGCCA 亚细胞定位及双分子荧光互补载体构建
Construction of yeast subcellular localization and bimolecular fluorescence complementation vector
221-ScPVA12-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTGACTTCATCAGATAGCCCAGGATG
ScPVA12-qF ATGGAAGAGGTCAAGTTGCGAGT 定量PCR
Real-time qPCR
ScPVA12-qR TGAATCCTCCAGCAGGTCCATCTAC
Actin-F CACGGCCACTGGAAGCA 内参基因
Reference gene
Actin-R TCCTCAG GGTTCCTGATGCC
eEF-1α-F TTTCACACTTGGAGTGAAGCAGAT 内参基因
Reference gene
eEF-1α-R GACTTCCTTCACAATCTCATCATAA
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
Detection of SCMV
SCMV-CP-R ACGCTACACCAGAAGACACT

图1

ScPVA12蛋白结构域分析 A: ScPVA12与AtVAP27-3氨基酸比对结果(红色标记为MSD, 黄色标记为CCD, 蓝色标记为TMD)。B: ScPVA12与AtVAP27-3结构域示意图(标尺为250个氨基酸残基)。"

图2

甘蔗ScPVA12与其他单子叶植物PVA12蛋白的氨基酸序列比对 高粱: SbPVA12 (XP_002445075.1); 南荻: MlPVA12 (CAD6266147.1); 玉米: ZmPVA12 (NP_001130606.1); 黍: PmPVA12(RLN03983.1); 谷子: SiPVA12 (XP_004972806.1); 二穗短柄草: BdPVA12 (XP_003573476.1); 野生二粒小麦: TdPVA12 (XP_037458000.1)。"

图3

ScPVA12与其他物种PVA12蛋白的系统进化树分析 群I为单子叶植物群, 其中亚群I-1为C4植物, 亚群群I-2为C3植物; 群II为双子叶植物群。"

图4

ScPVA12-YFP在本氏烟表皮细胞中的定位 A: ScPVA12-YFP亚细胞定位; B: ScPVA12-YFP和SCMV-P3N-PIPO-CFP亚细胞共定位。将3种荧光蛋白信号进行两两组合并使用伪彩进行观察共定位情况, 组合分别为ScPVA12-YFP (绿色伪彩)+HDEL-RFP (红色)、ScPVA12-YFP (红色伪彩)+P3N-PIPO-CFP (绿色伪彩)以及HDEL-RFP (红色)+P3N-PIPO-CFP (绿色伪彩)。标尺为25 μm。白色箭头标记共定位位置。"

图5

Y2H检测ScPVA12与SCMV-P3N-PIPO的互作 pTSU2-APP/pNubG-Fe65为阳性对照, pPR3-N/pNubG-Fe65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ScPVA12与SCMV-P3N-PIPO的互作 A: YN融合于ScPVA12的N末端, YC融合于SCMV-P3N-PIPO的C末端; B: YC融合于ScPVA12的C末端, YN融合于SCMV-P3N-PIPO的N末端。将YN-ScPVA12和P3N-PIPO-YC (A)、YN-P3N-PIPO和ScPVA12-YC (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。标尺为25 μm。白色箭头标记呈点状结构的荧光信号。"

图7

ScPVA12基因在甘蔗不同组织中的表达模式 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差异显著。"

图8

ScPVA12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差异显著。"

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