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^,*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

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^,*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

通讯作者: * 徐景升, E-mail: xujingsheng@126.com

第一联系人: E-mail: yushanzhai@126.com
收稿日期:2018-12-31接受日期:2019-01-19网络出版日期:2019-03-08

基金资助:

本研究由国家自然科学基金项目资助.31371688

Received:2018-12-31Accepted:2019-01-19Online:2019-03-08

Fund supported:

This study was supported by the National Natural Science Foundation of China.31371688

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.
Keywords：sugarcane;SCMV;chloroplast;NAD(P)H dehydrogenase complex;O subunit


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本文引用格式
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. 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[J]. 作物学报, 2019, 45(10): 1478-1487. doi:10.3724/SP.J.1006.2019.94002
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. Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of Sugarcane mosaic virus[J]. Acta Agronomica Sinica, 2019, 45(10): 1478-1487. doi:10.3724/SP.J.1006.2019.94002


甘蔗(Saccharum spp. hybrid)是我国乃至全世界最重要的糖料作物和能源作物之一, 甘蔗糖占我国食糖总量的92％, 占世界食糖总量的74%, 甘蔗乙醇占世界生物质燃料乙醇的60%^[1,2]。甘蔗花叶病是严重危害甘蔗生产的病毒性病害之一, 在全世界蔗区包括我国甘蔗主产区广西、云南、广东、海南等地普遍发生^{[3,4,5,6,7,8,9]}。甘蔗感染花叶病后, 叶片出现黄绿相间、大小不一的条纹或斑驳, 植株矮小, 分蘖减少, 蔗糖结晶率下降, 甘蔗产量损失3%~50%, 蔗糖损失6%~14% ^[6,9-11]。甘蔗花叶病的病原主要为甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)、高粱花叶病毒(Sorghum mosaic virus, SrMV)和甘蔗条纹花叶病毒(Sugarcane streak mosaic virus, SCSMV), 均属于马铃薯Y病毒科(Potyviridae)^[6,8,12-19], 其中SCMV和SrMV属于马铃薯Y病毒属(Potyvirus)^[20,21], SCSMV是新发现的病原, 2012年国际病毒分类委员会(International Committee on Taxonomy of Viruses, ICTV)将其立为该科新属, 即禾病毒属(Poacevirus)^[9,22-23]。SCMV、SrMV和SCSMV都是单链正义RNA病毒, 长度约为10 kb, 编码2个多聚蛋白, 经自身编码的P1、HC-Pro和NIa酶解后形成11个功能成熟的蛋白, 分别为P1、HC-Pro、P3、P3N-PIPO、6K1、CI、6K2、VPg、NIa、NIb和CP^{[12-17,22-24]}。此类病毒结构简单, 其基因组5′端没有帽子结构(m7GpppN, N为任意核酸), 病毒要完成复制, 必须通过其编码的VPg与真核翻译起始因子eIF4E共价连接, 才能启动病毒RNA翻译^[25,26]。VPg是马铃薯Y病毒科病毒的多功能蛋白, 除了参与病毒的翻译, 还参与病毒在寄主体内的复制、胞间移动、长距离运输及自噬等过程^[25,27-28]。

叶绿体中的光合系统由光合系统I (PSI)和光合系统II (PSII)组成。PSII反应中心被光能激发后, 裂解水分子释放氧气, 将来自水分子的电子经过PSII、质醌(PQ)、细胞色素b6/f复合体、质蓝素、PSI、铁氧还蛋白线性传递, 最终使NADP⁺还原, 并偶联产生跨类囊体膜质子梯度(ΔpH), 驱动ATP合成酶合成ATP^[29]。除了这条主要的线性光合电子传递链, 叶绿体中还有一条与线粒体复合体I介导的呼吸电子传递链类似的由NAD(P)H脱氢酶(NDH)复合体和质子末端氧化酶共同参与的围绕PSI循环电子途径, 循环电子途径只产生ATP, 不积累NADPH^{[30,31,32,33,34,35,36,37]}。叶绿体NDH复合体位于类囊体膜上^[38,39,40], 在调节ATP合成促进碳同化产物形成、防止光系统过还原、减少活性氧、光保护抗逆等方面具有重要的生物学功能^{[41,42,43,44,45,46,47]}。叶绿体NDH复合体至少有29个亚基, 构成NDH的5个亚复合体^[25,48-56]。NDH复合体O亚基属于亚复合体A成员, 对于维持NDH复合体的稳定和正常功能具有重要作用^[53,57]。

