CN100523178C - 中国葡萄属野生种华东葡萄芪合成酶基因序列及其应用 - Google Patents
中国葡萄属野生种华东葡萄芪合成酶基因序列及其应用 Download PDFInfo
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Abstract
本发明涉及中国葡属野生种华东葡萄芪合成酶基因序列及其应用，本发明采用mRNA差异显示技术(mRNA differential display reverasetranscriptional-PCR，DDRT-PCR)结合末端快速扩增技术(Rapid amplificationof cDNA ends，RACE)克隆了中国葡萄属野生种华东葡萄(Vitispseudoreticulata)抗病相关基因-芪合成酶(stilbene synthase)基因家族成员7个，其大小分别为1288bp、1343bp、1411bp、1468bp、1492bp、1506bp和1556bp，它们均编码392个氨基酸。该基因编码产物芪合成酶能把1分子香豆酰辅酶A(P-Coumaroyl-CoA)和3分子丙二酰辅酶A(Malonyl-CoA)转化为白藜芦醇。白藜芦醇作为一种植保素不仅抵抗病菌，还有抗氧化、预防心血管病和防癌、降血脂等作用。本发明涉及通过转基因方法使小麦、番茄、水稻、玉米、大豆、油菜等植物具有合成白藜芦醇的能力，提高这些植物的抗病性和增加其营养保健功能。
Description
技术领域
本发明涉及植物抗病基因克隆及基因工程技术领域，特别涉及中国葡萄属野生种芪合成酶基因全长序列及其应用。
背景技术
白藜芦醇(Resveratrol)，化学名为3，4’，5-三羟基芪(3，4’，5-Trihydrolystilbene)，是植物体内一种天然的二苯乙烯类多酚物质。1924年就被发现，1974年在葡萄属植物中找到了该物质。目前已知白藜芦醇还存在于桑树、花生、买麻藤、朝鲜槐等12种31个属的72种植物中，就其含量而言葡萄属植物位居第一。在葡萄属植物中，中国野生葡萄部分种白藜芦醇的含量较高，如毛葡萄(Vitis quinquangularis)商-24、83-4-96和华东葡萄(Vitis pseudoreticulata)白河-35-1等。白藜芦醇作为葡萄属植物的抗逆性物质——一种植保素(phytoalexin)具有抗病菌的作用。Kreps研究发现Seyve Villard、Portugalske Modre、雷司令和米勒吐尔高等品种的叶片接种灰霉病病原菌(Botrytis cinerea)后，品种的抗病性与白藜芦醇的含量成正相关。Fregoni研究发现植物白藜芦醇含量高者对灰霉病的抗性强。在葡萄受到灰色葡萄孢(Botrytis cinerea)侵染后，距离坏死果实较近且没有受到侵染果实中的白藜芦醇含量很高，它对灰色葡萄孢、金黄色葡萄球菌、肺炎双球菌等均有明显抑制作用。Giesbrech等从哥伦比亚买麻藤(Gnetunpariculatium)中提取的白藜芦醇具有抗金黄色葡萄球菌(Slaphylococucsaureus)、表皮葡萄球菌(Sepidermidis)和耻垢分枝杆菌(Mycobacteriam Smegmtics)的作用。此外，白藜芦醇还有抗氧化、预防心血管病和防癌、降血脂等作用。
芪合成酶(Stilbene synthase)是白藜芦醇生物合成过程中起关键作用的酶。Schoppner和kindl(J.Biol.Chem.，1924)研究发现，芪合成酶能把1分子香豆酰辅酶A(P-Coumaroyl-CoA)和3分子丙二酰辅酶A(Malonyl-CoA)转化为白藜芦醇。Melchior等(FEBS Letters，1990)研究发现芪合成酶基因在植物中以基因家族存在，已从松树、花生、河岸葡萄(Vitis riparia)、欧洲葡萄(Vitis vinifera)等植物中分离了芪合成酶基因。
1992年美国波斯顿Dena-Farber癌症研究所的两位科学家P.Liang和A.D Pardee建立的mRNA差异显示技术(mRNA differential display reversetranscription-PCR，DDRT-PCR)(Science，1992)，由于该技术具有RNA用量少、灵敏度高，操作简便快速和可分析低丰度mRNA等特点，因而在探讨抗病机制及克隆新基因等方面得到了广泛的应用。
发明内容
本发明的目的在于，提供中国葡属野生种华东葡萄芪合成酶基因序列及其编码的芪合成酶活性蛋白应用。
为了实现上述目的，本发明首次采用mRNA差异显示技术进行中国葡萄属野生种抗白粉病(Uncinula necator schw.Burr.)基因克隆，获得了白粉病抗性基因-芪合成酶基因cDNA片段T11AC/B0320-723的核苷酸序列。以该核苷酸序列为基础，采用cDNA末端快速扩增技术(Rapid amplification ofcDNA ends，RACE)(Frohman et al，Proc.Natl.Acad.Sci，1988)获得了芪合成酶基因家族成员7个cDNA全长核苷酸序列。
本发明首次通过基因聚合技术将中国葡萄属野生种特有的VPSTS7、VPSTS6、VPSTS5、VPSTS4、VPSTS3、VPSTS2、VPSTS1等7个芪合成酶基因转入感病的欧洲葡萄品种、小麦、番茄、水稻、玉米、大豆、油菜等植物提高抗病性，提高这些植物功能性成分白藜芦醇的含量，人们取食这些植物食品时，能达到预防心血管病和防癌、降血酯等目的，也为进一步研究中国葡萄野生种芪合成酶基因表达机理提供分子依据。
附图说明
图1是5’RACE与3’RACE的图片；
图2是3’RACE cDNA片断酶切鉴定结果图片；
图3是中国葡萄属野生种华东葡萄(vitis pseudoreticulata)STS1基因全长序列；
图4是中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS2基因全长序列；
图5是中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS3基因全长序列；
图6中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS4基因全长序列；
图7是中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS5基因全长序列
图8是中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS6基因全长序列；
图9是中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS7基因全长序列；
图10中国葡萄属野生种芪合成酶基因核苷酸聚类分析图片；
图11中国葡萄属野生种芪合成酶基因扩增结果图片，其中符号为：1.PCR产物，M.DNA Marker DGL 2000；
图12.重组表达载体的双酶切鉴定图片，图中符号为：1、2重组表达载体；3、4.BamHI/XhoI双酶切过的重组表达载体；M.Marker；
图13融合蛋白的表达与纯化图片，图中符号为：1.诱导转空pGEX-4T-1菌体总蛋白；2.诱导重组菌体总蛋白；3.诱导重组菌体裂解液上清蛋白；4.诱导重组菌体裂解液沉淀蛋白；5.洗涤过的包涵体；6、7.复性并纯化的融合蛋白；
图14是中间表达载体的构建图；
