,, 赵凯茜, 王跃进,西北农林科技大学园艺学院/旱区作物逆境生物学国家重点实验室/农业农村部西北地区园艺作物生物与种质创制重点实验室,陕西杨凌 712100Expression of Stilbene Synthase Genes from Chinese Wild Vitis quinquangularis and Its Effect on Resistance of Grape to Powdery Mildew
DING Xi,, ZHAO KaiXi, WANG YueJin,College of Horticulture, Northwest A & F University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture and Rural Affairs, Yangling 712100, Shaanxi通讯作者:
责任编辑: 岳梅
收稿日期:2020-04-20接受日期:2020-06-5网络出版日期:2021-01-16
| 基金资助: |
Received:2020-04-20Accepted:2020-06-5Online:2021-01-16
作者简介 About authors
丁茜,E-mail：
摘要
关键词：
Abstract
Keywords：
PDF (2979KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
丁茜, 赵凯茜, 王跃进. 中国野生毛葡萄芪合酶基因表达及对葡萄抗白粉病的影响[J]. 中国农业科学, 2021, 54(2): 310-323 doi:10.3864/j.issn.0578-1752.2021.02.007
DING Xi, ZHAO KaiXi, WANG YueJin.
开放科学（资源服务）标识码（OSID）：
0 引言
【研究意义】葡萄是世界各地广泛栽培的果树[1]。据世界粮农组织（FAO）2017年数据统计（http:// www.fao.org/home/zh/）,全球葡萄种植面积和总产量分别为693.14万公顷、7 427.66万吨。目前,葡萄生产中主栽品种为欧洲葡萄（Vitis vinifera）,但该品种抗病能力差,易感白粉病[2],病菌侵染对葡萄产量和质量造成巨大影响[3]。生产上常用化学药剂防治真菌病害,而杀菌剂的使用会增加生产成本,降低果实品质,长期使用还会影响环境[4]。因此,抗病育种是葡萄育种的主要目标之一[5]。利用转基因技术抗病育种,能够避免常规杂交育种出现的杂交不亲和及周期长的缺点。抗病育种需要有抗病种质资源,中国野生毛葡萄‘丹凤-2’（Vitis quinquangularis accession Danfeng-2）不仅抗病性强,并且白藜芦醇（resveratrol）含量高[6]。因此,将‘丹凤-2’中的抗病基因转入欧洲葡萄,提高其抗病性,获得抗病且白藜芦醇含量高的新品种,具有重要的理论和实践意义。【前人研究进展】研究表明,葡萄中的白藜芦醇是其特有的一种植保素[7],不仅可以增强植物抗病性[8],而且果实中的白藜芦醇对人体有抗氧化、抗衰老以及抗癌等作用[9,10,11]。芪合酶（stilbene synthase,STS）是白藜芦醇代谢途径的关键酶。因此,将芪合酶基因（STS）作为提高植物抗病性的目的基因,在葡萄[12]以及其他物种如猕猴桃[13]、苹果[14]、拟南芥[15]、水稻[16]、白杨[17]、苜蓿[18]等进行遗传转化,研究证明转入外源STS的过表达植株对逆境胁迫的抗性增强并且芪类物质积累。笔者所在课题组前期利用转基因技术,获得了过表达VpSTS29的无核白和霞多丽植株[19]、过表达VqSTS6、VqSTS26、VqSTS32、VqSTS12和VqSTS25的无核白植株,人工接种葡萄白粉病菌（Uncinula necator）后发现,转基因植株芪类物质含量和种类增加,并且对白粉病的抗性也有所增强[12,20-21]。【本研究切入点】以中国野生毛葡萄‘丹凤-2’在成熟果实中表达较高的VqSTS9和在幼叶中表达较高的VqSTS21为目的基因,构建植物过表达载体,用农杆菌介导法转入欧洲葡萄无核白（Vitis vinifera cv. Thompson Seedless）,研究基因表达特性以及抗病功能。【拟解决的关键问题】以无核白的分生愈伤组织为受体材料,通过农杆菌介导法获得过表达VqSTS9、VqSTS21的无核白植株,比较过表达植株在接菌前后STS表达水平及芪类物质含量和种类的差异,研究STS的表达特性以及抗病功能,为葡萄抗病育种提供参考。1 材料与方法
试验于2017—2019年在西北农林科技大学旱区作物逆境生物学国家重点实验室完成。1.1 试验材料
中国野生毛葡萄‘丹凤-2’和欧洲葡萄无核白均定植于西北农林科技大学葡萄种质资源圃,试验所用葡萄白粉病菌取自此资源圃。在西北农林科技大学旱区逆境生物学国家重点实验室组培室培养无核白组培苗和分生愈伤组织。1.2 VqSTS9与VqSTS21的生物信息学分析
在NCBI网站（https://www.ncbi.nlm.nih.gov/）分别下载VqSTS9（GenBank登录号：JQ868689）、VqSTS21（GenBank登录号：JQ868677）基因序列,用DNAMAN V6软件比对序列与测序结果,以此来确定序列;VqSTS9、VqSTS21的基因染色体定位信息登录欧洲葡萄基因组网（http://www.genoscope.cns.fr/externe/ Genome Browser/Vitis/）获得;使用MEGA 6软件聚类分析VqSTS9、VqSTS21与其他物种STS的氨基酸序列。1.3 VqSTS9和VqSTS21克隆与表达载体构建
提取中国野生毛葡萄‘丹凤-2’叶片的总RNA,参考E.Z.N.A Plant RNA Kit（Omega）试剂盒说明书进行。将RNA反转录合成cDNA,此过程参照FastKing RT Kit（With gDNase）试剂盒（天根KR116）说明书进行。以cDNA为模板,设计扩增基因的引物（表1）,分别在引物5′和3′端添加BamH I和Sal I酶切位点序列,利用同源序列克隆VqSTS9和VqSTS21,将目标条带大小正确的产物回收,产物连接pGEM-T-easy载体,转入大肠杆菌T0P10感受态细胞中,培养至长出菌斑,挑取单克隆菌斑,菌液PCR检测,阳性菌液送至奥科生物有限公司测序。用E.Z.N.A Plasmid Mini Kit I（Omega）质粒提取试剂盒提取测序结果正确的菌液中的重组质粒,重组质粒和植物表达载体pCAMBIA2300分别用BamHI/SalI双酶切,将酶切后的目的基因与表达载体连接,转入农杆菌（Agrobacterium tumefaciens）GV3101中,构建植物表达载体pCAMBIA35S::VqSTSs::GFP。Table 1
表1
表1本研究所用引物
Table 1
| 基因 Gene | 上游引物 Forward primer | 下游引物 Reverse primer | 目标大小 Target size (bp) |
