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作物学报 ›› 2024, Vol. 50 ›› Issue (7): 1855-1866.doi: 10.3724/SP.J.1006.2024.34191

• 研究简报 • 上一篇    下一篇

甘蔗类钙调素ScCML13与SCMV运动蛋白P3N-PIPO的互作研究

玉泉馨(), 杨宗桃, 张海, 程光远, 焦文迪, 曾康, 罗廷绪, 黄国强, 王璐, 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 / 教育部作物遗传育种与综合利用重点实验室, 福建福州 350002
  • 收稿日期:2023-11-14 接受日期:2024-01-31 出版日期:2024-07-12 网络出版日期:2024-02-23
  • 通讯作者: *徐景升, E-mail: xujingsheng@126.com
  • 作者简介: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)

Interaction between calmodulin-like ScCML13 of sugarcane and SCMV movement protein P3N-PIPO

YU Quan-Xin(), YANG Zong-Tao, ZHANG Hai, CHENG Guang-Yuan, JIAO Wen-Di, ZENG Kang, LUO Ting-Xu, HUANG Guo-Qiang, WANG Lu, XU Jing-Sheng*()   

  1. Fujian 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
  • Received:2023-11-14 Accepted:2024-01-31 Published:2024-07-12 Published online:2024-02-23
  • 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)

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摘要：

类钙调素(Calmodulin-like, CML)是植物特有的钙信号感受器蛋白之一, 参与植物生长发育和响应外界环境信号转导。甘蔗(Saccharum spp. hybrid)中CML应答甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)侵染尚未见报道。本研究从甘蔗热带种Badila (S. officinarum)中克隆了一个CML基因, 命名为ScCML13。该基因开放读码框(open reading frame, ORF)长度为519 bp, 编码172 aa。生物信息学分析表明, ScCML13属于稳定的亲水脂溶蛋白, 无跨膜结构域, 有4个结合Ca2+的EF-hand结构域; 系统进化树分析表明, 该蛋白在单子叶和双子叶植物中及单子叶C3和C4植物中具有明显分化; 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, ScCML13与SCMV的运动蛋白P3N-PIPO互作。亚细胞定位试验表明ScCML13定位于内质网和细胞核。共定位试验发现ScCML13会干扰SCMV-P3N-PIPO的质膜(plasma membrane)或胞间连丝(plasmodesmata)定位。实时荧光定量PCR (Real Time Quantitative PCR, RT-qPCR)分析发现, ScCML13基因主要在甘蔗叶片中表达, 在节间和根中的相对表达量低; ScCML13基因在感染SCMV后2 h显著上调, 随后下调至与对照组相比的水平, 而在感染后期上调。

关键词: 类钙调素, 甘蔗花叶病毒, P3N-PIPO, 蛋白互作

Abstract:

Calmodulin-like (CML), one of the calcium signal receptor proteins unique to plants, is involved in plant growth and development and in response to environmental signal transduction. However, the response of CML to Sugarcane mosaic virus (SCMV) infection in sugarcane (Saccharum spp. hybrid) has not been reported. In the present study, a CML gene was cloned from a Badila (S. officinarum) and designated as ScCML13. The open reading frame (ORF) of ScCML13 gene is 519 bp in length and encodes a protein with 172 aa in length. Bioinformatics analysis showed that ScCML13 was a stable hydrophilic lipoprotein in with no transmembrane domain and contains 4 Ca2+-binding EF-hand domains. Phylogenetic tree analysis showed that the ScCML13 protein differentiated in monocotyledon and dicotyledon plants and in monocotyledonous C3 and C4 plants. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments indicated that ScCML13 interacted with P3N-PIPO, the movement protein of SCMV. Subcellular localization assays demonstrated that ScCML13 was localized to the endoplasmic reticulum and nucleus, whereas the co-localization assays showed that ScCML13 interfered with the localization of SCMV-P3N-PIPO to plasma membrane or plasmodesmata. The RT-qPCR showed that ScCML13 gene was mainly expressed in sugarcane leaves, but relatively low in internodes and roots. The ScCML13 gene was significantly up-regulated at 2 h upon SCMV infection, and then down-regulated to the levels compared with the control group, while up-regulated at the later stage of infection.