图15是芪合成酶基因植物表达载体的构建图。
图16是OLYMPUS DP70荧光显微镜(蓝色滤光片)下观察结果，A：转化芪合成酶基因(STS)葡萄；B：未转化小麦；C：转化芪合成酶基因(STS)小麦。
以下结合附图对本发明作进一步详细描述。
具体实施方式
本发明获得的中国葡萄属野生种华东葡萄芪合成酶基因序列具体操作步骤如下：
A.采用改进的SDS/酚法(果树学报，张今今等，2003，20(3)：178-181)于接种当天，即第0天，及接种后1、3、5、7、9d采样提取中国葡萄属野生种华东葡萄白河-35-1叶片总RNA，反转录合成cDNA第一链。以cDNA第一链为模板，用dT11GG、dT11AG、dT11AC、dT11CC、dT11CG等5个锚定引物与Operon公司生产的S421～S440及B0301～B0326共46个随机引物组成了引物对(primer pairs)230个，进行了PCR扩增。经6％聚丙烯酰胺凝胶电泳分析DDRT-PCR产物、银染后，锚定引物5’TTTTTTTTTTTAC3’(dT11AC)与随机引物5’CATCAATCGC3’组成的引物对PCR扩增获得了中国葡萄野生种抗白粉病基因表达差异的cDNA片段T11AC/B0320-700，该片段在未接种的材料中没有表达，而在接种葡萄白粉病病原菌后表达；
B.用煮沸法(分子克隆实验指南第二版，J.萨姆布鲁克等著，金冬雁等译，科学出版社，1998)回收该差异cDNA片段，二次PCR扩增后，回收该差异cDNA片段并与pGEM-T easy载体连接，连接产物转化大肠杆菌DH5α感受态细胞，在含有Amp(100μg·ml-1)和X-gal/IPTG的LB平板上培养，蓝白斑筛选鉴定挑选白色菌落培养。用UNIQ-10柱式质粒小量抽提试剂盒回收质粒，用EcoR I酶切鉴定有外源DNA片段插入的阳性克隆。将阳性克隆送大连宝生生物公司测序，测序结果表明，差异cDNA片段T11AC/B0320-700核苷酸序列全长723bp；
C.以接种葡萄白粉病病原菌7d提取的中国葡萄属野生种华东葡萄白河-35-1叶片总RNA为模板，逆转录分别合成5’RACE与3’RACE cDNA第一链。用5’RACE引物GSP15’TTCGGAAGGTCCTCGGAATGTAACAGC3’与3’RACE引物GSP25’CAGGTGGAACTGTCCTTCGAACCGC3’分别进行了5’RACE与3’RACE PCR扩增。将5’RACE PCR产物7.0μl进行1.2％琼脂糖凝胶电泳获得一条大小约750bp(图1)，克隆测序后该cDNA片段实际大小为768bp。
取3’RACE PCR产物7.0μl进行1.2％琼脂糖凝胶电泳，获得了大小介于900～1000bp的5个cDNA片段(图1)。用DNA胶回收试剂盒将这5条cDNA片段分别回收、克隆，获得大量白斑重组子，每个cDNA片段克隆产物随机挑选5个白斑克隆进行酶切鉴定，5’-1的5个酶切产物产生了大不一致的1条片段，5’-2的5个酶切产物产生了大小不同的3条片段，5’-3、5’-4与5’-5的5个分别产生了大小不同的2条酶切片段，共产生了大小不同的cDNA片段10个(图2)。将产生这10个cDNA的重组克隆测序，序列大小分别为983bp、933bp、919bp、895bp、839bp、770bp、715bp、491bp、476bp、351bp；
D.由于所获得的5’RACE与3’RACE的cDNA片段有113bp的重叠，因此，将3’RACE获得的10个cDNA片段分别与5’RACE的1个cDNA片段拼接，除去重叠序列后得到10个cDNA序列，其大小分别为924bp、1049bp1064bp、1288bp(图3)、1343bp(图4)、1411bp(图5)、1468bp(图6)、1492bp(图7)、1506bp(图8)与1556bp(图9)。
E.将这10个cDNA序列联网到www.ncbi.nlm.nih.gov进行开放阅读框架分析，其中cDNA片段1556bp、1506bp、1492bp、1468bp、1411bp、1343bp与1288bp具有完整的开放阅读框架，均编码392个氨基酸，分别命名为VPSTS7、VPSTS6、VPSTS5、VPSTS4、VPSTS3、VPSTS2、VPSTS1；cDNA片段1064bp、1049bp与924bp具有不完整开放阅读框架。这样，我们就获得了中国葡萄属野生种芪合成酶基因家族成员7个cDNA全长序列。这7个基因核苷酸序列同源性较高，介于90％-100％之间(图10)。
经在GenBank上检索，它们与Vitis riparia芪合成酶基因(登录号为AB046373，全长1618bp)、Vitis labrusca芪合成酶基因(登录号为AB046374，全长1618bp)、Parthenocissus henryana芪合成酶基因(登录号为AY094615，全长1179bp)、Cissus rhombifolia芪合成酶基因(登录号为AY094616，全长1179bp)、Parthenocissus quinquefolia芪合成酶基因(登录号为AY094617，全长1179bp)的同源性约为94％，证明为新的中国葡萄属野生种特有的芪合成酶基因。
本发明的实施例1：
采用改进SDS/酚法(果树学报，2003，20(3)：178-181)，提取经白粉病病原菌诱导后的中国野生葡萄华东葡萄白河-35-1叶片总RNA。反转录合成cDNA第一链。根据已获得的中国葡萄属野生种芪合成酶基因核苷酸序列设计一对PCR引物：5‘TAGGATCCATGGCTTCAGTTGAGGAAAT3’与5‘GCCTCGAGTTAATTTGTAACCATAGG3’。在25μl反应混合液中包含cDNA第一链2.5μl、GSP1(10μm)1μl、GSP2(10μm)1μl、1.0μl dNTPs Mix(10mM)、1.0μl Taq DNA polymerase、PCR Buffer 2.5μl、PCR-Grade wat er34.5μl，混匀后上浮25μl矿物油。PCR扩增程序为94℃ 30s，68℃ 30s，72℃ 2min，35个循环；72℃ 10min。反应结束后，进行1.2％琼脂糖凝胶电泳回收该cDNA片断(图11)，并与pGEM-T easy载体连接，连接产物转化大肠杆菌DH5α感受态细胞，在含有Amp(100μg·ml-1)和X-gal/IPTG的LB平板上培养，蓝白斑筛选挑选白色菌落培养。用UNIQ-10柱式质粒小量抽提试剂盒回收质粒，用EcoR I酶切鉴定有外源DNA片段插入的阳性克隆。测序后该cDNA片段实际大小为1179bp，序列分析表明该cDNA片段为中国葡萄属野生种芪合成酶基因。
本发明的实施例2：
将实施例1获得的重组克隆载体双酶切后切下的目的片段与经同样双酶切过的原核表达载体pGEX-4T-1进行连接，转化感受态的大肠杆菌BL21细胞，提取质粒，BamHI/XhoI酶切鉴定，筛选阳性重组菌(图12)。重组菌经过IPTG诱导表达，离心，重悬于上样缓冲液(50mM Tris-HCl pH6.8，2％SDS，0.1％溴酚兰，10％甘油，100mM DTT)，沸水中煮3-5分钟，进行SDS-PAGE电泳。同时设定IPTG浓度梯度(0.1mM，0.2mM，0.3mM，0.4mM)及时间对照(1h，2h，3h，4h，5h，6h)，确定最佳表达条件为IPTG 0.3mM，30℃诱导5小时。