叶绿体是绿色植物光合作用的场所, 同时也是马铃薯Y病毒的复制场所, 马铃薯Y病毒的6K2诱导内质网形成囊泡, 移动到叶绿体形成叶绿体-6K2复合物, 为病毒基因组复制提供场所^[58]。在前期研究工作中, 我们以SCMV编码蛋白VPg为诱饵, 利用酵母双杂交(yeast two hybrid, Y2H)技术从甘蔗Badila叶片的cDNA酵母文库^[59]中筛选到了一个具有完整开放读码框(open reading frame, ORF)的基因; 该基因的ORF为471 bp, 编码长度为156 aa的蛋白。根据Blastp同源比对分析推测该基因编码叶绿体NDH复合体的O亚基, 命名为ScNdhO。本研究克隆了该基因, 并进行了生物信息学、组织特异性与应答SCMV侵染的表达模式分析, 利用Y2H和BiFC研究了ScNdhO与SCMV-VPg的互作关系, 并对其在SCMV侵染中的作用做了探讨, 以期为甘蔗抗花叶病育种提供基础数据和实验依据。

1 材料与方法

1.1 材料及处理方法

SCMV-FZ1病毒株系、甘蔗品种Badila和本氏烟由福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室提供。采用腋芽快繁技术培养Badila组培苗。组培苗长至15~25 cm、完全展开叶出现4~5片时, 摩擦接种SCMV, 设置3个重复, 每个重复3株, 对照植株使用磷酸缓冲液(pH 7.0)摩擦接种, 使用SCMV-CP基因特异引物(表1)检测接种是否成功。分别在接种后0 h、4 h、8 h、12 h、1 d、2 d、5 d、7 d取样, 研究目的基因应答SCMV侵染的表达模式。组培苗分蘖期时随机选取9株长势一致的植株, 分成3组, 每组3株。取其心叶、+1叶(甘蔗植株由上到下第一个有可见肥厚带的叶片)、+1叶的叶鞘、侧芽、蔗茎和白色嫩根, 用于研究目的基因的空间表达特性。取样后立即用液氮速冻, 置-80℃冰箱保存备用。

Table 1
表1
表1本研究使用的引物
Table 1Primers used in this study

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

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1.2 总RNA提取和cDNA合成

采用TRIzol (Invotrigen, USA)试剂按照说明书提取样品总RNA, 使用1.0%琼脂糖凝胶电泳检测RNA质量。使用Nanodrop (Thermo Scientific, USA)测定RNA的浓度, 按照Prime Script RT Reagent Kit使用说明书, 将RNA反转录成cDNA。

1.3 ScNdhO基因的克隆及生物信息学分析

挑取阳性酵母克隆测序, 获得目的基因的序列。利用NCBI的ORF finder (https://www.ncbi.nlm. nih.gov/orffinder/)软件, 对所获的基因序列进行开放阅读框分析。根据序列信息设计特异PCR引物, 以Badila叶片的cDNA模板, 扩增该基因的ORF并测序验证。然后将该基因克隆至pMD19-T载体, 转化E. coli DH5α 感受态细胞。

利用ProtParam (http://expasy.org/tools/protparam. html)预测蛋白的一级结构、理化性质; 利用Prabi (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html)、SignalP 4.1 (http://www. cbs.dtu.dk/services/SignalP/)和TMHMM 2.0 (http:// www.cbs.dtu.dk/services/TMHMM/)分别预测分析其二级结构、信号肽和跨膜特性; 通过NCBI 中的CDD (conserved domain database)数据库(https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)预测蛋白保守结构域; 用 Blastp在线工具查找ScNdhO的同源氨基酸序列, 并使用DNAMAN7.0软件多重比对同源氨基酸序列, 使用ClustalX和MEGA 6.0的ML (maximum likelihood, LG+G)法构建系统进化树。

1.4 ScNdhO的RT-qPCR表达分析

根据ScNdhO基因的ORF序列, 设计特异性实时荧光定量PCR引物, 以供试材料的cDNA为模板, 以GAPDH基因和eEF-1α基因(表1)为内参基因^[60], 按照SYBR Green PCR Master Mix Kit (Roche)说明书配制反应体系。实时荧光定量PCR扩增程序为50℃ 2 min; 95℃ 10 min; 95℃ 15 s、60℃ 1 min, 循环40次。设置每个样品3次重复, 以无菌水作对照, 采用2^-ΔΔCt算法进行基因表达水平分析, 使用统计软件SPSS 12.0对基因表达差异进行显著性分析。