|---|---|---|---|
| VqSTS9 (vector construction, PCR detection) | F: CGGGATCCATGGCTTCAGTCGAGGAA TTTAGAAACG | R: GCGTCGACATTTGTAACCGTAGGAAT GCTATGCAGC | 1179 |
| VqSTS21 (vector construction, PCR detection) | F: CGGGATCCATGGCTTCAGTCGAGGAA ATTAGAAACG | R: GCGTCGACATTTGTAACCATAGGAAT GCTATGCAACAC | 1179 |
新窗口打开|下载CSV
1.4 无核白葡萄的遗传转化
用无核白组培苗的单芽茎段,在培养基上诱导出用于遗传转化受体材料的分生愈伤组织,遗传转化用农杆菌介导法。转化的具体步骤：恒温摇床上二次活化农杆菌菌液（180 r/min,28℃）,活化好的菌液,室温下5 500 r/min离心10 min,去上清,菌液重悬液（1/2 MS+20 g·L-1蔗糖,200 μmol·L-1乙酰丁香酮）重悬两次,180 r/min,28℃恒温培养2 h,转化用的菌液浓度OD600=0.7—1.0。将培养好的分生愈伤组织切小块,用活化好的适宜浓度的菌液侵染20 min,置于共培养培养基（IM3+200 μmol·L-1乙酰丁香酮,pH 5.8）上暗培养48 h,之后,用脱菌液（1/2 MS+20 g·L-1蔗糖+200 mg·L-1 Carb）洗脱10 min,重复两次,再用无菌水4—5次,置于筛选培养基（IM3+300 mg·L-1 Cef+75 mg·L-1 Kan+200 mg·L-1 Carb,pH 5.8）光培养,每个月切去褐化组织并且更换培养基一次,之后将愈伤组织转移至诱导分化培养基上促进分化长芽,将长至2—5 cm的芽置于生根培养基中进行生根成苗培养,方法参考文献[22]。1.5 转基因植株的鉴定
通过PCR、Western blot对抗性植株进行检测鉴定,两种检测结果均为阳性的抗性苗确定其为转基因植株,对其进行扩繁、炼苗移栽。检测方法步骤：提取转基因植株叶片DNA（CTAB法）,进行PCR检测,引物如表1所示。Western blot检测,先提取转基因植株叶片的蛋白质,提取方法：取叶片约0.5 g,在液氮中研磨成粉末,装于2.0 mL离心管中,加入250 μL蛋白裂解液,混匀,置冰上15 min,离心20 min,吸取上清,加入上样缓冲液,混匀,沸水中煮5 min,冷却至室温后,离心3 min,待用。参考HEART SDS-PAGE凝胶配制试剂盒说明书制备SDS-PAGE胶,将提取好的蛋白质点样跑胶;将带有蛋白的胶切下用半干转膜法将蛋白胶中的蛋白转至PVDF膜上;用一抗杂交目的蛋白,二抗放大目的蛋白的信号,用带GFP标签鼠单克隆抗体作为一抗,用HRP-羊抗小鼠IgG（H+L）作为二抗;二抗孵育结束后,洗掉膜表面的抗体,膜上加显色液,用BIO RAD Universal Hood II成像。1.6 芪合酶代谢相关基因的表达分析
通过qRT-PCR分析过表达植株中STS以及其代谢相关基因的表达,分别包括VqSTS9与VqSTS21、PAL、RSGT、MYB14、MYB15、CHS。qRT-PCR的模板为转基因植株叶片的cDNA,提取方法参照1.3,VqSTS9、VqSTS21定量引物设计参考SHI等[23],其余5个基因定量引物设计参照CHENG等[12],内参基因为VvGAPDH,反应体系参考NovoStart?SYBR qPCR SuperMix Plus说明书,用三步法进行qRT-PCR分析,3次生物学重复,每个生物学重复进行3次技术重复。1.7 转基因植株抗病性分析
1.7.1 人工接种白粉病菌 用压片法[24]对生长状态良好的转基因和野生型（wild type,WT）植株接种白粉病菌。操作方法：选生长状态良好的叶片进行接种,首先给叶片表面喷无菌水,然后将有白粉病菌的葡萄叶片在待接种叶片表面上覆盖轻轻摩擦完成接种。1.7.2 白粉病菌侵染过程观察 剪取白粉病菌侵染的叶片置于离心管中,加入台盼兰染液,使叶片完全浸泡在染液中,沸水中煮15 min,室温冷却后,倒掉染液,加入水合氯醛溶液浸泡清洗,至叶片无明显蓝色,在显微镜下观察记录白粉病菌的生长情况。
1.7.3 qRT-PCR分析接菌前后植株STS表达 在接菌后0、1、2、3、4、5、6、7 d分别取野生型和转基因植株的接菌叶片,提取cDNA,进行qRT-PCR分析, 方法参照1.6。
1.7.4 高效液相色谱法（HPLC）分析 对接菌0 d和7 d的野生型和转基因植株叶片,用HPLC检测接菌前后植株中芪类物质的种类及含量。样品中的目标物质通过标准曲线上的保留时间来确定,含量通过标准峰面积和样品峰面积的比值来计算,方法参照ZHOU等[25]。
1.8 VqSTS9、VqSTS21在接种白粉病菌后的表达分析
通过压片法[24]对中国野生毛葡萄‘丹凤-2’叶片接种白粉病菌,以无菌水作为对照,在处理后0、12、24、48、72、96、120和144 h取样,进行qRT-PCR分析,方法参照1.6。1.9 数据统计与分析
原始数据采用Microsoft Excel计算,数值均表示为“平均值±标准误”,采用Origin8.0软件作图,通过SPSS软件one-way ANOVA（Tukey test）分析检验差异结果（*P<0.05,**P<0.01）。2 结果
2.1 VqSTS9、VqSTS21在白粉病菌诱导条件下的表达
VqSTS9、VqSTS21均可以响应白粉病菌的诱导表达,在接菌后12 h表达量达到最高峰,与未接菌叶片相比,相对表达量分别提高了17与12倍。之后下降,120 h表达量再次上调,之后又有所下降。两个基因的表达模式相似,但表达水平不同,VqSTS9在接种12 h相对表达量高于VqSTS21（图1）。图1
新窗口打开|下载原图ZIP|生成PPT图1VqSTS9（A）、VqSTS21（B）在白粉病菌诱导下的表达
Fig. 1The expression of VqSTS9 (A), VqSTS21 (B) induced by U. necator
2.2 VqSTS9、VqSTS21 序列分析
利用同源序列法克隆得到VqSTS9和VqSTS21基因序列（图2-A）,经序列分析,可得出VqSTS9、VqSTS21 CDS序列长度为 1 179 bp,蛋白质分子量为43 kD,编码392个氨基酸。以欧洲葡萄‘PN40024’（Vitis vinifera cv. Pinot Noir）基因组数据为参考依据,对VqSTS9、VqSTS21进行染色体定位分析（图2-B）和氨基酸序列比对分析（图2-C）。分析得出VqSTS9反向定位于16号染色体16 710 284—16 711 818位置,对应欧洲葡萄VvSTS6,VqSTS9与VvSTS6的氨基酸有4个位点不同。VqSTS21反向定位于16号染色体16 491 600—16 493 131位置,对应欧洲葡萄VvSTS2,VqSTS21与VvSTS2无氨基酸位点的差异。通过聚类分析不同植物的STS（图2-D）,发现VqSTS9与VvSTS6最相似,相似度为98.72%;VqSTS21与VvSTS2最相似,相似度为100%,其次是与不同物种的高粱（Sorghum bicolor）,相似度分别为73.26%、73.78%。图2