重组原核表达载体转化表达菌，37℃下0.3mM IPTG诱导5小时，经上样处理，进行SDS-PAGE电泳检测，与经同样诱导转化空质粒的菌样品总蛋白对比，重组表达菌在分子量65KD左右有一条特异表达带，证明目的基因在该表达系统获得了表达，经灰度扫描分析，目的蛋白表达量占菌体总蛋白21％(图13)。目的蛋白主要以包涵体形式表达，经过表达条件优化后仍不能改善目的蛋白可溶性，因此通过包涵体变性溶解，梯度复性，使目的蛋白能够与GST融合蛋白纯化介质结合、洗脱，从而使目的融合蛋白获得纯化(图13)。
本发明的实施例3：
将实施例1获得的重组克隆载体用BamHI/SacI切下1.1kb的STS基因片段，并定向插入经BamHI/SacI双酶切的中间表达载体(图14)上，构建植物表达载体(图15)。将构建好的植物表达载体经改进冻融法转化农杆菌，在附加卡那霉素和庆大霉素的YEB培养基上筛选，挑取抗性克隆，提取质粒DNA进行PCR鉴定，特异的扩增出1.1kb的条带，表明已成功构建了芪合成酶基因植物表达载体，并导入根癌农杆菌GV3101。
从YEB平板上挑取GV3101单菌落(携带目的基因的双元载体pWR-STS)，接种到20mlYEB液体培养基(附加Kan 60ug/ml，Gent 20ug/ml)，200rpm，28℃24h(0D600＝0.6)；取15ml菌液，4℃，5000rpm，5min收集沉淀，并用MS液体培养基(附加1mg/l BAP，0.1mg/l 2，4-D)重悬沉淀，并用上述培养基稀释至OD600＝0.6，加入AS，终浓度为20mg/l；从无菌试管苗取叶片，切成40mm2的叶盘，将其浸入事先准备好的农杆菌菌液中培养10min；取出叶盘置于无菌滤纸上，去除滤纸表面多余的农杆菌菌液；将上述叶盘转入MS培养基(附加1mg/l BAP，0.1mg/l2，4-D和1％PVPP)共培养3d(暗培养)；将共培养3d后的叶盘转入筛选培养基：MS(固)+1mg/l BAP+0.1mg/l 2，4-D+1％PVPP+15ug/ml Hyg B+400ug/lCef；两个月后进行GUS活性分析，取在选择培养基上培养2个月的愈伤组织进行GUS组织化学染色；同时可进行绿色荧光蛋白检测(图16)；生根培养基：NN69+1mg/l BAP+0.1mg/l IBA，4℃暗培养2周，然后25℃，16h光培养，8h暗培养，最后获得芪合成酶基因表达的转基因烟草和转基因葡萄新材料和新品种。
本发明的实施例4：
将实施例3获得的芪合成酶基因植物表达载体导入根癌农杆菌GV3101，采用根癌农杆菌法将芪合成酶基因成功导入番茄、大豆、油菜等优良品种，获得芪合成酶基因表达的转基因番茄、大豆、油菜等新种质和新材料(方法同实例3)。
采用基因枪法将芪合成酶基因导入玉米、小麦、水稻等优良品种。材料的预处理：在基因枪轰击前先将小麦外植体转入含渗透剂0.125M甘露醇和0.125M山梨糖醇的诱导培养基，将产生的淡黄色新鲜愈伤组织在转化前4~6h进行渗透处理，轰击材料集中置于培养皿(直径9cm)中心不大于3cm的范围内，25℃，暗培养。用70％乙醇对枪室和气体加速管消毒灭菌；将飞盘和可裂圆片在100％乙醇中浸泡5min消毒，然后在超净台中吹干；分别移取5ul金粉微粒悬浮液于每个飞盘(已安庄在固定环上)，在超净台中吹干(5min)。从A管(对照)中移取金粉微粒悬浮液，用于准备两个飞盘(供轰击平板1和2之用)，每个飞盘5ul；从B管中移取金粉微粒悬浮液各准备三个飞盘(分别用于轰击平板3～5)，每个飞盘5ul。一次可从每管金粉微粒悬浮液中取样准备5个飞盘，视试验设计而定；打开氦气瓶阀，调节压力调节器(在氦气瓶阀的另一端)，使发射压力达到1300psi(即高于可裂圆片承压值200～300psi)。
开动真空泵；将可裂圆片放在固定盖内，旋紧；将飞盘装好(带有微粒的一面朝下)，然后将它们放入枪室内(从上往下数第二层)；将含小麦愈伤组织的培养皿放在样品支架上，插入枪室内第五层，关上枪室门；打开真空阀，当真空度达到25～28英寸汞柱(inches of Hg)时，按下“Fire”按钮并保持此状态，直至听见气体破碎可裂圆片的声音为止；消除真空态，将样品取出；从步骤6开始重复操作，完成所有样品的处理；将用未包被DNA的金粉微粒轰击过的细胞(平板1和2)在非选择性培养基上23℃下暗黑培养48小时；将用包被pWR-STS质粒DNA的金粉微粒轰击过的细胞(平板3、4和5)在23℃下暗培养24小时；将轰击后的愈伤组织继续在渗透培养基中培养16h，然后转到原培养基中过渡培养2周获得转基因小麦植株(图16)。使这些不含白藜芦醇的植物也具有合成白藜芦醇的能力，提高他们对真菌病害的抗病性，同时也提高这些物种的营养保健功能，使人们通过吃这些事物，可以防止血小板凝聚、清除自由基、抗过敏、抗肿瘤等。此外，这些转基因材料也可以作为进一步通过杂交育种选育抗病和提高这些物种营养保健功能的种质。
基因序列
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS1基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC TGAGAATAAT GCAGGAGCAC GGGTTCTCGT GGTGTGCTCT GAGATCACCG
661 TTGTTACATT CTGTGGGCCT TCTGAAACTG CTTTGGACTC TTTAGTTGGC CAAGCCCTTT
721 TTGGTGATGG TTCTGCAGCT GTAATTGTTG GATCAGATCC AGATCTCTCG ATTGAACAAC
781 CACTCTTCCA GCTAGTCTCG GCAGCCCAAA CATTCATCCC CAATACCCAA GGTGCTATTG
841 CAGGCAACTT ACGTGAAGTG GGTCTCACCT TTCATTTATG GCCTAATGTG CCAACTTTGA
901 TCTCCGAAAA TATAGAGAAA TGCTTAACTC AAGCTTTTGA CCCAATTGGA ATCAGCGATT
961 GGAATTCCTT ATTTTGGATT GCTCATCCAG GTGGCCCAGC TATTCTTGAT GCAGTTGAAG
1021 CAAAATTGAG TTTGGATAAA CAAAAACTTA AAGCAACAAG ACATGTTCTA AGTGAATATG
1081 GGAACATGTC AAGGGCTTGT GTCTTGTTTA TTATGGACGA GATGAGAAAG AAATCGTTGA
1141 AGGAAGAAAA AACAACAACA GGTGAAGGAT TGGATTGGGG CGTCTTGTTT GGCTTTGGAC
1201 CAGGCCTAAC CATCGAGACT GTTGTCCTCC ACAGTATTCC TAGGGATTCC AATTGAGTGA
1261 AAAAAAAAAA AAAAAAAAAA AAAAAAAA
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS2基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GCAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT TCGTGGTCCT TCTGAAGATG CTTTGGACTC TTTAGTTGGC CAAGCCCTTT