1.5 载体构建

用ScNdhO目的片段, 以绿色荧光蛋白(green fluorescence protein, GFP)为标记, 构建ScNdhO蛋白的亚细胞定位载体ScNdhO-GFP。用SCMV的VPg编码序列, 以红色荧光蛋白(mCherry fluorescence protein, mCherry)为标记, 构建VPg的亚细胞定位载体VPg-mCherry。将ScNdhO基因构建到酵母表达载体 pGADT7中, 获得ScNdhO的捕获载体(GAL4 activation domain, AD) pGADT7-ScNdhO。利用Gateway技术, 将ScNdhO基因构建到BiFC的YC载体中, 得到ScNdhO-YC载体。所有构建的载体都经过测序验证。SCMV-VPg的Y2H诱饵载体(GAL4 DNA binding domain, BD) pGBKT7-SCMV-VPg、BiFC的YN载体SCMV-VPg-YN来自本课题组前期研究工作^[17]。

1.6 ScNdhO的亚细胞定位

将含有ScNdhO-GFP植物表达载体的农杆菌注射入健康的本氏烟叶片, 48 h后在激光共聚焦显微镜(Leica TCS SP5II)下观察本氏烟叶片表皮细胞中ScNdhO蛋白的定位, GFP的激发光波长为488 nm, 捕获波长为505~555 nm。mCherry的激发光波长为552 nm, 捕获波长为590~630 nm。叶绿素自荧光的激发光波长为552 nm, 捕获波长为650~680 nm。

1.7 Y2H验证ScNdhO与SCMV-VPg的互作

将pGADT7-ScNdhO、pGBKT7-SCMV-VPg组合, pGADT7-T、pGBKT7-53组合(阳性对照), pGADT7-T、pGBKT7-Lam组合(阴性对照), 分别转化酵母菌株Y2HGold, 30℃ 250 r min^-1培养至OD₅₄₆为0.6~0.8。将转化后的酵母细胞按照1、10^-1和10^-2梯度稀释, 分别在缺少亮氨酸(Leu)和色氨酸(Trp)的固体酵母合成限定基本培养基(SD/-Leu/-Trp)固体培养基上培养, 30℃倒置培养4~5 d。待菌落长到直径为3~4 mm左右后, 挑取单菌落转移到1 mL缺少亮氨酸(Leu)、色氨酸(Trp)、组氨酸(His)和腺嘌呤(Ade)的液体酵母合成限定基本培养基(SD/-Leu/- Trp/-His/-Ade)中培养。如果酵母生长, 将其转移到SD/-Leu/-Trp/-His/-Ade固体培养基上培养, 菌落生长表明蛋白之间存在互作。

1.8 BiFC验证ScNdhO与SCMV-VPg的互作

参照朱海龙等^[18]的方法将ScNdhO-YC与SCMV-VPg-YN等比例共转农杆菌EH105菌株, 将其注射入健康的本氏烟叶片中。正常条件下培养2~3 d后取样, 在激光共聚焦显微镜(Leica TCS SP5II)下观察拍照。YFP的激发光波长为514 nm, 捕获波长为530~590 nm?

2 结果与分析

2.1 ScNdhO基因的生物信息学分析

在前期研究中, 以SCMV-VPg为诱饵从甘蔗叶片cDNA酵母文库中筛选到1个具有全长ORF的基因, ORF长度为471 bp, 编码156个氨基酸。根据该基因序列设计特异PCR引物(表1), 从甘蔗中克隆了该基因, 测序结果与阳性酵母质粒测序结果相同。使用BlastP进行氨基酸序列比对, 发现该基因编码蛋白与高粱叶绿体NAD(P)H脱氢酶复合体O亚基(XP_002456968.1)同源性高达89%, 与玉米的O亚基(NP_001349312.1)同源性为89%, 因此将该基因命名为ScNdhO (GenBank登录号为MK280691)。

