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作物学报 ›› 2019, Vol. 45 ›› Issue (10): 1478-1487.doi: 10.3724/SP.J.1006.2019.94002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗NAD(P)H脱氢酶复合体O亚基基因克隆及其与甘蔗花叶病毒VPg互作

翟玉山,赵贺,张海,邓宇晴,程光远,杨宗桃,王彤,彭磊,徐倩,董萌,徐景升()   

  1. 福建农林大学国家甘蔗工程技术研究中心 / 农业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:2018-12-31 接受日期:2019-01-19 出版日期:2019-10-12 网络出版日期:2019-09-10
  • 通讯作者: 徐景升
  • 基金资助:
    本研究由国家自然科学基金项目资助(31371688)

Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of Sugarcane mosaic virus

ZHAI Yu-Shan,ZHAO He,ZHANG Hai,DENG Yu-Qing,CHENG Guang-Yuan,YANG Zong-Tao,WANG Tong,PENG Lei,XU Qian,DONG Meng,XU Jing-Sheng()   

  1. Sugarcane Research & Development Centre, China Agricultural Technology System, Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
  • Received:2018-12-31 Accepted:2019-01-19 Published:2019-10-12 Published online:2019-09-10
  • Contact: Jing-Sheng XU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31371688)

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摘要：

NAD(P)H脱氢酶(NDH)复合体介导循环电子传递, 对于维持叶绿体高效的光合作用具有重要作用。甘蔗(Saccharum spp. hybrid)中NDH复合体应答及参与甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)的侵染尚未见报道。本研究克隆了甘蔗NDH复合体的O亚基, 命名为ScNdhO, 其开放读码框(open reading frame, ORF)长度为471 bp, 编码长度为156 aa的蛋白。生物信息学分析表明, ScNdhO为稳定的亲水性蛋白, 不存在信号肽, 无跨膜结构域; 蛋白二级结构元件多为无规则卷曲, 具有典型的NDH复合体O亚基结构域; 系统进化树分析表明, 该蛋白属于NDH复合体O亚基蛋白家族。实时荧光定量 PCR分析发现, ScNdhO基因的表达具有明显的组织特异性, 在成熟甘蔗叶片中的表达量最高, 在茎中的表达量最低, 在根中几乎不表达; ScNdhO基因在SCMV侵染早期上调表达, 后期下调表达。亚细胞定位分析表明, ScNdhO定位于叶绿体。酵母双杂交(yeast two hybrid, Y2H)和双分子荧光互补(bimolecular fluorescence complementation, BiFC)实验表明, ScNdhO与SCMV-VPg互作。推测ScNdhO被SCMV选择性利用, 可能参与SCMV基因组复制及花叶病症状的产生。

关键词: 甘蔗, SCMV, 叶绿体, NAD(P)H脱氢酶复合体, O亚基

Abstract:

NAD(P)H dehydrogenase (NDH) complex mediates cyclic electron transports, playing key role in efficient photosynthesis in chloroplast. The involvement of NDH complex in Sugarcane mosaic virus (SCMV) infection of sugarcane (Saccharum spp. hybrid) has not been reported. In this study, we isolated the coding sequence of the subunit of the NAD(P)H dehydrogenase complex from sugarcane and designated it as ScNdhO. The open reading frame (ORF) of ScNdhO is 471 bp and encodes a 156 aa length protein. Bioinformatics analysis showed that ScNdhO is a stable hydrophilic protein with no signal peptide and transmembrane domain. The secondary structure of ScNdhO is composed of mostly random coilα-helices, with a typical domain of NDH complex O subunit. Phylogenetic tree analysis showed that ScNdhO belongs to the NDHO supperfamily. Real-time quantitative PCR analysis showed that ScNdhO gene was tissue specific in sugarcane, with the lowest expression level in roots or stem, and the highest in leaf. The expression of ScNdhO was upregulated in the early stage of SCMV infection, but downregulated with time going. Subcellular location assays showed that ScNdhO was located in chloroplast. ScNdhO interacted with the VPg from SCMV as demonstrated by yeast two hybrid and bimolecular fluorescence complementation assays. We proposed that ScNdhO should be selectively employed by SCMV and involved in the mosaic symptom.

Key words: sugarcane, SCMV, chloroplast, NAD(P)H dehydrogenase complex, O subunit

表1

本研究使用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Strategy
ScNdhO-F ACCAAGCCGAGCAATCCCTC 基因克隆
ScNdhO-R CACCGTGTCCTGACCCCAAG Gene cloning
ScNdhO-AD-F GGAATTCCATATGATGGAAGCCCTCGCCTCG 酵母双杂交捕获载体构建
ScNdhO-AD-R CGGAATTCTATCCTCTCGTAGTCGAGCTTG Vector generation for Y2H
ScNdhO-YC-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAAGCCCTCGCCTCG 双分子荧光互补载体构建
ScNdhO-YC-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTATCCTCTCGTAGTCGAGCTTG Vector generation for BiFC
ScNdhO-qF CGATGAAGAAAGGGCAGA 定量PCR
ScNdhO-qR TGTAGAATGGCGGGTGTC Real-time-qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因
GAPDH-R TCCTCAG GGTTCCTGATGCC Reference gene
eEF-1α-F TTTCACACTTGGAGTGAAGCAGAT 内参基因
eEF-1α-R GACTTCCTTCACAATCTCATCATAA Reference gene
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV

图1

ScNdhO蛋白的氨基酸序列同源性分析和系统进化树分析"

图2

ScNdhO-GFP在本氏烟表皮细胞中的定位 ScNdhO-GFP的定位如箭头所示; Up row: GFP对照; Middle row: ScNdhO-GFP定位; Down row: ScNdhO-GFP与VPg-mCherry共定位; bar = 50 μm。"

图3

ScNdhO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)。Leaf roll: 心叶; Leaf: 正一叶; Sheath: 叶鞘; Bud: 腋芽; Stem: 茎; Root: 根。"

图4

ScNdhO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)。"

图5

Y2H检测ScNdhO与SCMV-VPg的互作 pGADT7-T和pGBKT7-53组合作为阳性对照, pGADT7-T和pGBKT7-Lam组合作为阴性对照。SD/-Leu/-Trp: 缺少亮氨酸(Leu)和色氨酸(Trp)的酵母合成限定基本培养基; SD/-Leu/-Trp/-His/-Ade: 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ScNdhO与SCMV-VPg的互作 ScNdhO融合于YFP的C末端, SCMV-VPg融合于YFP的N末端, 然后在本氏烟叶片中瞬时表达, 48 h后激光共聚焦观察。bar = 50 μm。"

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