721 TTGGTGATGG GTCTGCAGCT GTAATCGTTG GATCTGATCC AGATATCTCG ATTGAACGAC
781 CACTCTTTCA ACTTGTTTCA GCAGCCCAAA CATTTATTCC TAATTCAGCA GGTGCTATTG
841 CAGGCAACTT ACGTGAAGTG GGTCTCACCT TTCATTTGTG GCCTAATGTG CCTACCTTGA
901 TCTCCGAAAA CATTGAGAAT TGTTTGACTA AGGCTTTTGA CCCAATTGGT ATTAGTGATT
961 GGAATTCCTT ATTCTGGATT GCTCATCCAG GTGGCCCAGC TATTCTCGAC GCAGTTGAAG
1021 CAAAAGTCGG TTTAGATAAA CAGAAACTCA AAGCAACAAG GCATATTCTA AGTGAATATG
1081 GGAACATGTC AAGTGCATGT GTCTTATTTA TTTTGGACGA GATGAGAAAG AAATCGCTCA
1141 AGGAAGGCAA AACGACAACA GGTGAAGGAT TGGATTGGGG TGTCTTGTTT GGCTTTGGGC
1201 CAGGCCTAAC CATTGAGACC GTTGTGCTCC ATAGCGTTGG TACAGATTCA AACTAGGTGA
1261 AATGATGGGA AAAGTTATTC ATGAGTCATG TTTTATTAAT CAAATAAAAG GTAGTGAAAA
1321 AAAAAAAAAA AAAAAAAAAA AAA
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS3基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GCAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT TCGAGGACCT TCCGAAGATG CTTTGGATTC TTTAGTTGGT CAAGCTCTTT
721 TTGGTGATGG GTCTGCAGCT GTGATTGTTG GATCAGATCC AGATGTCTCA ATTGAAAGAC
781 CCCTCTTCCA GCTCGTCTCA GCAGCCCAAA CGTTTATTCC TAATTCAGCA GGTGCCATTG
841 CGGGTAACTT ACGTGAGGTG GGACTCACTT TTCACTTGTG GCCTAATGTG CCTACATTGA
901 TTTCCGAGAA TATAGAGAAA TACTTGACTC AAGCTTTTGA TCCACTTGGT ATTAGCGATT
961 GGAACTCGTT ATTTTGGATT GCTCACCCTG GTGGCCCTGC AATTCTTGAT GCAGTTGAAG
1021 CAAAACTCAA TTTAGATAAA AAGAAACTTG AAGCAACGAG GCATGTGCTA AGTGAGTATG
1081 GAAACATGTC AAGTGCATGT GTATTGTTTA TTTTGGATGA GATGAGGAAG AAATCCCTTA
1141 AGGGGGAAAA GGCCACCACA GGTGAAGGAT TGGATTGGGG AGTATTATTT GGTTTTGGGC
1201 CGGGCTTAAC CATAGAGACT GTTGTGTTGC ATAGCATTCC TATGGTTTCC AATTGAGTGC
1261 AAAAGAGTAA GAAAAATGAC AAATGATCCC TATCTTATTG AATTAAGGAG GTGTTAATAA
1321 AGATATATAC ATATATCTAC AAAGTTAATA ATACTCCTAA ATTTTTTTAT CCCTATCATA
1381 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS4基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GCAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT TCGTGGTCCT TCCGAAGATG CTTTGGACTC TTTAGTTGGC CAAGCCCTTT
721 TTGGTGATGG GTCTGCAGCT GTAATCGTTG GATCTGATCC AGATATCTCG ATTGAACGAC
781 CACTCTTTCA ACTTGTTTCA GCAGCCCAAA CATTTATTCC TAATTCAGCA GGTGCTATTG
841 CAGGCAACTT ACGTGAAGTG GGTCTCACCT TTCATTTGTG GCCTAATGTG CCTACCTTGA
901 TCTCCGAAAA CATTGAGAAT TGTTTGACTA AGGCTTTTGA CCCAATTGGT ATTAGTGATT
961 GGAATTCCTT ATTCTGGATT GCTCATCCAG GTGGCCCAGC TATTCTCGAC GCAGTTGAAG
1021 CAAAAGTCGG TTTAGATAAA CAGAAACTCA AAGCAACAAG GCATATTCTA AGTGAATATG
1081 GGAACATGTC AAGTGCATGT GTCTTATTTA TTTTGGACGA GATGAGAAAG AAATCGCTCA
1141 AGGAAGGCAA AACGACGACA GGTGAAGGAT TGGATTGGGG TGTCTTGTTT GGCTTTGGGC
1201 CAGGCCTAAC CATTGAGACC GTTGTGCTCC ATAGCGTTGG TACAGATTCA AACTAAGTGA
1261 AATGATGGGA AAAGTTATTC ATGAGTCATG TTTTATTAAT CAAATAAAAG GTAGTAAAGA
1321 TAATGAATTG TTATTTTACG ATTGTTGGAG GACCTCCTCA AAGACCTCAA GTTAAAGTCA
1381 TTGTTTATTC TTGTGTATGT TATCAAAATG AAGGAATAAT ATATCCGAGT TTTTGTTGAA
1441 AAAAAAAAAA AAAAAAAAAA AAAAAAAA
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS5基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GCAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT CCGAGGACCT TCCGAAGATG CTTTGGATTC TTTAGTTGGT CAAGCCCTTT
721 TTGGCGATGG GTCTGCAGCT GTGATTGTTG GATCAGATCC AGATGTCTCG ATTGAACGGC