ProtParam分析表明, ScNdhO蛋白的分子量为16.93 kD, 等电点为9.30; 不稳定系数为39.54, 为稳定蛋白; 脂溶指数为88.46, 总平均疏水性是-0.164, 可能是亲水蛋白。二级结构预测表明, ScNdhO中无规则卷曲结构所占的比例最高, 为57.69%; 延伸链所占比例最低, 为18.59%; α-螺旋占23.72%。SignalP 4.1分析表明, ScNdhO蛋白不含信号肽, 是非分泌蛋白。TMHMM 2.0分析表明, ScNdhO蛋白无明显的跨膜区域。保守结构域分析表明, ScNdhO属于NDH复合体O亚基超基因家族, 佐证了ScNdhO为甘蔗NDH复合体的O亚基。

2.2 ScNdhO的氨基酸同源性分析和系统进化树分析

使用NCBI网站的Blastp搜索ScNdhO的同源序列, 对甘蔗及其他物种NDH复合体O亚基的氨基酸序列的同源性分析表明, ScNdhO蛋白的物种分化比较明显, 地钱(Marchantia polymorpha)及单细胞的集胞蓝藻(Cianobacteria synechocystis)与高等植物明显分离, 形成群I; 双子叶植物番茄(Solanum lycopersicum)、烟草(Nicotiana tabacum)、拟南芥(Arabidopsis thaliana)、葡萄(Vitis vinifera)、三叶杨(Populus trichocarpa)、大豆(Glycine max)、苜蓿(Medicago sativa)形成群II; 单子叶植物高粱(Sorghum bicolor)、玉米(Zea mays)、谷子(Setaria italica)、水稻(Oryza sativa)、二穗短柄草(Brachypodium distachyon)、大麦(Hordeum vulgare)形成群III; 在单子叶植物群III中, C₄植物形成明显的亚群III-2, C₃植物形成亚群III-1 (图1)。这表明, ScNdhO在遗传进化上具有明显的种属差异性, 与物种进化程度相关。

图1

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图1ScNdhO蛋白的氨基酸序列同源性分析和系统进化树分析

Fig. 1Deduced amino acid sequence and the phylogenic tree of ScNdhO protein

2.3 ScNdhO的亚细胞定位

由图2可知, 对照GFP在烟草表皮细胞中大量表达, 绿色荧光信号在细胞质、质膜和细胞核中均有分布; ScNdhO-GFP融合蛋白的绿色荧光信号与叶绿体的自荧光信号重合, 表明其主要定位于叶绿体; ScNdhO-GFP与VPg-mCherry共定位于叶绿体, 表明SCMV-VPg有可能与ScNdhO-GFP互作于叶绿体。

图2

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图2ScNdhO-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。
Fig. 2Subcellular localization of ScNdhO fused with GFP in the epidermal cells of N. benthamiana

The ScNdhO-GFP was labled by arrow; Up row: GFP control; Middle row: localization of ScNdhO-GFP; Down row: colocalization of ScNdhO-GFP with VPg-mCherry; bar = 50 μm.

2.4 ScNdhO基因的组织特异性表达及应答SCMV侵染的表达模式

图3表明, ScNdhO基因的表达具有明显的组织特异性, 在不同组织中的表达量差异显著: 在成熟叶片中相对表达量最高, 心叶和叶鞘中次之, 侧芽较少, 茎中微量表达, 根中几乎不表达。使用SCMV-CP基因特异引物(表1)检测SCMV接种甘蔗, 扩增出目的片段, 表明接种成功。SCMV侵染对ScNdhO基因表达影响显著, 与对照相比, ScNdhO基因在侵染早期显著上调, 然后明显下调表达, 第7天的表达量低于对照(图4)。

图3

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图3ScNdhO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。
Fig. 3Expression profile of ScNdhO in different sugarcane tissues

The error bars represent the standard error of each treating group (n = 3).

图4

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图4ScNdhO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)。
Fig. 4Expression profile of ScNdhO under the infection of SCMV

The error bars represent the standard error of each treating group (n = 3).