新窗口打开|下载原图ZIP|生成PPT图2VqSTS9、VqSTS21的克隆、序列与聚类分析
A：VqSTS9、VqSTS21目的基因克隆VqSTS9 and VqSTS21 gene cloning;B：VqSTS9、VqSTS21的染色体定位Chromosome localization of VqSTS9, VqSTS21;C：VqSTS9、VqSTS21与欧洲葡萄氨基酸序列比对Amino acid sequence alignment of VqSTS9, VqSTS21 and STS from V. vinifera;D：VqSTS9、VqSTS21与不同物种STS氨基酸序列聚类分析,包括欧洲葡萄、松叶兰、樟子松、云杉、高粱、花生、桑、何首乌、大黄Cluster analysis of amino acid sequence of VqSTS9, VqSTS21 and STS from V. vinifera, Psilotum nudum, Pinus sylvestris, Picea abies, Sorghum bicolor, Arachis hypogaea, Morus alba, Fallopia multiflora, Rheum palmatum。登录号The accession number：VqSTS9（AFM56666.1）、VqSTS21（AFM56657.1）、VvSTS6（NP_001267934.1）、VvSTS2（XP_003634068.1）、PnSTS（BAA87924）、PsylSTS（CAA43165）、PaSTS（AEN84236.1)、SbSTS（AAL49965）、AhSTS（BAA78617）、MaSTS（ARM20004.1）、FmSTS（AFP97667.1）、RpSTS（AFX68803.1）
Fig. 2Cloning, sequence and cluster analyses of VqSTS9, VqSTS21
2.3 VqSTS9、VqSTS21克隆及表达载体的构建
利用同源序列法从中国野生毛葡萄‘丹凤-2’克隆得到VqSTS9、VqSTS21,构建用于本次遗传转化的植物过表达载体pCAMBIA35S::VqSTS9::GFP、pCAMBIA35S::VqSTS21::GFP（图3-A）。BamH Ι/Sal Ι双酶切目的基因与表达载体的重组质粒（图3-B）,将重组质粒转入农杆菌 GV3101中,菌液PCR检测（图3-C）。图3
新窗口打开|下载原图ZIP|生成PPT图3VqSTS9、VqSTS21基因克隆与植物表达载体构建
A：两个表达载体示意图Schematic diagram of two expression vectors。B：VqSTS9、VqSTS21基因连接表达载体pCAMBIA2300的双酶切检测VqSTS9, VqSTS21 genetic connection expression vector pCAMBIA2300 double enzyme detection,M：DNA Marker;1：BamH Ι单酶切表达载体质粒The BamH Ι single enzyme expression vector plasmid;2：Sal Ι单酶切表达载体质粒The Sal Ι single enzyme expression vector plasmid;P：pCAMBIA2300的空载体质粒对照Empty vector pCAMBIA2300 as control;CK：空白对照Blank control (ddH2O);3—5：BamH Ι和Sal Ι双酶切植物表达载体 pCAMBIA35S::VqSTS9::GFP,3个重复Double enzyme of BamH Ι and Sal Ι plant expression vector pCAMBIA35S: :VqSTS9: :GFP, three repeats;6—8：BamH Ι和Sal Ι双酶切植物表达载体pCAMBIA35S::VqSTS21::GFP,3个重复Double enzyme of BamH Ι and Sal Ι plant expression vector pCAMBIA35S::VqSTS21::GFP, three repeats。C：过表达载体转化农杆菌的菌液PCR检测PCR detection of Agrobacterium-transformed by overexpression vector,M：DNA Marker;P：阳性质粒对照Positive plasmid control;1—3：农杆菌转化pCAMBIA35S::VqSTS9::GFP载体,3个重复Agrobacterium-transformed pCAMBIA35S::VqSTS9::GFP vector, three repeats;4—6：农杆菌转化pCAMBIA35S::VqSTS21::GFP载体, 3个重复Agrobacterium-transformed pCAMBIA35S::VqSTS21::GFP vector, three repeats
Fig. 3VqSTS9 and VqSTS21 gene cloning and plant expression vector construction
2.4 欧洲葡萄无核白遗传转化及抗性植株检测
2.4.1 遗传转化 取野生型（WT）无核白组培苗的单芽茎段,在培养基上诱导分生愈伤组织,用此愈伤组织作为转基因的受体材料。将目的基因通过农杆菌介导法转入愈伤组织中,通过再生途径,诱导愈伤组织分化出抗性芽,并通过生根培养长成抗性植株。其过程如下（图4-A）：生长状态良好的愈伤组织（a）;将其切块,用活化好的农杆菌侵染15—20 min,期间不断摇晃使其与菌液充分接触,随后倒掉菌液,多余的菌液用滤纸吸去,愈伤组织放入共培养培养基（b）中,培养48 h;共培养结束后,脱菌,放入筛选培养基中（c）,每20—25 d继代一次,切除褐色部分,保留绿色部分继续进行培养,培养50—60 d后（d）,放入筛选诱导分化培养基中,诱导愈伤组织长出抗性芽（e）;将抗性芽切下放入生根培养基中诱导生根（f）;生根的抗性芽继续在培养基中长大成苗（g）;抗性苗检测结果为阳性的,剪取单芽茎段大量扩繁（h）,长至成苗（i）,练苗移栽（j）。图4
新窗口打开|下载原图ZIP|生成PPT图4无核白遗传转化过程（A）以及转基因植株的鉴定（B）