781 CTCTTTTCCA ACTAGTTTCA GCAGCCCAAA CGTTTATTCC TAATTCAGCA GGTGCCATTG
841 CGGGTAACTT ACGTGAGGTG GGACTCACCT TTCACTTGTG GCCTAATGTG CCTACTTTGA
901 TTTCCGAGAA CATAGAGAAA TGCTTGACTC AAGCTTTTGA TCCACTTGGT ATTAGCGATT
961 GGAACTCGTT ATTTTGGATT GCTCATCCAG GTGGCCCTGC AATTCTTGAT GCAGTTGAAG
1021 CAAAACTCAA TTTAGATAAA AAGAAACTTG AAGCAACAAG ACATGTGTTG AGTGAGTATG
1081 GTAACATGTC AAGTGCATGT GTGTTGTTTA TTTTGGATGA GATGAGGAGG AAATCATTGA
1141 AGGGGGAAAA TGGCACCACA GGTGAAGGAT TGGATTGGGG AGTATTGTTC GGTTTTGGAC
1201 CAGGCTTGAC CATCGAGACC GTTGTGCTGC ATAGCGTACC TATGATTACA AATTGAGTGA
1261 AGAATGATAA GAAAAGTTAT ATACAAGCCA TGTCTTATTG TATTAAGGAG GTGTTAATGA
1321 AGATATATAT ATATCTCCAA AGCTTGTAAT AATCTTACAA TAATTTATTC CTATCATAAA
1381 ATTGACGGAT TGTAATTTAG TGATCATTGG AGATTGTAGA GACCTCAAGT TAAAAAAGTG
1441 AGACTTCAAG TTCTTGTCTT AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS6基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GTAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT TCGTGGTCCT TCCGAAGATG CTTTGGACTC TTTAGTTGGC CAAGCCCTTT
721 TTGGTGATGG GTCTGCAGCT GTAATCGTTG GATCTGATCC AGATATCTCG ATTGAACGAC
781 CACTCTTTCA ACTTGTTTCA GCAGCCCAAA CATTTATTCC TAATTCAGCA GGTGCTATTG
841 CAGGCAACTT ACATGAAGTG GGTCTCACCT TTCATTTGTG GCCTAATGTG CCTACCTTGA
901 TCTCCGAAAA CATTGAGAAT TGTTTGACTA AGGCTTTTGA CCCAATTGGT ATTAGTGATT
961 GGAATTCCTT ATTCTGGATT GCTCATCCAG GTGGCCCAGC TATTCTCGAC GCAGTTGAAG
1021 CAAAAGTCGG TTTAGATAAA CGGAAACTCA AAGCAACAAG GCATATTCTA AGTGAATATG
1081 GGAACATGTC AAGTGCATGT GTCTTGTTTA TTTTGGACGA GATGAGAAAG AAATCGCTCA
1141 AGGAAGGAAA GACAACCACA GGTGAAGGAT TGGATTGGGG TGTTTTGTTT GGCTTTGGGC
1201 CAGGCTTGAC CATTGAGACC GTTGTGCTAC ATAGCGTTCA AATGGATTCA AACTAAGTAA
1261 AATGATAGGA AAAACGATTC ACGAGCCATG TTCTATTAAC CAAATATAGG TAGTAAAGAT
1321 ATATCACCAT GAAATCATCT TTACTCAGAA TTTATTATTT GTTATTTATC GTTGTTGGAG
1381 GACTTTTTAA GAGCCCTCAA GTTAAAATCA TTGTTTATCC TTGTGTATGT TATCAAAATG
1441 AAAGAAATAA TGGATTTCTA TTTTGTTTAA AAAAAAAAGA AAAAAAAAAA AAAAAAAAAA
1501 AAAAAA
中国葡萄属野生种华东葡萄(Vitis pseudoreticulata)STS7基因全长序列
1 AACCCAGCTC CAAGAACGCT TCTCTTTCCT TCCTCAACTT AATCTTAAGC TTTCATTTCA
61 GTACGTAGCT GGCATCAATG GCTTCAGTTG AGGAAATTAG AAACGCTCAA CGTGCCAAGG
121 GTCCGGCCAC CATCCTAGCC ATTGGCACAG CTACTCCCGA CCACTGTGTC TACCAGTCTG
181 ATTATGCTGA TTACTATTTC AGGGTCACTA AGAGCGAGCA CATGACTGAG TTGAAGAAGA
241 AGTTCAATCG CATATGTGAC AAATCAATGA TAAAAAAGCG TTACATTCAT TTGACTGAAG
301 AAATGCTTGA AGAACATCCA AACATTGGTG CTTATATGGC TCCATCTCTT AACATACGCC
361 AAGAGATTAT CACAGCTGAG GTACCTAAGC TTGGTAAGGA AGCAGCATTG AAGGCACTTA
421 AAGAGTGGGG CCAGCCAAAG TCCAAGATCA CCCACCTTGT ATTTTGTACA ACCTCTGGTG
481 TAGAAATGCC TGGTGCTGAT TATAAACTTG CTAATCTCTT GGGTCTTGAA ACTTCAGTTA
541 GAAGAGTTAT GTTGTACCAT CAAGGGTGCT ATGCAGGTGG AACTGTCCTT CGAACCGCTA
601 AGGATCTTGC AGAGAATAAT GCAGGAGCAC GAGTTCTTGT GGTGTGCTCT GAGATCACTG
661 TTGTTACATT TCGCGGCCCT TCCGAAGATG CTTTGGACTC TTTAGTTGGC CAAGCCCTTT
721 TTGGTGATGG GTCTGCAGCT GTAATCGTAG GATCAGATCC GGATATCTCA ATTGAACGAC
781 CACTCTTCCA GCTTGTCTCA GCAGCCCAAA CATTTATTCC TAATTCTGCA GGTGCCATTG
841 CAGGAAACTT ACGTGAGGTG GGACTCACCT TTCATTTGTG GCCCAATGTG CCCACTTTAA
901 TTTCTGAGAA CATAGAGAAA TGTTTGACTC AGGCTTTTGA CCCACTTGGT ATTAGCGATT
961 GGAACTCGTT ATTTTGGATT GCTCACCCAG GTGGCCCTGC AATTCTTGAT GCAGTTGAAG
