中国野生毛葡萄芪合成酶基因表达与抗白粉病分析
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摘要
【目的】葡萄是世界性重要果树,欧洲葡萄品种因其优质高产被广泛栽培,但其突出缺点是抗病性弱,尤其易受白粉病危害。葡萄中芪合成酶(stilbene synthase,STS)的代谢产物白藜芦醇是植物体内具有抗病功能的植保素,果实中的白藜芦醇对人具有保健作用。中国野生毛葡萄‘丹凤-2’抗病性强且白藜芦醇含量高。论文旨在研究中国野生毛葡萄‘丹凤-2’芪合成酶基因(STS)及其功能,应用于抗病育种来提高欧洲葡萄抗病性及果实中芪类物质含量。【方法】同源克隆中国野生毛葡萄‘丹凤-2’芪合成酶基因VqSTS26和VqSTS32,构建pCAMBIA35S::VqSTSs::GFP过表达载体;以器官发生途径诱导的无核白分生愈伤组织作为受体材料,采用农杆菌介导法进行遗传转化,获得转基因葡萄植株;分别从转录水平和代谢产物水平比较转基因与野生型无核白在自然生长条件与人工接种葡萄白粉病菌(Uncinula necator)诱导下STS表达及芪类物质产生与积累的差异;通过显微观察白粉病菌在转基因与野生型无核白叶片上的生长发育进程,统计孢子萌发、菌丝生长与分生孢子梗形成数目,对转基因植株进行抗病性分析。【结果】通过PCR检测和Western blot鉴定,获得了稳定转化VqSTS26植株8株和稳定转化VqSTS32植株5株。在自然生长条件下实时荧光定量PCR分析表明VqSTS26、VqSTS32转基因无核白STS的表达量显著提高,芪合成酶上游基因PAL与下游基因RSGT的表达量上调,而与芪合成酶存在底物竞争关系的CHS表达下调;液相色谱分析表明芪类物质主要以糖苷的反式云杉新苷形式存在,VqSTS26、VqSTS32转基因无核白芪类物质的含量极显著高于野生型无核白。STS的表达及其产物合成受白粉病菌诱导,随白粉病菌诱导,STS表达量在1—2 dpi时显著升高,至7 dpi时表达量达最高;诱导表达产生的芪类物质由原来的反式云杉新苷新增加了反式白藜芦醇和葡萄素,且含量增加;转基因植株在STS表达量、芪类物质的积累方面均极显著高于野生型无核白。显微观察白粉病菌在葡萄叶片上的生长发育状态,对比野生型无核白,转基因植株白粉病菌生长受到抑制,菌丝发育更迟,7 dpi时转基因葡萄叶片上的分生孢子梗数量低于野生型无核白。【结论】过表达中国野生毛葡萄‘丹凤-2’VqSTS26和VqSTS32可以提高无核白中STS的表达量,促进芪类物质的形成与积累,抑制转基因无核白叶片上白粉病菌的生长。因此,中国野生毛葡萄‘丹凤-2’与其携带的STS及其产物,是定向改良欧洲葡萄品种白粉病抗性与芪类物质含量的重要种质资源与基因资源。
【Objective】 Grapevine is an important fruit crop in the world. European grape is one of the most important cultivated grape varieties for its high quality and high yield. However, it is susceptible to fungal diseases, especially the powdery mildew. Resveratrol is a metabolite of stilbene synthase(STS) in grape. It is known as a phytoalexin and plays an important role in disease resistance. Besides, resveratrol in fruits has a health-care effect on humans. Chinese wild Vitis quinquangularis ‘Danfeng-2'displays resistance to powdery mildew and high resveratrol content. The objective of this study is to analyze the function of STS from Chinese wild V. quinquangularis ‘Danfeng-2', and to apply it to disease-resistance breeding to improve European grapes' disease-resistance and stilbenoids content. 【Method】 VqSTS26 and VqSTS32 were obtained by homologous cloning from Chinese wild V. quinquangularis ‘Danfeng-2', and the pCAMBIA35 S:: Vq STSs:: GFP overexpression vector was constructed. The meristem callus of V. vinifera Thompson Seedless was induced from the organogenesis, which was used as the receptor material. The transgenic grapes were obtained by agrobacterium-mediated genetic transformation. The expression of STS at transcriptional level and the content of stilbenoids at metabolite level were compared between transgenic plants and wild-type Thompson Seedless under natural growth conditions and Uncinula necator induction. The growth and development of U. necator on transgenic and wild-type grapes' leaves were observed by microscope. The spore germination, hyphal growth and conidiophore formation were counted, and the powdery mildew resistance of transgenic plants was analyzed. 【Result】 Eight transgenic plants with VqSTS26 and five transgenic plants with VqSTS32 were successfully obtained by PCR detection and Western blot identification. Real-time quantitative PCR analysis(qRT-PCR) showed that the expression of STS was significantly increased in transgenic grapes under natural condition, and the expression of its upstream gene PAL and downstream gene RSGT was up-regulated, while the expression of CHS was down-regulated due to the substrate competition with stilbene synthase. Liquid chromatographic analysis showed that the stilbenoids content in transgenic grapes was higher than that in wild-type Thompson Seedless, mainly existed in the form of trans-piceid. The expression of STS and its product synthesis were induced by U. necator. The expression of STS in transgenic grapes was significantly increased at 1-2 dpi(days post induction) under U. necator induction and was highest at 7 dpi, while trans-resveratrol and viniferin were added from original trans-piceid. The STS expression and its products' content in transgenic plants displayed excellently higher levels than those in wild-type Thompson Seedless under U. necator induction. U. necator developmental process on grape leaves was observed by microscope. It was found that the growth of U. necator on transgenic grapes was inhibited compared with that of wild-type Thompson Seedless, and the development of hyphae was later than that on wild-type Thompson Seedless. The number of conidiophore on transgenic grape leaves at 7 dpi was lower than that on wild-type Thompson Seedless. 【Conclusion】 Overexpression of VqSTS26 and VqSTS32 from ‘Danfeng-2' in Thompson Seedless can improve the expression of STS, promote the formation and accumulation of stilbenoids, and inhibit the growth of U. necator on transgenic Thompson Seedless leaves. Therefore, Chinese wild V. quinquangularis ‘Danfeng-2' and its carrying STS, as well as its products are important germplasm resources and genetic resources for the improvement of resistance to powdery mildew and content of stilbenoids in European grape varieties.