Key words: calmodulin-like protein, Sugarcane mosaic virus, P3N-PIPO, protein interaction

表1

本研究所使用引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Strategy
ScCML13-F ATGAGTTTCAACCAGTCTACTGTCAAG 基因克隆
ScCML13-R CTAGTAGCCATAGCTAGTCCTCCT Gene cloning
221-ScCML13-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGTTTCAACCAGTCTAC 亚细胞定位及双分子荧光互补载体构建
Vectors generation for subcellular localization and BiFC
221-ScCML13-R GGGGACCACTTTGTACAAGAAAGCTGGGTCGTAGCCATAGCTAGTCCTCC
pPR3-ScCML13-F GCAGAGTGGCCATTACGATGAGTTTCAACCAGTCTACT 酵母双杂交载体构建
pPR3-ScCML13-R ATTCTCGAGAGGCCGCTAGTAGCCATAGCTAGTCCT Vector generation for Y2H
ScCML13-qF TCGAGGAGTTTGAGCACATGATGAC 定量PCR
ScCML13-qR TTACACCCAGCTCTTTGGCAATCC Real-time-qPCR
Actin-qF CTGGAATGGTCAAGGCTGGT 内参基因[64]
Actin-qR TCCTTCTGTCCCATCCCTACC Reference gene[64]
eEF-1α-qF TTCACACTTGGAGTGAAGCAGAT 内参基因[65]
eEF-1α-qR GACTTCCTTCACAATCTCATCATAA Reference gene[65]
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测[66]
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV[66]

图1

单子叶物种CML13同源比对及ScCML13蛋白结构域分析 ScCML13: 甘蔗(OR575430); SbCML13: 高粱(XP_021307808.1); LrCML13: 硬直黑麦草(XP_047066070.1); BdCML13: 二穗短柄草(XP_003562666.1); OsCML13: 水稻(NP_001409202.1)。ScCML13含有4个典型的EF-hand结构域, 分别为EF-hand I (31~59)、EF-hand II (67~95)、EF-hand III (104~132)和EF-hand IV (140~168), 所有EF-hand的长度均为29 aa。"

图2

ScCML13与其他物种CML蛋白的系统进化树分析 ScCML13: 甘蔗(OR575430)。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

Y2H检测ScCML13与SCMV-P3N-PIPO蛋白互作 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4

BiFC检测ScCML13与SCMV-P3N-PIPO蛋白互作 A: cYFP融合于SCMV-P3N-PIPO的N末端, nYFP融合于ScCML13的C末端; B: cYFP融合于ScCML13的N末端, nYFP融合于SCMV-P3NPIPO的C末端。标尺为25 μm。"

图5

ScCML13在本氏烟表皮细胞中的定位及其与SCMV-P3N-PIPO共定位 A: ScCML13在本氏烟表皮细胞中的定位; B: SCMV-P3N-PIPO单独定位, 白色箭头指向PD; C: ScCML13与SCMV-P3N-PIPO共定位。ScCML13-YFP使用绿色伪彩标识, SCMV-P3N-PIPO-CFP使用紫色伪彩标识, HDEL-mCherry为内质网标记, H2B-mCherry为细胞核标记。白色箭头指向与单独定位时具有明显差异的SCMV-P3N-PIPO-CFP荧光信号。标尺为25 μm。"

图6

ScCML13基因在甘蔗中的组织特异性表达分析及其应答SCMV侵染的表达模式 A: ScCML13基因在甘蔗中的组织特异性表达分析; B: ScCML13基因应答SCMV侵染的表达模式。t测验检测差异显著性, 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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