1021 CAAAACTCAA GTTAGATAAA AAGAAACTCG AAGCAACGAG GCATGTGCTA AGTGAGTATG
1081 GAAACATGTC AAGTGCATGT GTGTTGTTTA TTTTGGATGA GATGAGAAAG AAATCCCTTA
1141 AGGGGGAGAG GGCCACCACA GGTGAAGGAT TGGATTGGGG AGTATTATTC GGTTTTGGAC
1201 CAGGCTTGAC TATTGAAACT GTTGTGTTGC ATAGCATTCC TATGGTTACA AATTAAGTGA
1261 AGGAAAAGAG AATGGTCCCT TCAATGTCCT ATTATGTTGA ATAGGAGTAA GGTATTTATC
1321 TCCGAAACTA AATTATACTC TTAAACTATT TTATTATTTT TTCTAAATTT AGATTGTAAT
1381 CTAGTGATTG TTAGGCCCTC TTGGTGAGCT CAAATTAAAC GGTTGAGTTT CAAGTTCAGA
1441 CTGTTTTATT ATCTTGAAGA TTCCCCAAAC ATTGTAATGT TGTGTTCATA TGAACATTGT
1501 TGAAAAGTAA ATAAAAGAAA TATTGGAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA
Claims (3)
1.中国葡萄属野生种华东葡萄芪合成酶基因序列，其特征在于，该基因是STS1基因、STS2基因、STS3基因、STS4基因、STS5基因、STS6基因或者STS7基因，其中，STS1基因的1288bp全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc tgagaataat gcaggagcac gggttctcgt ggtgtgctct gagatcaccg 660
ttgttacatt ctgtgggcct tctgaaactg ctttggactc tttagttggc caagcccttt 720
ttggtgatgg ttctgcagct gtaattgttg gatcagatcc agatctctcg attgaacaac 780
cactcttcca gctagtctcg gcagcccaaa cattcatccc caatacccaa ggtgctattg 840
caggcaactt acgtgaagtg ggtctcacct ttcatttatg gcctaatgtg ccaactttga 900
tctccgaaaa tatagagaaa tgcttaactc aagcttttga cccaattgga atcagcgatt 960
ggaattcctt attttggatt gctcatccag gtggcccagc tattcttgat gcagttgaag 1020
caaaattgag tttggataaa caaaaactta aagcaacaag acatgttcta agtgaatatg 1080
ggaacatgtc aagggcttgt gtcttgttta ttatggacga gatgagaaag aaatcgttga 1140
aggaagaaaa aacaacaaca ggtgaaggat tggattgggg cgtcttgttt ggctttggac 1200
caggcctaac catcgagact gttgtcctcc acagtattcc tagggattcc aattgagtga 1260
aaaaaaaaaa aaaaaaaaaa aaaaaaaa； 1288
STS2基因1343bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gcaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt tcgtggtcct tctgaagatg ctttggactc tttagttggc caagcccttt 720
ttggtgatgg gtctgcagct gtaatcgttg gatctgatcc agatatctcg attgaacgac 780
cactctttca acttgtttca gcagcccaaa catttattcc taattcagca ggtgctattg 840
caggcaactt acgtgaagtg ggtctcacct ttcatttgtg gcctaatgtg cctaccttga 900
tctccgaaaa cattgagaat tgtttgacta aggcttttga cccaattggt attagtgatt 960
ggaattcctt attctggatt gctcatccag gtggcccagc tattctcgac gcagttgaag 1020
caaaagtcgg tttagataaa cagaaactca aagcaacaag gcatattcta agtgaatatg 1080
ggaacatgtc aagtgcatgt gtcttattta ttttggacga gatgagaaag aaatcgctca 1140
aggaaggcaa aacgacaaca ggtgaaggat tggattgggg tgtcttgttt ggctttgggc 1200
caggcctaac cattgagacc gttgtgctcc atagcgttgg tacagattca aactaggtga 1260
aatgatggga aaagttattc atgagtcatg ttttattaat caaataaaag gtagtgaaaa 1320
aaaaaaaaaa aaaaaaaaaa aaa； 1343
STS3基因1411bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gcaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt tcgaggacct tccgaagatg ctttggattc tttagttggt caagctcttt 720
ttggtgatgg gtctgcagct gtgattgttg gatcagatcc agatgtctca attgaaagac 780
ccctcttcca gctcgtctca gcagcccaaa cgtttattcc taattcagca ggtgccattg 840
cgggtaactt acgtgaggtg ggactcacttttcacttgtg gcctaatgtg cctacattga 900
tttccgagaa tatagagaaa tacttgactc aagcttttga tccacttggt attagcgatt 960
ggaactcgtt attttggatt gctcaccctg gtggccctgc aattcttgat gcagttgaag 1020
caaaactcaa tttagataaa aagaaacttg aagcaacgag gcatgtgcta agtgagtatg 1080