【Objective】 Grapevine is an important fruit crop in the world. European grape is one of the most important cultivated grape varieties for its high quality and high yield. However, it is susceptible to fungal diseases, especially the powdery mildew. Resveratrol is a metabolite of stilbene synthase(STS) in grape. It is known as a phytoalexin and plays an important role in disease resistance. Besides, resveratrol in fruits has a health-care effect on humans. Chinese wild Vitis quinquangularis ‘Danfeng-2'displays resistance to powdery mildew and high resveratrol content. The objective of this study is to analyze the function of STS from Chinese wild V. quinquangularis ‘Danfeng-2', and to apply it to disease-resistance breeding to improve European grapes' disease-resistance and stilbenoids content. 【Method】 VqSTS26 and VqSTS32 were obtained by homologous cloning from Chinese wild V. quinquangularis ‘Danfeng-2', and the pCAMBIA35 S:: Vq STSs:: GFP overexpression vector was constructed. The meristem callus of V. vinifera Thompson Seedless was induced from the organogenesis, which was used as the receptor material. The transgenic grapes were obtained by agrobacterium-mediated genetic transformation. The expression of STS at transcriptional level and the content of stilbenoids at metabolite level were compared between transgenic plants and wild-type Thompson Seedless under natural growth conditions and Uncinula necator induction. The growth and development of U. necator on transgenic and wild-type grapes' leaves were observed by microscope. The spore germination, hyphal growth and conidiophore formation were counted, and the powdery mildew resistance of transgenic plants was analyzed. 【Result】 Eight transgenic plants with VqSTS26 and five transgenic plants with VqSTS32 were successfully obtained by PCR detection and Western blot identification. Real-time quantitative PCR analysis(qRT-PCR) showed that the expression of STS was significantly increased in transgenic grapes under natural condition, and the expression of its upstream gene PAL and downstream gene RSGT was up-regulated, while the expression of CHS was down-regulated due to the substrate competition with stilbene synthase. Liquid chromatographic analysis showed that the stilbenoids content in transgenic grapes was higher than that in wild-type Thompson Seedless, mainly existed in the form of trans-piceid. The expression of STS and its product synthesis were induced by U. necator. The expression of STS in transgenic grapes was significantly increased at 1-2 dpi(days post induction) under U. necator induction and was highest at 7 dpi, while trans-resveratrol and viniferin were added from original trans-piceid. The STS expression and its products' content in transgenic plants displayed excellently higher levels than those in wild-type Thompson Seedless under U. necator induction. U. necator developmental process on grape leaves was observed by microscope. It was found that the growth of U. necator on transgenic grapes was inhibited compared with that of wild-type Thompson Seedless, and the development of hyphae was later than that on wild-type Thompson Seedless. The number of conidiophore on transgenic grape leaves at 7 dpi was lower than that on wild-type Thompson Seedless. 【Conclusion】 Overexpression of VqSTS26 and VqSTS32 from ‘Danfeng-2' in Thompson Seedless can improve the expression of STS, promote the formation and accumulation of stilbenoids, and inhibit the growth of U. necator on transgenic Thompson Seedless leaves. Therefore, Chinese wild V. quinquangularis ‘Danfeng-2' and its carrying STS, as well as its products are important germplasm resources and genetic resources for the improvement of resistance to powdery mildew and content of stilbenoids in European grape varieties.
