CN1034426C - 黄瓜花叶病毒的壳蛋白基因 - Google Patents
黄瓜花叶病毒的壳蛋白基因 Download PDFInfo
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Abstract
提供了黄瓜花叶病毒株C(CMV-C)的壳蛋白基因，制备它的方法，用它制取基因转移植物和包含这种基因的基因转移植物的方法。
Description
本发明涉及黄瓜花叶病毒株C(CMV-C)的壳蛋白基因，更具体说，本发明涉及一种制备所说的基因并把它掺入转移载体中的方法，还涉及将它用于生产对CMV病毒感染具有抗性的转化植物细胞和转化植株。
黄瓜花叶病毒(CMV)是一种单链(+)RNA植物病毒，它具有一个在功能上被区分的基因组。这种病毒基因组含有4种称为RNA1-4的RNA；分别为3389核苷酸、3035核苷酸、2193核苷酸和1027核苷酸(Peden and Symons，1973；Gould and Symons，1982；Rezaian等人，1984；Rezaian等人，1985)。对于感染来说只需要RNA1-3，因为RNA-3也编码由RNA4编码的壳蛋白(Peden和Symons，1973)。CMV RNA通过转译从RNA1产生一个95KDal的多肽，从RNA2产生一个94KDal多肽(Gordon等人，1983)，并从RNA3产生两种多肽，它的5＇端编码一种35KDal多肽，而3″端编码一种24.5KDal的多肽(Gould和Symons，1982)。该24.5KDal多肽与由RNA4编码的多肽是相同的，并且是壳蛋白。
CMV壳蛋白基因转移并整合到植物基因组中后；它不含有其表达所必需的信号。必须通过基因工程使它的转译起始密码(ATG)上游含有一个组成启动子，以及在转译终止密码下游加入多聚腺苷酸(PolyA)信号(AATAAA)。有好几种在植物中起作用的启动子可以利用，但是，我们认为最好的启动子是由CaMV、Ti基因的胭脂碱合酶(Bevan等人，1983)和章鱼碱合酶(Depicker等人，1982)以及大豆贮存蛋白(云扁豆蛋白)基因得到的那些组成启动子。从这些基因得到的多聚腺苷酸加入信号也适于我们的用途。
EP223452描述了对病毒病有抗性的植物以及产生这种植物的方法。其他文献还有An，G.等人(1985)的“用于高等植物转化的新的克隆载体”EMBOJ.4：277-284；Dodds，J.A.等人(1985)的“黄瓜花叶病毒株之间的交叉保护作用：宿主及接种物类型对于病毒粒子积累及攻击株的双链RNA的影响”Virology144：301-309；Pietrzak，M.等人(1986)的“用新的植物表达载体转化原生质体后两种细菌抗生素抗性基因在植物中的表达”Nuc.Acids Res.14：5857-5868。
本发明提供了：
从黄瓜花叶病毒株C(CMV-C)得到的壳蛋白基因。
一种含有从CMV-C得到的壳蛋白基因、花椰菜花叶病毒35S基因的启动子、以及花椰菜花叶病毒35S基因或菜豆(PhaseolusVulgavis)种子贮存蛋白基因的多聚腺苷酸信号的植物转化载体。
一种含有植物转化载体的细菌细胞，该载体含有得自CMV-C的壳蛋白基因、花椰菜花叶病毒35S基因的启动子、以及花椰菜花叶病毒35S基因或菜豆贮存蛋白基因的多聚腺苷化信号。
一种含有得自CMV-C的壳蛋白基因、花椰菜花叶病毒35S基因的启动子。以及花椰菜花叶病毒基因或菜豆种子贮存蛋白基因(云扁豆蛋白)的多腺苷化信号的转化植物细胞。
一种包含转化细胞的植物，该转化细胞含有CMV-C壳蛋白基因、花椰菜花叶病毒35S启动子、花椰菜花叶病毒基因或菜豆种子贮存蛋白基因(云扁豆蛋白)的多腺苷化信号。本发明的转化植物包括甜菜、柑桔类水果、玉米、黄瓜、辣椒、土豆、大豆、南瓜、西红柿。特别是葫芦科(如南瓜、黄瓜等)以及茄科(辣椒、西红柿等)的植物。
一种产生抗病毒植物的方法，包括繁殖一种表达黄瓜花叶病毒株C的壳蛋白基因的植物。特别是产生葫芦科和茄科植物的方法。
图1至图5用来表示本发明的构成。采用一些常规方法图示质粒和DNA片段：
(1)单线图代表环状和线状的双链DNA。
(2)星号(*)表示所代表的分子是环状的，没有星号表示该分子是线状的。
(3)功能成分的天然分界之间的结合用沿水平线的垂直线表示。
(4)指出基因或功能成分。
(5)基因和限制位点之间的距离不按比例，除非另有指出，这些图仅仅表示它们的相对位置。