gaaacatgtc aagtgcatgt gtattgttta ttttggatga gatgaggaag aaatccctta 1140
agggggaaaa ggccaccaca ggtgaaggat tggattgggg agtattattt ggttttgggc 1200
cgggcttaac catagagact gttgtgttgc atagcattcc tatggtttcc aattgagtgc 1260
aaaagagtaa gaaaaatgac aaatgatccc tatcttattg aattaaggag gtgttaataa 1320
agatatatac atatatctac aaagttaata atactcctaa atttttttat ccctatcata 1380
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a； 1411
STS4基因1468bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gcaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt tcgtggtcct tccgaagatg ctttggactc tttagttggc caagcccttt 720
ttggtgatgg gtctgcagct gtaatcgttg gatctgatcc agatatctcg attgaacgac 780
cactctttca acttgtttca gcagcccaaa catttattcc taattcagca ggtgctattg 840
caggcaactt acgtgaagtg ggtctcacct ttcatttgtg gcctaatgtg cctaccttga 900
tctccgaaaa cattgagaat tgtttgacta aggcttttga cccaattggt attagtgatt 960
ggaattcctt attctggatt gctcatccag gtggcccagc tattctcgac gcagttgaag 1020
caaaagtcgg tttagataaa cagaaactca aagcaacaag gcatattcta agtgaatatg 1080
ggaacatgtc aagtgcatgt gtcttattta ttttggacga gatgagaaag aaatcgctca 1140
aggaaggcaa aacgacgaca ggtgaaggat tggattgggg tgtcttgttt ggctttgggc 1200
caggcctaac cattgagacc gttgtgctcc atagcgttgg tacagattca aactaagtga 1260
aatgatggga aaagttattc atgagtcatg ttttattaat caaataaaag gtagtaaaga 1320
taatgaattg ttattttacg attgttggag gacctcctca aagacctcaa gttaaagtca 1380
ttgtttattc ttgtgtatgt tatcaaaatg aaggaataat atatccgagt ttttgttgaa 1440
aaaaaaaaaa aaaaaaaaaa aaaaaaaa； 1468
STS5基因1492bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gcaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt ccgaggacct tccgaagatg ctttggattc tttagttggt caagcccttt 720
ttggcgatgg gtctgcagct gtgattgttg gatcagatcc agatgtctcg attgaacggc 780
ctcttttcca actagtttca gcagcccaaa cgtttattcc taattcagca ggtgccattg 840
cgggtaactt acgtgaggtg ggactcacct ttcacttgtg gcctaatgtg cctactttga 900
tttccgagaa catagagaaa tgcttgactc aagcttttga tccacttggt attagcgatt 960
ggaactcgtt attttggatt gctcatccag gtggccctgc aattcttgat gcagttgaag 1020
caaaactcaa tttagataaa aagaaacttg aagcaacaag acatgtgttg agtgagtatg 1080
gtaacatgtc aagtgcatgt gtgttgttta ttttggatga gatgaggagg aaatcattga 1140
agggggaaaa tggcaccaca ggtgaaggat tggattgggg agtattgttc ggttttggac 1200
caggcttgac catcgagacc gttgtgctgc atagcgtacc tatgattaca aattgagtga 1260
agaatgataa gaaaagttat atacaagcca tgtcttattg tattaaggag gtgttaatga 1320
agatatatat atatctccaa agcttgtaat aatcttacaa taatttattc ctatcataaa 1380
attgacggat tgtaatttag tgatcattgg agattgtaga gacctcaagt taaaaaagtg 1440
agacttcaag ttcttgtctt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa； 1492
STS6基因1506bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gtaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt tcgtggtcct tccgaagatg ctttggactc tttagttggc caagcccttt 720
ttggtgatgg gtctgcagct gtaatcgttg gatctgatcc agatatctcg attgaacgac 780
cactctttca acttgtttca gcagcccaaa catttattcc taattcagca ggtgctattg 840
caggcaactt acatgaagtg ggtctcacct ttcatttgtg gcctaatgtg cctaccttga 900
tctccgaaaa cattgagaat tgtttgacta aggcttttga cccaattggt attagtgatt 960
ggaattcctt attctggatt gctcatccag gtggcccagc tattctcgac gcagttgaag 1020
caaaagtcgg tttagataaa cggaaactca aagcaacaag gcatattcta agtgaatatg 1080
