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作物学报 ›› 2022, Vol. 48 ›› Issue (2): 332-341.doi: 10.3724/SP.J.1006.2022.14001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗类泛素蛋白UBL5应答SCMV侵染及其与SCMV-6K2的互作

杨宗桃(), 刘淑娴, 程光远, 张海, 周营栓, 商贺阳, 黄国强, 徐景升*()   

  1. 福建农林大学国家甘蔗工程技术研究中心 / 农业农村部福建甘蔗生物学与遗传育种重点实验室 / 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:2021-01-05 接受日期:2021-04-14 出版日期:2022-02-12 网络出版日期:2021-06-16
  • 通讯作者: 徐景升
  • 作者简介:E-mail: fafuyangzongtao@163.com
  • 基金资助:
    本研究由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(CXZX2018026);福建省科技厅引导性项目资助(2017N0003)

Sugarcane ubiquitin-like protein UBL5 responses to SCMV infection and interacts with SCMV-6K2

YANG Zong-Tao(), LIU Shu-Xian, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng*()   

  1. Sugarcane Research & Development Center, China Agricultural Technology System, Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
  • Received:2021-01-05 Accepted:2021-04-14 Published:2022-02-12 Published online:2021-06-16
  • Contact: XU Jing-Sheng
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31971991);the Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2018026);Guiding Project of Science and Technology Department of Fujian Province(2017N0003)

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摘要：

泛素化修饰在蛋白功能调控、生长发育和逆境应答中具有重要作用。类泛素蛋白(ubiquitin-like proteins, UBLs)是泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)的重要组分。本课题组前期利用酵母双杂交(yeast two-hybrid, Y2H)技术从甘蔗(Saccharum spp. hybrid)中分离鉴定了1个与甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)编码蛋白6K2互作的类泛素蛋白UBL5, 命名为ScUBL5, 长度为73 aa。本研究利用双分子荧光互补(bimolecular fluorescence complementation, BiFC)试验进一步证实了ScUBL5与SCMV-6K2互作; 生物信息学分析表明, ScUBL5为稳定的亲水性非分泌蛋白, 无信号肽和跨膜结构。系统进化树分析表明, ScUBL5具有明显的种属特异性。亚细胞定位分析表明, ScUBL5定位于细胞质和细胞核。实时荧光定量PCR分析发现, ScUBL5基因的表达具有明显的组织特异性, 在完成形态建成的正一叶、第7叶、根和第8节间中的相对表达量显著高于未成熟的心叶和第3节间; SCMV侵染对ScUBL5基因表达影响显著, ScUBL5基因在侵染早期显著上调, 侵染后期下调但显著高于对照。

关键词: 甘蔗, 类泛素蛋白, 甘蔗花叶病毒, 6K2

Abstract:

Ubiquitylation plays key roles in the regulation of protein function, growth and development, and response to stress. Ubiquitin-like proteins (UBLs) are the main components of the ubiquitin-proteasome system (UPS). In our previous study, the UBL5 homologue was isolated from sugarcane (Saccharum spp. hybrid) by yeast two-hybrid (Y2H) with the 6K2 of Sugarcane mosaic virus (SCMV) as bait, and then designated as ScUBL5 with 73 aa in length. In the present study, the interaction of ScUBL5 with the SCMV-6K2 was further confirmed by bimolecular fluorescence complementation assays (BiFC). Bioinformatics analysis showed that ScUBL5 is a stable hydrophilic non-secretory protein without signal peptide or transmembrane domain. Phylogenetic tree analysis showed that ScUBL5 is species specific. Subcellular localization analysis showed that ScUBL5 is localized in cytoplasm and nucleus. ScUBL5 gene shows obvious tissue specificity in sugarcane by real-time quantitative PCR analysis. The expression levels of ScUBL5 gene in the established morphogenesis tissues such as the 1st leaf, the 7th leaf, the 8th internode and the root were significantly higher than those in the immature tissues such as leaf roll and the 3rd internode. The expression of ScUBL5 gene is significantly affected by SCMV infection. ScUBL5 was significantly upregulated in the early stage of SCMV infection, then downregulated but significantly higher than the control at the late stage of SCMV infection.

Key words: sugarcane, ubiquitin-like protein, Sugarcane mosaic virus, 6K2

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
221-ScUBL5-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCAGTCCATGCT 双分子荧光互补载体构建
Vector generation for BiFC
221-ScUBL5-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTCTTCGTCCTCGC
ScUBL5-qF CTCCATCATCGGCGAGATCATCAC 定量PCR
Real-time-qPCR
ScUBL5-qR GGCGGCGACGAAGAAGAAGAG
GAPDH-F CACGGCCACTGGAAGCA 内参基因
Reference gene
GAPDH-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

基于SWISS-MODEL的ScUBL5蛋白三维建模 ScUBL5 (MN167908): 甘蔗的UBL5; SbUBL5 (XP_002448973.1): 高粱的UBL5; OsUBL5 (XP_015619383.1): 水稻的UBL5; AtUBL5 (NP_176030.1): 拟南芥的UBL5。"

图2

ScUBL5的氨基酸序列 水稻: OsUBL5 (XP_015619383.1); 甘蔗: ScUBL5 (QHD26889.1); 二穗短柄草: BdUBL5 (XP_003578938.1); 高粱: SbUBL5 (XP_002448973.1); 拟南芥: AtUBL5 (NP_199045.1); 烟草: NtUBL5 (XP_016510251.1); 小麦: TaUBL5 (AJA91631.1)。"

图3

不同物种UBL5蛋白的系统进化分析"

图4

ScUBL5-YFP在本氏烟表皮细胞中的定位 标尺为25 μm。"

图5

BiFC检测ScUBL5与SCMV-6K2的互作 A: YC融合于ScUBL5的C末端, YN融合于SCMV-6K2的N末端; B: YN融合于ScUBL5的N末端, YC融合于SCMV-6K2的C末端。将6K2-YN和ScUBL5-YC (A), 6K2-YC和ScUBL5-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。标尺为25 μm。"

图6

ScUBL5基因在甘蔗不同组织中的表达模式 LR: 心叶; +1 L: 正一叶; +7 L: 正七叶; +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时差异显著。"

图7

ScUBL5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时差异显著。"

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