在实施本发明中所采用的大部分重组DNA的方法是该领域中专业人员所熟知的标准方法。它们在例如EP223452中有详细描述，该专利被作为本文的参考文献。酶是由商业来源得到的，并按销售商建议的方式或该领域中已知的其他变化方式使用。各种试剂、缓冲剂和培养条件也都是该领域中的专业人员公知的。包括这些标准技术的一般参考资料有以下文献：R.Wu，ed(1979)Methods inEnzymology Vol.68：J.H.Miller(1972)Experiments in MolecularGenetics；T.Maniatis等人(1982)Molecular Cloning：ALaboratory Manual；D.M.Glovered.(1985)DNA CloningVol.II；所有这些都被作为本文的参考文献。
实例1CMV RNA的分离
在烟草植物中繁殖黄瓜花叶病毒株C(CMV-C)，并用Lot等人(Annals of Phytopathology4：25，1972)的方法分离出RNA。
实例2 CMV-C的克隆
(a)双链cDNA3的合成
将全部CMV-CRNA多腺苷化以提供一个寡聚dT引物的退火位点。反应缓冲液如下：5μl1MTrispH7.9；1μl1MMgCl2；2.5μl0.1MMnCl2；5μl5μNaCl；0.5μl100mMATP；18μl2.8mg/ml牛血清清蛋白，3.2μl这种缓冲液与2μgCMV-C的总RNA混合，加入3.8μl水和1μl多腺苷酸聚合酶，并将反应混合物在37℃保温10分钟。
将所得到的多腺苷化的RNA用于Polites和Marotti(Biotechniques4：514，1986)的cDNA合成方案中，所不同的是用0.75mMKcl代替50mMNaCl，以及用130μci/100μl32P-dcTP代替10-50μCi/100μl。
在ds-cDNA被合成后，用G-100柱层析纯化，用乙醇沉淀，并悬浮在20μl1×EcoRI甲基化酶缓冲液中(100nMNaCl，100mMTris-HClpH8.0，1mMEDTA，80μMS-腺苷基蛋氨酸，100μg/ml牛血清清蛋白)。取出2μl等分液用于随后的凝胶分析，对该反应混合物追加1μl32mM的S-腺苷基蛋氨酸和1μl(20单位)EcoRI甲基化酶，该反应在37℃下保温30分钟，并通过在70℃保温10分钟停止反应。
从上述反应物中取出2μl，并加入1μl(5单位)大肠杆菌DNA聚合酶I的Klenow片段。反应在37℃保温10分钟，然后用酚/氯仿提取，再用乙醇沉淀。用70％乙醇、再用70％乙醇/0.3M醋酸钠洗涤沉淀。
干燥这些沉淀物并重新悬浮在8μl0.5μg/μl的磷酸化的EcoRI连接子中(得自Collaborative Research，Inc，128Spring Street，Lexington，MA02173)。加入1μl10X连接酶缓冲液(800mMTris-HClpH8.0，200mMMgCl2，150mMDTT，10mMATP)以及1μlT4DNA连接酶(4单位/μl)，反应在15℃下保温过夜。
通过在65℃保温10分钟停止连接反应。加入60μl水，10μl10XEcoRI盐(900mMTrispH8.0，100mMMgCl2，100mMNaCl)以及10μlEcoRI(10单位/μl)，反应在37℃下保温1小时(开始时取出5μl的等分液用于随后的凝胶分析)。用酚/氯仿停止该反应，并用氯仿提取，取出5μl的等分液用于凝胶分析，将剩下的一半冻干后备用。另一半通过G-100柱层析纯化，含有cDNA的G-100组分通过丁醇提取进行浓缩，用乙醇沉淀，并再悬浮在10μl水中。取出3μl用于以后的分析，然后加入1μlλgtll臂(得自StratageneCo.，3770TandySt，San Diego，CA92121)，1μl10X连接酶缓冲液以及1μlT4DNA连接酶，反应在15℃下保温过夜。
按照包装提取剂制造商(Gigapack Plus，也是得自Stratagene)所建议的步骤将得到的连接了的λgtll/cDNA分子包装起来，这就得到重组λ噬菌体，按照本领域专业人员公知的方法将它进行平皿培养。
对由纯化的CMV白叶株(得自Dr.D.Gonsalves，Cornell Uni-versity，Geneva，NY)RNA4得到的单链cDNA进行放射性标记，然后用它进行杂交以鉴别含有壳蛋白基因的λ克隆。这种RNA4的单链cDNA以如下方式合成：如上面对总CMV-CRNA描述的那样使RNA4多腺苷化，所不同的是用5.8μgRNA4。以Po-lites和Marotti(Biote chniques4：514，1986)描述的方法合成第一链，所不同的是不含无放射性的dCTP，而代之以260μCi/100μl的放射性dcTP。