2.5 ScNdhO与SCMV-VPg的互作验证

Y2H实验结果显示, 在四缺培养基上, pGADT7-ScNdhO和pGBKT7-SCMV-VPg共转的酵母菌株Y2HGold可以生长并呈现蓝色, 阳性对照可以正常生长并呈现蓝色, 阴性对照无菌落(图5), 说明ScNdhO与SCMV-VPg互作。BiFC实验结果与Y2H实验结果一致, ScNdhO-YC与SCMV-VPg-YN互作产生黄色荧光信号, 并与叶绿体的红色荧光信号重叠, 表明互作于叶绿体(图6)。

图5

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图5Y2H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。
Fig. 5Y2H assay for protein-protein interactions between ScNdhO and SCMV-VPg

The positive and negative controls are yeast cotransformants with pGADT7-T plus pGBKT7-53 and pGADT7-T plus pGBKT7-Lam, respectively. SD/-Leu/-Trp: synthetic defined yeast minimal medium lacking Leu and Trp; SD/-Leu/-Trp/-His/-Ade: synthetic defined yeast minimal medium lacking Leu, Trp, His, and Ade.

图6

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图6BiFC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。
Fig. 6BiFC assay for protein-protein interactions between ScNdhO and VPg from SCMV

ScNdhO was fused to the C-terminal half of YFP, while SCMV-VPg was fused to the N-terminal half of YFP. ScNdhO-YC and SCMV-VPg-YN were transiently coexpressed in N. benthamiana leave. The fluorescent signal was monitored by confocal microscopy at 48 hpi. bar = 50 μm.

3 讨论

抗花叶病是甘蔗育种的重要指标之一^[61,62,63], 但是有关甘蔗花叶病病原即SCMV、SrMV和SCSMV侵染甘蔗及诱发花叶病症状的分子机制研究却鲜见报道^[12,17-18]。本研究首次克隆了甘蔗叶绿体NAD(P)H脱氢酶(NDH)复合体的O亚基基因ScNdhO, 首次报道了ScNdhO与SCMV-VPg互作并应答SCMV侵染。本研究对于阐明SCMV侵染甘蔗导致花叶病症状的分子机制研究具有重要意义。

叶绿体NDH复合体在碳水化合物同化、逆境应答等过程中具有重要作用^{[31,36,41-42,44,57]}。线性电子传递中ATP/NADPH比值是固定的, 低于或接近卡尔文循环所需的比值1.5^[64], 但是在逆境或者同化物大量合成的情况下, 循环电子途径必须介入供应ATP。对于C₄植物而言, 光合作用所需的ATP/ NADPH比值至少要达到2.5。与C₃光合作用相比, C₄光合作用需要额外的2分子ATP用于1,5 -二磷酸核酮糖羧化酶/加氧酶(Rubisco)浓缩CO₂, 这2分子额外的ATP就是由围绕PSI的循环电子传递提供的, 现已在玉米、黄顶菊等C₄植物中鉴定了NDH复合体介导的循环电子传递和生产ATP^{[33-34,65-67]}。进化分析也表明, C₃植物与C₄植物的NdhO分成不同的亚群。甘蔗叶片是主要的光合器官, 完全展开的正一叶是光合作用最旺盛的叶片, 本研究中ScNdhO在正一叶中的表达量最高, 推测叶绿体NDH复合体通过循环电子传递为CO₂同化提供能量。NDH复合体O亚基对NDH复合体的正确组装和行使功能很重要, 敲除NdhO烟草或拟南芥的突变体, 亚复合体A无法组装, 进而完全瓦解NDH介导的循环电子传递^[57,68]。ScNdhO在SCMV侵染早期上调表达, 一方面有可能通过这种互作使SCMV复制复合体定位于叶绿体, 完成病毒基因组复制; 另一方面则是寄主对SCMV侵染的抗性反应。在SCMV侵染后期ScNdhO下调表达, 本研究推测SCMV侵染影响了NDH复合体的稳定, 干扰了循环电子传递, 使光合作用处于逆境状态, 导致叶绿素减少^[10], 产生花叶症状, 进而影响碳水化合物合成。

