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作物学报 ›› 2021, Vol. 47 ›› Issue (8): 1522-1530.doi: 10.3724/SP.J.1006.2021.04194

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗PsbR亚基应答SCMV侵染及其与SCMV-6K2的互作

张海(), 程光远, 杨宗桃, 刘淑娴, 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:2020-08-25 接受日期:2020-11-13 出版日期:2021-08-12 网络出版日期:2020-12-22
  • 通讯作者: 徐景升
  • 作者简介:E-mail: zhanghai940410@163.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(CXZX2018026);福建省科技厅引导性项目(2017N0003)

Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2

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

  1. National Engineering Research Center for Sugarcane/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/Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2020-08-25 Accepted:2020-11-13 Published:2021-08-12 Published online:2020-12-22
  • Contact: XU Jing-Sheng
  • Supported by:
    National Natural Science Foundation of China(31971991);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：

光系统II (Photosystem II, PSII)的PsbR亚基对于放氧复合体(oxygen-evolving complex)的组装和稳定具有至关重要的作用。本课题组前期克隆了甘蔗(Saccharum spp. hybrid)的PsbR亚基编码基因, 命名为ScPsbR, 并利用酵母双杂交技术(yeast two hybrid, Y2H)验证了ScPsbR与甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)编码蛋白6K2的互作。本研究通过生物信息学分析表明, ScPsbR具有典型的PsbR亚基结构域, 无信号肽, 具有1个跨膜结构域, 为稳定的疏水性蛋白。系统进化树分析表明, 该蛋白在C3和C4植物中存在明显的分化。亚细胞定位试验表明, ScPsbR定位于叶绿体且与SCMV-6K2共定位。双分子荧光互补(bimolecular fluorescence complementation, BiFC)试验进一步验证了ScPsbR与SCMV-6K2的互作。实时荧光定量PCR检测表明, ScPsbR基因表达具有显著的组织特异性, 在根和茎中表达极少, 未成熟叶片和初衰叶中次之, 成熟叶片中相对表达量最高; SCMV侵染显著影响ScPsbR基因表达, ScPsbR基因在侵染0~12 h显著上调, 侵染1~5 d下调至略低于对照的水平, 但差异不显著, 侵染7~15 d显著下调。

关键词: PsbR亚基, 甘蔗花叶病毒, 6K2, 蛋白互作

Abstract:

The PsbR subunit of photosystem II (PSII) plays a vital role in the assembly and stability of the oxygen-evolving complex. In the previous study, we cloned the coding sequence of the PsbR subunit from sugarcane (Saccharum spp. hybrid) and designated it as ScPsbR. The interaction between ScPsbR and 6K2 protein encoded by Sugarcane mosaic virus (SCMV) was verified by yeast two-hybrid technology. In this study, bioinformatics analysis indicated ScPsbR protein was found to possess a canonical subunit domain of PsbR and a transmembrane domain without signal peptide, and be a stable hydrophobic protein. Phylogenetic tree analysis indicated obvious divergence between C3 and C4 plants for the PsbRs. Subcellular localization experiments suggested that ScPsbR was localized and co-localized with SCMV-6K2 to the chloroplast. The interaction of ScPsbR with the SCMV-6K2 was further verified by bimolecular fluorescence complementation assays. Real-time quantitative PCR results indicated that ScPsbR gene was tissue-specific in sugarcane plants. There was almost no expression of ScPsbR gene in the roots or stems, with increased expression level in the senescing leaves and immature leaves, and the highest expression level in mature leaves. However, the expression level of ScPsbR was significantly changed under the challenged of SCMV. During the infection of SCMV, ScPsbR was significantly up-regulated at 0-12 hour(s) and reduced to a level slightly lower than that of the control at 1-5 days with no significant differences, then significantly down-regulated in 7-15 days.

Key words: PsbR subunit, Sugarcane mosaic virus, 6K2, 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
221-ScPsbR-F
221-ScPsbR-R		 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCCTCCATGATCTC
GGGGACCACTTTGTACAAGAAAGCTGGGTCGCCGGAGAGGGCGCTGGTGT		 双分子荧光互补载体构建
Vector generation for BiFC
ScPsbR-qF
ScPsbR-qR		 TCCATGATCTCGTCGTCAGCTCTG
ACGGTCAGGCCACCTCCAATC		 定量PCR
Real-time-qPCR
GAPDH-F
GAPDH-R		 CACGGCCACTGGAAGCA
TCCTCAG GGTTCCTGATGCC		 内参基因
Reference gene
eEF-1α-F
eEF-1α-R		 TTTCACACTTGGAGTGAAGCAGAT
GACTTCCTTCACAATCTCATCATAA		 内参基因
Reference gene
SCMV-CP-F
SCMV-CP-R		 TACAGAGAGACACACAGCTG
ACGCTACACCAGAAGACACT		 SCMV检测
Detection of SCMV

图1

ScPsbR基因的核苷酸序列及其编码的氨基酸序列 *: 终止密码子。黑色下画线部分为转运肽。红色下画线部分为跨膜结构域。转运肽和成熟蛋白之间的边界用箭头标出。"

图 2

甘蔗ScPsbR与其他单子叶植物PsbR蛋白的氨基酸序列比对 SbPsbR (XP_002444002.1): 高粱; ZmPsbR (XP_020408082.1): 玉米; PhPsbR (XP_025821160.1): 黍; SiPsbR (XP_004972630.1): 谷子; BdPsbR (XP_010234446.1): 二穗短柄草; OsPsbR (AAB46718.1): 水稻。"

图3

ScPsbR与其他物种PsbR蛋白的系统进化树分析"

图4

ScPsR-YFP在本氏烟表皮细胞中的定位 A: ScPsbR-YFP亚细胞定位; B: ScPsbR-YFP和6K2-CFP亚细胞共定位。白色箭头表示叶绿体, 标尺为25 μm。"

图5

BiFC检测ScPsbR与SCMV-6K2的互作 A: YC融合于ScPsbR的C末端, YN融合于SCMV-6K2的N末端; B:YN融合于ScPsbR的N末端, YC融合于SCMV-6K2的C末端。将YN-6K2和ScPsbR-YC(A), YN-ScPsbR和6K2-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。白色箭头表示具有互作荧光信号的叶绿体。标尺为25 μm。"

图6

ScPsbR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)。LR: 心叶; +1 L: 正一叶; +7 L: 正七叶; +3 I: 第三节间; +8 I: 第八节间; R: 根。柱上不同小写字母表示在5%水平差异显著。"

图7

ScPsbR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)。柱上不同小写字母表示在5%水平差异显著。"

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