A：无核白遗传转化过程The genetic transformation of Thompson Seedless,a：无核白分生愈伤组织Meristem callus;b：侵染后的愈伤组织与农杆菌共培养The infected callus was co-cultured with A. tumefaciens;c：转化后的愈伤组织筛选培养Callus screening culture after transformation;d：筛选60 d后的愈伤组织Callus screening culture after 60 days;e：愈伤组织诱导出抗性芽Callus induced resistant buds;f：抗性芽生根培养Rooting culture of resistant bud;g：抗性芽长成植株Resistant buds grow into plants;h：抗性植株的继代扩繁Secondary propagation of resistant plants;i：继代后长成植株Plant growth after succession;j：转基因植株移栽Transplanting of transgenic plants。B：转基因植株的鉴定Identification of transgenic plants,a：转基因植株Transgenic plants;b：PCR检测抗性植株PCR was used to detect resistant plants,M为Marker,P为质粒M was Marker, and P was plasmid;c：Western blot检测抗性植株Western blot was used to detect resistant plants
Fig. 4The genetic transformation of Thompson Seedless (A) and identification of transgenic plants (B)
2.4.2 抗性植株检测 遗传转化共获得无核白抗性植株419株,转VqSTS9、VqSTS21的抗性植株分别有223和196株。用PCR检测外源、内源STS和Western blot检测VqSTSs-GFP融合蛋白在植株中的表达（图4-B）。两种水平的检测结果均为阳性的植株有7株,过表达VqSTS9和VqSTS21的无核白植株分别有4和3株,转化的阳性率分别为1.79%、1.53%。
2.5 转基因植株芪合酶代谢相关基因的表达分析
VqSTS9和VqSTS21分别在转基因株系中表达上调,其中OEVqSTS9-L2和OEVqSTS21-L2表达量最高,分别是野生型的5.6、12.3倍;MYB14表达上调,其中OEVqSTS9-L3和OEVqSTS21-L1表达量最高,分别是野生型的4.3、25.0倍;RSGT表达显著上调,其中OEVqSTS21-L2表达量最高,是野生型的16.8倍;CHS表达量下调。VqSTS、PAL、MYB14、MYB15、RSGT在大部分植株中表达量均上调,其中OEVqSTS9植株中,VqSTS9和RSGT表达量高,而在OEVqSTS21植株中,MYB14、MYB15表达量高,表达量最高的是OEVqSTS21-L2植株中的MYB15。上述结果表明过表达VqSTS后,VqSTS及其相关基因中正向调控芪类物质合成的转录因子均不同程度上调表达,而与芪合酶存在底物竞争关系的CHS下调表达（图5）。图5
新窗口打开|下载原图ZIP|生成PPT图5野生型和转基因无核白植株芪合酶代谢相关基因表达
A：4个转 VqSTS9株系的qRT-PCR 分析qRT-PCR analysis of 4 VqSTS9 transgenic lines;B：3个转 VqSTS21株系的qRT-PCR分析qRT-PCR analysis of three VqSTS21 transgenic lines;OEVqSTS-Ls为转基因株系OEVqSTS-Ls were different transgenic lines
Fig. 5STS metabolic pathway related gene expression in WT and transgenic lines
2.6 转基因无核白植株抗白粉病分析
2.6.1 人工接种白粉病菌后转基因植株的抗病性表现 为了研究转基因植株对白粉病的抗性,对转VqSTS9和 VqSTS21无核白与野生型植株接种白粉病菌（图6-A）,显微镜观察并统计接菌后 1—3 d每100个孢子的萌发数、初级菌丝与次级菌丝的数量,以及接菌7 d后分生孢子梗的数量（表2）,染色结果（图6-B）显示在接种白粉病菌1 d后,植株叶片观察到接种的分生孢子,与野生型相比,转基因植株叶片孢子数量较少;接菌2 d和3 d后,转基因植株叶片初级、次级菌丝数少于野生型植株;在接菌7 d后,与野生型植株相比,转基因植株分生孢子梗的数量少。上述结果表明过表达VqSTS9和VqSTS21无核白植株对白粉病的抗性增强。图6
新窗口打开|下载原图ZIP|生成PPT图6转基因植株抗白粉病分析
A：用于接种白粉病菌的野生型无核白和转基因植株、与接种白粉病菌7 d的野生型和转基因的葡萄叶片Wildtype Thompson Seedless and transgenic plants used for U. necator induction and leaves with induction 7 dpi；B：显微观察葡萄叶片上白粉病菌的生长。标尺=100 μm The growth progress of U. necator on grape leaves was observed microscopically. Scale bar=100 μm
Fig. 6Analysis of the resistance to powdery mildew of transgenic plants
Table 2
表2
表2每100个孢子萌发数、初级菌丝数、次级菌丝数和分生孢子梗数统计
Table 2
| 株系 Line | 1 dpi | 2 dpi | 3 dpi | 7 dpi | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 萌发数 Germination | 初级菌丝数 Primary hyphae | 次级菌丝数 Secondary hyphae | 萌发数 Germination | 初级菌丝数 Primary hyphae | 次级菌丝数 Secondary hyphae | 萌发数Germination | 初级菌丝数 Primary hyphae | 次级菌丝数 Secondary hyphae | 分生孢子 梗数 Conidiophore | |
| WT | 35.67±2.05a | 16.67±1.25a | 2.33±0.47a | 25.67±1.70a | 10.33±1.25d | 36.67±1.70a | 14.67±1.25d | 8.67±1.25c | 45.00±1.63a | 203.33±5.31a |
| OEVqSTS9-L1 | 21.33±2.36c | 5.67±1.25d | 0 | 14.67±1.25cd | 12.67±2.05cd | 5.33±1.25b | 22.33±2.49bc | 15.33±1.25a | 13.67±1.25c | 61.33±3.86e |