植物病毒结构简单, 必须与寄主因子互作才能完成系统性侵染^[25,26]。本研究发现ScNdhO与SCMV-VPg互作, 因此推测ScNdhO被SCMV选择性利用。NDH复合体约有30个亚基, SrMV和SCSMV与SCMV的基因组结构非常相似, 他们的VPg有可能与ScNdhO或者其他亚基互作执行相应的生物学功能。病毒与寄主是协同进化的, 病毒对寄主侵染的最终目的是繁殖和传播^[69]。由昆虫作为媒介传播的植物病毒中至少50%是由蚜虫以非持续性方式传播的^[70,71]。蚜虫普遍嗜好发病植株, 比如雀麦花叶病毒属(Bromovirus)的菜豆黄花叶病毒(Bean yellow mosaic virus, BYMV)和蚕豆斑驳病毒(Broad bean mottle virus, BBMV), 豌豆耳突花叶病毒属(Enamovirus)的豌豆耳突花叶病毒(Pea enation mosaic virus, PEMV), 侵染后造成叶片黄化, 吸引豌豆蚜^[72], 与SCMV同属的小西葫芦黄花叶病毒(Zucchini yellow mosaic virus, ZYMV)侵染造成的叶片黄化植株吸引棉蚜^[73]。SCMV是由多种蚜虫非持续性方式传播的^[6], SCMV-VPg与ScNdhO互作并在侵染后期降低其表达, 可能对叶绿体产生胁迫, 造成叶片黄化, 吸引蚜虫便于病毒传播。病毒与寄主的互作是十分复杂的, 花叶病症状的产生可能是多方面因素的结果, 比如SCSMV-P3与甘蔗 Rubisco大亚基互作可能干扰Rubisco的功能而影响叶绿体的稳定性^[18]。SCMV-VPg与ScNdhO互作的具体生物学功能还有待进一步研究。

4 结论

从甘蔗中克隆到叶绿体NAD(P)H脱氢酶(NDH)复合体的O亚基基因ScNdhO (GenBank登录号为MK280691), 其ORF长度为471 bp, 编码长度为156 aa的蛋白。ScNdhO蛋白定位于叶绿体, 在完全展开的功能叶片中表达量最高。ScNdhO蛋白与SCMV-VPg互作。ScNdhO基因响应SCMV侵染, 在侵染早期上调表达, 在侵染后期则下调表达。

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为探索甘蔗条纹花叶病毒与甘蔗的互作机制,利用酵母双杂交技术筛选与SCSMV互作的甘蔗因子基因,利用PCR技术克隆了SCSMV的<em>HC</em>-<em>Pro</em>、<em>P3N</em>-<em>PIPO</em>、<em>CP</em>和<em>VPg</em>的编码区,构建到酵母双杂交诱饵载体pGBKT7上,获得了pGBKT7-<em>HC</em>-<em>Pro</em>、pGBKT7-<em>P3N</em>-<em>PIPO</em>、pGBKT7-<em>CP</em>和pGBKT7-<em>VPg</em> 4个诱饵载体。结果显示,将重组质粒转化酵母Y2HGold酵母菌株后,酵母菌株在SD/-Trp平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性;在SD/-Trp/X-&alpha;-gal平板上长出白色菌落未变蓝,在SD/-Leu、SD/-Trp/-Ade、SD/-Trp/-His和SD/-Trp/X-&alpha;-gal/AbA平板上不能生长,表明重组诱饵质粒表达产物对<em>MEL1</em>、<em>ADE2</em>、<em>HIS3</em>和<em>AUR1</em>-<em>C</em>报告基因无自激活作用。构建的诱饵表达载体可以作为诱饵用于文库筛选,为探索SCSMV侵染机制和发病机理奠定了基础。
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为探索甘蔗条纹花叶病毒与甘蔗的互作机制,利用酵母双杂交技术筛选与SCSMV互作的甘蔗因子基因,利用PCR技术克隆了SCSMV的<em>HC</em>-<em>Pro</em>、<em>P3N</em>-<em>PIPO</em>、<em>CP</em>和<em>VPg</em>的编码区,构建到酵母双杂交诱饵载体pGBKT7上,获得了pGBKT7-<em>HC</em>-<em>Pro</em>、pGBKT7-<em>P3N</em>-<em>PIPO</em>、pGBKT7-<em>CP</em>和pGBKT7-<em>VPg</em> 4个诱饵载体。结果显示,将重组质粒转化酵母Y2HGold酵母菌株后,酵母菌株在SD/-Trp平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性;在SD/-Trp/X-&alpha;-gal平板上长出白色菌落未变蓝,在SD/-Leu、SD/-Trp/-Ade、SD/-Trp/-His和SD/-Trp/X-&alpha;-gal/AbA平板上不能生长,表明重组诱饵质粒表达产物对<em>MEL1</em>、<em>ADE2</em>、<em>HIS3</em>和<em>AUR1</em>-<em>C</em>报告基因无自激活作用。构建的诱饵表达载体可以作为诱饵用于文库筛选,为探索SCSMV侵染机制和发病机理奠定了基础。

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