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作物学报 ›› 2020, Vol. 46 ›› Issue (11): 1722-1733.doi: 10.3724/SP.J.1006.2020.04030

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗PsbS亚基应答甘蔗花叶病毒侵染及其与6K2蛋白的互作研究

张海(), 刘淑娴, 杨宗桃, 王彤, 程光远, 商贺阳, 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:2020-02-10 接受日期:2020-04-15 出版日期:2020-11-12 网络出版日期:2020-04-27
  • 通讯作者: 徐景升
  • 作者简介:E-mail:zhanghai940410@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)

Sugarcane PsbS subunit response to Sugarcane mosaic virus infection and its interaction with 6K2 protein

ZHANG Hai(), LIU Shu-Xian, YANG Zong-Tao, WANG Tong, CHENG Guang-Yuan, SHANG He-Yang, 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:2020-02-10 Accepted:2020-04-15 Published:2020-11-12 Published online:2020-04-27
  • Contact: Jing-Sheng XU
  • 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)

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摘要：

非光化学猝灭(non-photochemical quenching, NPQ)是高等植物主要的光保护调节机制, 光合系统II (Photosystem II, PSII)的PsbS亚基在NPQ起关键作用。甘蔗(Saccharum spp. hybrid)中PSII PsbS亚基应答甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)的侵染尚未见报道。本课题组在前期研究中克隆了甘蔗的PsbS亚基编码基因, 命名为ScPsbS, 该基因开放读码框(open reading frame, ORF)长度为798 bp, 编码长度为265个氨基酸的蛋白。生物信息学分析表明, ScPsbS为稳定的疏水性蛋白, 具有叶绿体定位信号, 有4个跨膜结构域; 具有典型的PsbS亚基结构域。系统进化树分析表明, ScPsbS在单子叶、双子叶以及单子叶的C3和C4植物中存在明显的分化。亚细胞定位分析表明, ScPsbS定位于叶绿体且与SCMV-6K2部分共定位于叶绿体。双分子荧光互补(bimolecular fluorescence complementation, BiFC)试验进一步证实了ScPsbS与SCMV-6K2互作。实时荧光定量PCR分析发现, ScPsbS基因的表达具有明显的组织特异性, 在成熟叶片中相对表达量最高, 未成熟叶片和初衰叶中次之, 茎和根中几乎不表达; SCMV侵染对ScPsbS基因表达影响显著, ScPsbS基因在侵染早期显著上调, 侵染后期没有明显的差异。

关键词: 甘蔗, PsbS亚基, 甘蔗花叶病毒, 6K2, 光保护

Abstract:

Non-photochemical quenching (NPQ) is the main mechanism of photoprotective regulation in higher plants. The PsbS subunit of Photosystem II (PSII) plays a key role in NPQ. The involvement of PSII PsbS subunit in Sugarcane mosaic virus (SCMV) infection of sugarcane (Saccharum spp. hybrid) has not been reported. In the previous research, we cloned the coding sequence of the PsbS subunit from sugarcane and designated it as ScPsbS. ScPsbS had an open reading frame (ORF) length of 798 bp and encoded a protein of 265 aa. Bioinformatics analysis showed that ScPsbS was a stable hydrophobic protein with chloroplast localization signals and four transmembrane domains. The ScPsbS protein possesses a typical domain of PsbS protein. Phylogenetic tree analysis showed that ScPsbS was divergent between monocotyledons and dicotyledons, or C3 plants and C4 plants. Subcellular localization analysis showed that ScPsbS was located in chloroplasts and partially colocalized with SCMV-6k2 in chloroplasts. The interaction of ScPsbS with the SCMV-6K2 was further confirmed by bimolecular fluorescence complementation assays (BiFC). ScPsbS gene showed obvious tissue specificity in sugarcane tested by real-time quantitative PCR analysis. ScPsbS gene had highest expression in mature leaves, followed by immature leaves and leaves beginning to senesce, and hardly expressed in stems and roots. The expression of ScPsbS gene was significantly affected by SCMV infection, with significant upregulation in the early stage of SCMV infection, and no significant affection in the later stage of SCMV infection.

Key words: sugarcane, PsbS subunit, Sugarcane mosaic virus, 6K2, light protection

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-ScPsbS-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCAGTCCATGCT 双分子荧光互补载体构建
221-ScPsbS-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTCTTCGTCCTCGC Vector generation for BiFC
ScPsbS-qF CTCCATCATCGGCGAGATCATCAC 定量PCR
ScPsbS-qR GGCGGCGACGAAGAAGAAGAG Real-time-qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因
GAPDH-R
eEF-1α-F
eEF-1α-R		 TCCTCAG GGTTCCTGATGCC
TTTCACACTTGGAGTGAAGCAGAT
GACTTCCTTCACAATCTCATCATAA		 Reference gene
内参基因
Reference gene
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV

图1

基于SWISS-MODEL的ScPsbS、ZmPsbS、OsPsbS和AtPsbS蛋白三维建模 ZmPsbS (NP_001105228.2): 玉米的PsbS亚基; OsPsbS (XP_ 015621169.1): 水稻的PsbS亚基; AtPsbS (NP_973971.1): 拟南芥的PsbS亚基。"

图2

ScPsbS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PsbS结构域; 红色标记的氨基酸为靶向叶绿体前体蛋白切割位点的保守结构域。↓: 切割位点。"

图3

甘蔗ScPsbS与其他单子叶植物PsbS蛋白的氨基酸序列比对 高粱: SbPsbS (XP_002456704.1); 玉米: ZmPsbS (NP_ 001105228.2); 黍: PhPsbS (XP_025818476.1); 谷子: SiPsbS (XP_ 004970722.1); 水稻: OsPsbS (XP_015621169.1); 二穗短柄草: BdPsbS (XP_003564708.1); 小麦: TaPsbS (CDM85166.1)。"

图4

ScPsbS与其他物种PsbS蛋白的系统进化树分析"

图5

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

图6

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

图7

ScPsbS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。"

图8

ScPsbS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)。"

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