| OEVqSTS9-L2 | 16.67±1.70d | 8.00±2.45cd | 1.33±1.25a | 11.33±1.25d | 14.67±0.94bc | 6.67±1.70b | 21.67±1.70c | 11.67±1.25b | 16.33±0.47bc | 77.00±3.56d |
| OEVqSTS21-L2 | 26.67±2.05b | 10.67±1.70bc | 1.33±1.89a | 19.00±1.63b | 17.33±1.25ab | 7.33±1.25b | 27.33±1.25a | 14.33±1.70ab | 16.33±2.49bc | 87.33±3.68c |
| OEVqSTS21-L3 | 22.33±1.7bc | 13.00±0.82b | 0.67±0.94a | 17.33±1.70bc | 19.33±1.25a | 8.33±1.25b | 25.67±0.47ab | 16.00±0.82a | 18.00±1.63b | 124.67±4.11b |
新窗口打开|下载CSV
2.6.2 白粉病菌诱导下转基因植株STS的表达与产物分析 对转基因植株与野生型植株接种白粉病菌,通过qRT-PCR分析接菌前后STS表达量的变化（图7-A、7-B）。结果表明在白粉病菌诱导后,STS响应其诱导表达上调,转基因植株中STS表达量显著升高,且与同时期的野生型植株相比始终保持较高水平。在OEVqSTS9-L1和OEVqSTS9-L2株系中,STS相对表达量分别在接菌后3 d和4 d达到峰值,是同时期野生型表达量的15.8倍与13.8倍;在OEVqSTS21-L2和OEVqSTS21-L3株系中,STS相对表达量分别在接菌后5 d和3 d达到峰值,是同时期野生型表达量的18.6倍与45.8倍。
图7
新窗口打开|下载原图ZIP|生成PPT图7白粉病菌诱导下过表达植株VqSTS9、VqSTS21相对表达量以及芪类物质含量和种类
A、B：接菌后 STS相对表达量Relative expression of STS after inoculation;C、D：接菌0 d和7 d后芪类物质含量和种类检测The contents and types of stilbenes after inoculation 0 d and 7 d
Fig. 7The relative expression levels of VqSTS9 and VqSTS21 and the contents and types of stilbenes in overexpressed plants induced by U. necator
接菌0 d和7 d,分析转基因植株与野生型植株中芪类物质含量与种类的变化（图7-C、7-D）。结果表明接菌前过表达无核白植株中检测到反式云杉新苷和反式白藜芦醇,而野生型植株只检测到反式云杉新苷;接菌7 d后,野生型植株的芪类物质种类在反式云杉新苷基础上增加了白藜芦醇（13.14 μg·g-1 DW）,一些过表达植株中检测到葡萄素。与接菌前相比,接菌 7 d 后芪类物质总含量显著提高,其中OEVqSTS9-L1植株比OEVqSTS9-L2芪类物质的含量和种类多,OEVqSTS9-L1的糖苷和白藜芦醇含量分别为接菌前的2.7倍和2.4倍,葡萄素含量为8.2 μg·g-1 DW;植株OEVqSTS21-L2比OEVqSTS21-L3芪类物质含量多,OEVqSTS21-L2的糖苷和白藜芦醇含量分别为接菌前的1.6倍和2.1倍,葡萄素含量为18.3 μg·g-1 DW。
3 讨论
要解决欧洲葡萄不抗病的问题,需要培育抗病品种,而抗病育种中抗病种质资源尤为关键。早期,研究者试图从欧洲葡萄品种中寻找抗病物质。1976、1979年,LANGCAKE等相继鉴定出葡萄中的白藜芦醇及衍生物[26,27];1984年,SCH?PPNER等首次分离与纯化了合成白藜芦醇的关键酶芪合酶[28]。关于葡萄转芪合酶基因,最早研究是在1990年,转花生STS的烟草原生质体在紫外灯诱导下检测到白藜芦醇[29];研究表明,白藜芦醇不仅作为植保素具有抗病性,还具有生物学保健功能如抗癌、抗氧化、抗衰老等[8,9,10]。而芪合酶是白藜芦醇合成途径中的关键酶,可以响应并提高植物的抗病性[30]。因此,转芪合酶基因到其他植物成了研究的热点与重要领域,作为转基因受体的如番茄[31]、豌豆[32]、油菜[33]、大麦[34]、小麦[35]、葡萄[12]、番木瓜[36]等。笔者所在课题组前期也获得过表达STS的无核白植株[12,20-21]。研究证明转入外源STS,过表达植株对逆境胁迫的耐性增强并且使芪类物质积累。葡萄已被作为转基因STS及其产物白藜芦醇研究的模式植物,被广泛地用作抗性候选基因研究。将葡萄中STS作为改良植物抗性能力的目的基因,利用转基因技术在葡萄植物种内以及其他物种间进行遗传转化,从而提高植株抗病性。有研究表明,在植物体内,芪类物质有白藜芦醇、云杉新苷、葡萄素和紫檀芪[37],这些衍生物具有与白藜芦醇相似的作用[38],如葡萄素等能抑制霜霉菌菌丝生长[39]。本研究中,随着接种时间的增长,转STS植株芪类物质的量和种类均有所增加,特别是葡萄素的积累,说明转入的基因与获得的转基因植株具有抗病性。这与笔者课题组前期研究结果一致[12,40]。
在植物体内,通过苯丙氨酸代谢途径合成白藜芦醇[41]。合成包括一系列的酶反应,其中芪类化合物合成的关键酶是STS,它能够催化香豆酰-CoA和丙二酰-CoA合成白藜芦醇[42]。本研究通过qRT-PCR技术对转基因植株中与白藜芦醇合成相关的转录因子基因（MYB14、MYB15）、STS、苯丙氨酸解氨酶基因（PAL）、白藜芦醇糖基转移酶基因（RSGT）和查尔酮合成酶基因（CHS）进行了表达分析。结果表明在转基因株系中,在携带CaMV35S启动子植物表达载体增强基因表达情况下,与野生型相比,STS不同程度地表达上调,这与CHENG等[12]研究结果一致。CaMV35S属于组成型启动子,其整合到双子叶植物基因组后,能够启动在不同器官、发育时期外源基因高强度的表达,但其活性在单子叶植物中受到限制[43]。PAL作为苯丙氨酸代谢途径中的第一个关键酶,能够催化合成香豆酸辅酶A,在转VqSTS21的植株中表达上调,可能是由于STS表达上调增加了对香豆酸辅酶A的消耗。转基因植株中RSGT表达上调,其可催化白藜芦醇糖基化,转化为云杉新苷,由于STS表达上调使得白藜芦醇合成增加,导致RSGT表达上调。CHS与STS有共同的前体底物香豆酰辅酶A,转基因植株中CHS表达下调,可能是因为两者存在底物竞争关系[44]。H?LL等[45]研究发现,MYB14和MYB15能够正调控芪类物质的合成。本研究中,MYB14和MYB15不同程度地上调。因此,过表达VqSTS9和VqSTS21不仅提高了转基因植株STS的表达,还引起白藜芦醇合成途径中其他酶基因（PAL、RSGT）和相关转录因子基因（MYB14、MYB15）的上调表达,从而促进芪类物质的积累。