引文
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[2]贺普超.葡萄学.北京:中国农业出版社,1999.HE P C.Viticulture.Beijing:China Agriculture Press,1999.(in Chinese)
[3]贺普超,王跃进,王国英,任志邦,和纯成.中国葡萄属野生种抗病性的研究.中国农业科学,1991,24(3):50-56.HE P C,WANG Y J,WANG G Y,REN Z B,HE C C.The studies on the disease-resistance of Vitis wild species originated in China.Scientia Agricultura Sinica,1991,24(3):50-56.(in Chinese)
[4]GOMèS E,COUTOS-THéVENOT P.Molecular aspects of grapevinepathogenic fungi interactions//ROUBELAKIS-ANGELAKIS K A.Grapevine Molecular Physiology and Biotechnology.2nd ed.Springer Science+Business Media B.V.,2009:407-428.
[5]RIAZ S,DOLIGEZ A,HENRY R J,WALKER M A,KOLE C.Grape.Fruits and Nuts,2007,39(4):63-101.
[6]BISSON L F,WATERHOUSE A L,EBELER S E,WALKER M A,LAPSLEY J T.The present and future of the international wine industry.Nature,2002,418(6898):696-699.
[7]WANG Y,LIU Y,HE P,CHEN J,LAMIKANRA O,LU J.Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species.Vitis,1995,4:131-138.
[8]ZHOU Q,DU Y,CHENG S,LI R,ZHANG J,WANG Y.Resveratrol derivatives in four tissues of six wild Chinese grapevine species.New Zealand Journal of Crop and Horticultural Science,2015,43(3):204-213.
[9]SCHNEE S,VIRET O,GINDRO K.Role of stilbenes in the resistance of grapevine to powdery mildew.Physiological and Molecular Plant Pathology,2008,72:128-133.
[10]SMOLIGA J M,BAUR J A,HAUSENBLAS H A.Resveratrol and health-A comprehensive review of human clinical trials.Molecular Nutrition and Food Research,2011,55(8):1129-1141.
[11]JANG M,CAI L,UDEANI G O,SLOWING K V,THOMAS C F,BEECHER C W W,FONG H H S,FARNSWORTH N R,KINGHORN A D,MEHTA R G,MOON R C,PEZZUTO J M.Cancer chemopreventive activity of resveratrol,a natural product derived from grapes.Science,1997,275(5297):218-220.
[12]RUPPRICH N,KINDL H.Stilbene synthases and stilbenecarboxylate synthases,I Enzymatic synthesis of 3,5,4-trihydroxystilbene from p-coumaroyl coenzyme A and malonyl coenzyme A.Hoppe-Seyler's Zeitschrift für Physiologische Chemie,1978,359(2):165-172.
[13]LISWIDOWATI,MELCHIOR F,HOHMANN F,SCHWER B,KINDL H.Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells.Planta,1991,183(2):307-314.
[14]HART J H.Role of phytostilbenes in decay and disease resistance.Annual Review of Phytopathology,1981,19:437-458.
[15]PARAGE C,TAVARES R,RéTY S,BALTENWECK-GUYOT R,POUTARAUD A,RENAULT L,HEINTZ D,LUGAN R,MARAIS G,AUBOURG S,HUGUENEY P.Structural,functional,and evolutionary analysis of the unusually large stilbene synthase gene family in grapevine.Plant Physiology,2012,160(3):1407-1419.
[16]HAIN R,BIESELER B,KINDL H,SCHR?DER G,ST?CKER R.Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol.Plant Molecular Biology,1990,15(2):325-335.
[17]HAIN R,REIF H J,KRAUSE E,LANGEBARTELS R,KINDLH,VORNAM B,WIESE W,SCHMELZER E,SCHREIER P H,ST?CKER R H,STENZEL K.Disease resistance results from foreign phytoalexin expression in a novel plant.Nature,1993,361(6408):153-156.
[18]STARK-LORENZEN P,NELKE B,H?N?LER G,MüHLBACH H P,THOMZIK J E.Transfer of a grapevine stilbene synthase gene to rice(Oryza sativa L.).Plant Cell Reports,1997,16(10):668-673.
[19]NICOLETTI I,DE ROSSI A,GIOVINAZZO G,CORRADINI D.Identification and quantification of stilbenes in fruits of transgenic tomato plants(Lycopersicon esculentum Mill.)by reversed phase HPLC with photodiode array and mass spectrometry detection.Journal of Agricultural and Food Chemistry,2007,55(9):3304-3311.
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