ggaacatgtc aagtgcatgt gtcttgttta ttttggacga gatgagaaag aaatcgctca 1140
aggaaggaaa gacaaccaca ggtgaaggat tggattgggg tgttttgttt ggctttgggc 1200
caggcttgac cattgagacc gttgtgctac atagcgttca aatggattca aactaagtaa 1260
aatgatagga aaaacgattc acgagccatg ttctattaac caaatatagg tagtaaagat 1320
atatcaccat gaaatcatct ttactcagaa tttattattt gttatttatc gttgttggag 1380
gactttttaa gagccctcaa gttaaaatca ttgtttatcc ttgtgtatgt tatcaaaatg 1440
aaagaaataa tggatttcta ttttgtttaa aaaaaaaaga aaaaaaaaaa aaaaaaaaaa 1500
aaaaaa； 1506
STS7基因1556bp的全长序列是：
aacccagctc caagaacgct tctctttcct tcctcaactt aatcttaagc tttcatttca 60
gtacgtagct ggcatcaatg gcttcagttg aggaaattag aaacgctcaa cgtgccaagg 120
gtccggccac catcctagcc attggcacag ctactcccga ccactgtgtc taccagtctg 180
attatgctga ttactatttc agggtcacta agagcgagca catgactgag ttgaagaaga 240
agttcaatcg catatgtgac aaatcaatga taaaaaagcg ttacattcat ttgactgaag 300
aaatgcttga agaacatcca aacattggtg cttatatggc tccatctctt aacatacgcc 360
aagagattat cacagctgag gtacctaagc ttggtaagga agcagcattg aaggcactta 420
aagagtgggg ccagccaaag tccaagatca cccaccttgt attttgtaca acctctggtg 480
tagaaatgcc tggtgctgat tataaacttg ctaatctctt gggtcttgaa acttcagtta 540
gaagagttat gttgtaccat caagggtgct atgcaggtgg aactgtcctt cgaaccgcta 600
aggatcttgc agagaataat gcaggagcac gagttcttgt ggtgtgctct gagatcactg 660
ttgttacatt tcgcggccct tccgaagatg ctttggactc tttagttggc caagcccttt 720
ttggtgatgg gtctgcagct gtaatcgtag gatcagatcc ggatatctca attgaacgac 780
cactcttcca gcttgtctca gcagcccaaa catttattcc taattctgca ggtgccattg 840
caggaaactt acgtgaggtg ggactcacct ttcatttgtg gcccaatgtg cccactttaa 900
tttctgagaa catagagaaa tgtttgactc aggcttttga cccacttggt attagcgatt 960
ggaactcgtt attttggatt gctcacccag gtggccctgc aattcttgat gcagttgaag 1020
caaaactcaa gttagataaa aagaaactcg aagcaacgag gcatgtgcta agtgagtatg 1080
gaaacatgtc aagtgcatgt gtgttgttta ttttggatga gatgagaaag aaatccctta 1140
agggggagag ggccaccaca ggtgaaggat tggattgggg agtattattc ggttttggac 1200
caggcttgac tattgaaact gttgtgttgc atagcattcc tatggttaca aattaagtga 1260
aggaaaagag aatggtccct tcaatgtcct attatgttga ataggagtaa ggtatttatc 1320
tccgaaacta aattatactc ttaaactatt ttattatttt ttctaaattt agattgtaat 1380
ctagtgattg ttaggccctc ttggtgagct caaattaaac ggttgagttt caagttcaga 1440
ctgttttatt atcttgaaga ttccccaaac attgtaatgt tgtgttcata tgaacattgt 1500
tgaaaagtaa ataaaagaaa tat tggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa。 1556
2.权利要求1所述的中国葡萄属野生种华东葡萄芪合成酶基因序列用于构建植物表达载体提高植物的抗病性的应用。
3.如权利要求2所述提高植物的抗病性的应用，其特征在于，通过基因工程使植物产生白藜芦醇，上述涉及的植物包括小麦、番茄、水稻、玉米、大豆、油菜。
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| CN102220350B (zh) * | 2010-04-15 | 2012-09-19 | 上海科爱生物技术有限公司 | 利用昆虫系统表达白藜芦醇芪合酶和制备白藜芦醇的方法 |
| US9840716B2 (en) * | 2012-12-17 | 2017-12-12 | Republic Of Korea (Management: Rural Development Administration) | Resveratrol-enriched rice and the use thereof |
| CN112410356B (zh) * | 2020-11-10 | 2023-07-18 | 杭州鑫旺途科技有限公司 | 来源于三叶青的白藜芦醇合酶基因rs及其应用 |
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| Title |
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| 中国葡萄属野生种抗白粉病基因克隆与序列分析. 王西平，65－72，77，西北农林科技大学. 2004 |
| 中国葡萄属野生种抗白粉病基因克隆与序列分析. 王西平，65－72，77，西北农林科技大学. 2004 * |
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