转基因技术是一种的重要分子育种手段,在葡萄中已有转化成功的例子。一般葡萄用于遗传转化的受体材料主要包括通过体细胞胚发生途径诱导的胚性愈伤组织和器官发生途径诱导的分生愈伤组织。胚性愈伤组织起源于单细胞,可避免嵌合体的形成,但其诱导时间较长,诱导率低,受季节影响,使得转基因效率较低;而器官再生是由多个细胞共同发育分化形成的,在遗传转化时会有嵌合体形成但其诱导时间短,再生成苗率高,受季节影响较小[46]。本研究基于课题组前期遗传转化体系进行。通过构建携带目的基因的植物过表达载体,转入欧洲葡萄无核白中,获得抗性植株。转基因定向改良性状是今后精准育种的重要途径之一,但葡萄遗传转化效率受多方面因素的影响,包括基因型、受体材料（类型、状态）、侵染菌液浓度、共培养时间、抗生素筛选浓度等。因此,优化遗传转化体系与提高遗传转化效率是今后葡萄转基因定向改良目标性状的瓶颈技术问题。
4 结论
中国野生毛葡萄‘丹凤-2’芪合酶基因VqSTS9、VqSTS21具有抗白粉病的功能,将VqSTS9、VqSTS21利用农杆菌转入欧洲葡萄无核白中,芪合酶基因表达产物种类与含量积累增加,表现出转基因植株对白病菌的抗性。因此,中国野生葡萄毛葡萄‘丹凤-2’是进行抗病育种的重要种质资源,VqSTS9、VqSTS21是重要的抗病基因资源,可进一步用作杂交亲本与欧洲葡萄品种杂交改进欧洲葡萄品种抗病性。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
[本文引用: 1]
DOI:10.1038/hortres.2015.20URLPMID:26504571 [本文引用: 1]
The most economically important disease of cultivated grapevines worldwide is powdery mildew (PM) caused by the ascomycete fungus Erysiphe necator. The majority of grapevine cultivars used for wine, table grape, and dried fruit production are derived from the Eurasian grape species Vitis vinifera because of its superior aroma and flavor characteristics. However, this species has little genetic resistance against E. necator meaning that grape production is highly dependent on the frequent use of fungicides. The integration of effective genetic resistance into cultivated grapevines would lead to significant financial and environmental benefits and represents a major challenge for viticultural industries and researchers worldwide. This review will outline the strategies being used to increase our understanding of the molecular basis of V. vinifera susceptibility to this fungal pathogen. It will summarize our current knowledge of different resistance loci/genes that have evolved in wild grapevine species to restrict PM infection and assess the potential application of these defense genes in the generation of PM-resistant grapevine germplasm. Finally, it addresses future research priorities which will be important in the rapid identification, evaluation, and deployment of new PM resistance genes which are capable of conferring effective and durable resistance in the vineyard.
URLPMID:18680422 [本文引用: 1]
DOI:10.1016/j.scienta.2013.06.031URL [本文引用: 1]
URLPMID:12167877 [本文引用: 1]
DOI:10.1007/s11240-014-0471-yURL [本文引用: 1]
DOI:10.1007/BF02124034URLPMID:844529 [本文引用: 1]
DOI:10.1038/361153a0URLPMID:8421520 [本文引用: 2]
Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.
[本文引用: 2]
DOI:10.1038/nrd2060URLPMID:16732220 [本文引用: 2]
Resveratrol, a constituent of red wine, has long been suspected to have cardioprotective effects. Interest in this compound has been renewed in recent years, first from its identification as a chemopreventive agent for skin cancer, and subsequently from reports that it activates sirtuin deacetylases and extends the lifespans of lower organisms. Despite scepticism concerning its bioavailability, a growing body of in vivo evidence indicates that resveratrol has protective effects in rodent models of stress and disease. Here, we provide a comprehensive and critical review of the in vivo data on resveratrol, and consider its potential as a therapeutic for humans.
DOI:10.1155/2016/3128951URLPMID:26798416 [本文引用: 1]
Natural stilbenes are an important group of nonflavonoid phytochemicals of polyphenolic structure characterized by the presence of a 1,2-diphenylethylene nucleus. Stilbenes have an extraordinary potential for the prevention and treatment of different diseases, including cancer, due to their antioxidant, cell death activation, and anti-inflammatory properties which associate with low toxicity under in vivo conditions. This review aims to discuss various approaches related to their mechanisms of action, pharmacological activities in animal models and humans, and potential chemoprevention in clinical studies. The biological activity of natural stilbenes is still incompletely understood. Furthermore, after administration to animals or humans, these molecules are rapidly metabolized. Thus pharmacokinetics and/or activities of the natural structures and their metabolites may be very different. Novel drug formulations have been postulated in order to improve stability and bioavailability, to minimize side effects, and to facilitate interaction with their domains in target proteins. These pharmacological improvements should lead stilbenes to become effective candidates as anticancer drugs.
DOI:10.1007/s00425-015-2459-1URLPMID:26781778 [本文引用: 7]
MAIN CONCLUSION: The stilbene synthase gene VqSTS6, from Chinese wild type Vitis quinquangularis accession Danfeng-2, increases the resveratrol content and pathogen resistance of transgenic plants of V. vinifera Thompson Seedless. This study successfully created transgenic plants of V. vinifera Thompson Seedless which overexpressed VqSTS6, cloned from Chinese wild type V. quinquangularis accession Danfeng-2. Western blot and qRT-PCR showed a variable range in transcript levels among transgenic lines. The resistance to powdery mildew (Uncinula necator) was particularly enhanced in lines most highly expressing VqSTS6. Compared with the non-transformed controls, trans-resveratrol and other stilbene compounds were significantly increased in the transgenic lines. The correlation between high resveratrol content and high pathogen resistance in transgenic grapes is discussed. We hypothesize that the fruit-specific, highly expressed gene VqSTS6 from Chinese wild V. quinquangularis accession Danfeng-2, is directly involved in the resveratrol synthesis pathway in grapes, and plays an important role in the plant's defense against pathogens. Genetic transformation of VqSTS6 explored the potential of the wild Chinese grape species for use in breeding, the results of which would raise both the disease resistance and the fruit quality of V. vinifera grapevines.
DOI:10.1007/s002990000203URLPMID:30754928 [本文引用: 1]
A stilbene synthase gene was isolated from three Vitis spp. (V. vinifera, V. labrusca and V. riparia). These genes were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into kiwifruit (Actinidia deliciosa) plants by Agrobacterium-mediated gene transfer. The introduced gene(s) were expressed and piceid (resveratrol-glucoside) rather than resveratrol was produced in the leaves of the transformants. Resveratrol produced by the action of the integrated gene(s) seems to be metabolized into piceid by an endogenous glycosyltransferase. Among the transformants obtained, the highest piceid content in the young leaves was 182 mug/g fresh weight. Although these transformants did not show resistance against Botrytis cinerea, which causes gray-mould disease, the fruits may have some beneficial effects on human health.
DOI:10.1021/jf060249lURLPMID:16787008 [本文引用: 1]
A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are available on piceid synthesis in transgenic apple fruit and the effects of its accumulation on levels of other phenolic compounds present in the fruit.
DOI:10.1093/pcp/pcj061URLPMID:16731548 [本文引用: 1]
Sorghum SbSTS1 was the first example of a stilbene synthase gene in monocots. Previously, we demonstrated that the gene was involved in defense responses. To examine its biochemical function in planta, SbSTS1 was overexpressed in transgenic Arabidopsis. Metabolite analysis revealed that cis-resveratrol glucoside (piceid) accumulated as the major stilbene in the transgenic lines. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode, up to 580 microg g(-1) FW of cis-piceid were detected in 2-week-old plants, which represent a convenient source of the cis-isomers for pharmacological investigations. Our results also suggested the presence of unknown stilbene isomerase activities in Arabidopsis.
DOI:10.1371/journal.pone.0057930URLPMID:23483945 [本文引用: 1]
Resveratrol has been clinically shown to possess a number of human health benefits. As a result, many attempts have been made to engineer resveratrol production in major cereal grains but have been largely unsuccessful. In this study, we report the creation of a transgenic rice plant that accumulates 1.9 microg resveratrol/g in its grain, surpassing the previously reported anti-metabolic syndrome activity of resveratrol through a synergistic interaction between the transgenic resveratrol and the endogenous properties of the rice. Consumption of our transgenic resveratrol-enriched rice significantly improved all aspects of metabolic syndrome and related diseases in animals fed a high-fat diet. Compared with the control animals, the resveratrol-enriched rice reduced body weight, blood glucose, triglycerides, total cholesterol, and LDL-cholesterol by 24.7%, 22%, 37.4%, 27%, and 59.6%, respectively. The resveratrol-enriched rice from our study may thus provide a safe and convenient means of preventing metabolic syndrome and related diseases without major lifestyle changes or the need for daily medications. These results also suggest that future transgenic plants could be improved if the synergistic interactions of the transgene with endogenous traits of the plant are considered in the experimental design.
DOI:10.1023/b:trag.0000034658.64990.7fURLPMID:15359598 [本文引用: 1]
When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.
DOI:10.1094/MPMI.2000.13.5.551URLPMID:10796021 [本文引用: 1]
Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.
[本文引用: 1]
URL [本文引用: 2]
URL [本文引用: 2]
[本文引用: 2]
[本文引用: 2]
[本文引用: 1]
DOI:10.1016/j.plaphy.2013.10.021URLPMID:24246671 [本文引用: 1]
Resveratrol is positively correlated with grapevine disease resistance and its consumption is also highly beneficial to human health. HPLC analyses showed that resveratrol content was significantly higher in most wild Chinese grapevines than in most European Vitis vinifera grapevine cvs. Fruit of the wild Chinese genotype Vitis quinquangularis Danfeng-2 contains much higher levels of resveratrol than some others. Because stilbene synthase is responsible for resveratrol biosynthesis, 41 full-length stilbene synthase genes were isolated from Danfeng-2 using the RACE method. A neighbor-joining tree of the STS family displayed high similarity between Danfeng-2 and V. vinifera cv. Pinot Noir. The content of the endogenous stilbene synthase family in tissues and the expression levels induced by powdery mildew were both higher in Danfeng-2 than in Pinot Noir. Moreover, expression in the berry was significantly higher than in the leaves. Our results demonstrated that resveratrol accumulation was consistent with endogenous STS gene expressions, and that both were higher in Danfeng-2 than in Pinot Noir. Therefore, STS genes and producing resveratrol from V. quinquangularis played more important role in Vitis resistance. Otherwise, the gene VqSTS6 was markedly higher than the other VqSTS genes in the six tissues/organs assayed by Real-time PCR, which will offer a useful basis for commercial application of resveratrol from Chinese wild grapes.
[本文引用: 2]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
URLPMID:6427224 [本文引用: 1]
Stilbene synthase ( resveratrol -forming) converts one molecule of rho- coumaroyl -CoA and three molecules of malonyl-CoA into 3,4',5- trihydroxystilbene . Following selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. The enzyme was found to be a dimer of estimated Mr = 90,000 exhibiting under denaturing conditions a subunit Mr of approximately 45,000. The isoelectric point was determined with pI = 4.8. The enzyme's high selectivity towards rho- coumaroyl -CoA (Km = 2 microM) as substrate qualified it as resveratrol -forming stilbene synthase. Structurally related CoA esters, e.g. dihydro-rho- coumaroyl -CoA and cinnamoyl-CoA, were converted less than 1/10 as efficiently as rho- coumaroyl -CoA. Malonyl-CoA (Km = 10 microM) could not be substituted by acetyl-CoA. The purified enzyme was free of chalcone synthase activity. Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.
DOI:10.1007/BF00036918URLPMID:2103451 [本文引用: 1]
A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groundnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.
URLPMID:21838877 [本文引用: 1]
DOI:10.1111/j.1467-7652.2004.00099.xURLPMID:17168899 [本文引用: 1]
Tomato (Lycopersicon esculentum Mill.) tissues were transformed with a grape (Vitis vinifera L.) stilbene synthase cDNA, transcriptionally regulated by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated new compounds, not present in either wild-type or vector-transformed plants. These were identified, by high-pressure liquid chromatography, as trans-resveratrol and trans-resveratrol-glucopyranoside. The amounts of trans-resveratrol and its piceid form were evaluated in the transgenic fruit. It was found that the content of the metabolite varied during fruit maturation to up to 53 microg/g fresh weight of total trans-resveratrol at the red stage of ripening. This metabolite accumulation was possibly dependent on a combination of sufficiently high levels of stilbene synthase and the availability of substrates. With the aim of verifing the metabolic impairment, the amounts of chlorogenic acid and naringenin in both transgenic and wild-type ripening fruit were compared and no dramatic variation in the synthesis profile of the two metabolites was noted. To our knowledge, no data are available on the assessment of the effects of the expression of the StSy gene on other antioxidant compounds present in tomato fruit. To establish whether the presence of a novel antioxidant molecule affected the redox regulation in transgenic tomato fruit cells, the effect of resveratrol accumulation on the naturally present antioxidant pool was analysed. We showed that, in transgenic fruit which accumulate trans-resveratrol, there is an increase in the levels of ascorbate and glutathione, the soluble antioxidants of primary metabolism, as well as in the total antioxidant activity. Conversely, the content of tocopherol and lycopene, which are membrane-located antioxidants, is not affected. Consistent with the increased antioxidant properties, the lipid peroxidation was lower in transformed than in wild-type fruit.
URLPMID:16802117 [本文引用: 1]
URLPMID:16160820 [本文引用: 1]
[本文引用: 1]
URLPMID:16173460 [本文引用: 1]
DOI:10.1007/s00425-004-1343-1URLPMID:15309535 [本文引用: 1]
The phytoalexin resveratrol (trans-3,5,4'-trihydroxy-stilbene), a natural component of resistance to fungal diseases in many plants, is synthesized by the enzyme trihydroxystilbene synthase (stilbene synthase, EC 2.3.1.95), which appears to be deficient or lacking in susceptible plants. Earlier workers isolated a stilbene synthase gene (Vst1) from grapevine (Vitis vinifera L.), which has subsequently been introduced as a transgene into a range of species to increase resistance of hosts to pathogens to which they were originally susceptible. Papaya (Carica papaya L.) is susceptible to a variety of fungal diseases, including root, stem, and fruit rot caused by the pathogen Phytophthora palmivora. Since resveratrol at 1.0 mM inhibited mycelium growth of P. palmivora in vitro, we hypothesized that papaya resistance to this pathogen might be increased by transformation with the grapevine stilbene synthase construct pVst1, containing the Vst1 gene and its pathogen-inducible promoter. Multiple transformed lines were produced, clonally propagated, and evaluated with a leaf disk bioassay and whole plant response to inoculation with P. palmivora. RNA transcripts of stilbene synthase and resveratrol glycoside were induced in plant lines transformed with the grapevine pVst1 construct shortly after pathogen inoculation, and the transformed papaya lines exhibited increased resistance to P. palmivora. The immature transformed plants appear normal and will be advanced to field trials to evaluate their utility.
URL [本文引用: 1]
URL [本文引用: 1]
[本文引用: 1]
URLPMID:21504880 [本文引用: 1]
[本文引用: 1]
URLPMID:22863370 [本文引用: 1]
DOI:10.1007/s00299-007-0307-xURLPMID:17340093 [本文引用: 1]
Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter.
DOI:10.1016/j.plaphy.2007.12.009URLPMID:18272377 [本文引用: 1]
As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure-function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.
URLPMID:24151295 [本文引用: 1]
[本文引用: 1]