这种标记的单链cDNA通过P6柱层析而纯化，并用于探测从上述λ噬菌体平皿得到的复制滤片。单链cDNA与好几个噬菌体克隆的DNA杂交，表明它们至少含有一部分CMV-C壳蛋白基因。培养几个这种λ克隆，并按该领域中专业人员公知的方法由它们分离出DNA。
实例3包含CMV-C壳蛋白基因的pUC19克隆的构建
用标准方法将上面实例中的几个克隆的cDNA转移到质粒载体pUC19中(得自Bethesd aResearch，P.O.Box6009，Gaithersburg，Md20877)。然后用Maxam和Gilbert(Methods inEnzymology65：499，1980)所描述的方法测定在pUC19中克隆的片段的序列。以这种信息为依据，鉴别出一个含有完整壳蛋白基因的克隆。这一克隆(称为pCMV9.9)序列示于图1中。
实例4用CaMV35S多腺苷化信号构建包含可在植物中表达的CMV-C壳蛋白基因的微T-DNA质粒
为了连接CaMV35S启动子和多腺苷化信号，一个从AccI位点(311位置)延伸到EcoRI位点(1421位置)的片段从pCMV9.9中切下，并连接到载体pDH51(Pietrzak等人，1986)(得自ThomasHohn，Friedrich Miescher Institute，P.O.Box2543，CH-4002，Basel，Switzerland)的多克隆位点上。这是通过下述操作完成的：对pCMV9.9进行完全EcoRI和部分AccI的消化，从适当的AccI-EcoRI片段上产生一个平头分子(利用大肠杆菌DNA聚合酶I的Klenow片段)，并将它连接到pDH51的SmaI位点上(图2)。这种克隆称为pDH51/CP19，用Maxam-Gilbert的方法测定其序列以证实它适于在植物中表达。
将这种能在植物中表达的壳蛋白基因转移至适于农杆菌介导的基因转移的载体中。从pDH51/CP19上切下一个EcoRI-EcoRI片段，并将它置于pUC1813质粒的EcoRI位点上(pUC1813是得自RobertKay，Dept，of Chemistry，Washington State University，Pullman，Washington)，产生出质粒pUC1813/CP19。通过HindIII的部分消化切下一个包含可在植物中表达的基因的1.8Kb片段，并将它连接到载体pGA482上(An等人，1985)(得自CynehungAn，Institute of Biological Chemistry，Washington StateUniversity)。
得到的质粒被称为pGA482/CP19H(图3)。从包含该基因的pUC1813克隆中切下一个部分的BamHI-BamHI3片段，将它插入pGA482/CP19的BglII位点上，从而将可在植物中表达的葡糖苷酸酶基因(得自Clontech Laboratories，Inc.，4055Fabian Way，PaloAlto，CA)加在pGA482/CP19/H中。这种最终的构建体称为pGA482/CP19/GUS(图3)，并通过Maxam-Gilbert定序法加以证实。
可以将这种质粒或它的衍生物转移到农杆菌株A208、C58、LBA4404、C58Z707、A4RS、A4RS(pRi278b)及其他菌株中。由ATCC(12301Parklawn Drive，Rockville，MD)可得到菌株A208、C54、LBA4404和A4RS，从F.Casse-Delbart博士(C.N.R.A.，Routade Saint Cyr，F78000，Versailles，France)处可得到A4RS(pRi278b)，由A.G.Hepburn博士(University ofIllinois，Urbana，IL)处可得到C58Z707。
实例5用云扁豆蛋白多腺苷化信号构建包含可在植物中表达的CMV壳蛋白基因的微T-DNA质粒
通过从pDH51/CP19上切下AVrII-Xba片段用云扁豆蛋白的多腺苷化信号代替35S的多腺苷化信号。这种消化去除了包含壳蛋白克隆3＇端的DNA区(然而CMV壳蛋白的所有编码区保持完整)、人为引入的A-残基片段和部分pDH51多连接子。用一个从含有云扁豆蛋白多腺苷化信号的pUC1813克隆得到的XbaI-CbaI片段代替AvrII-XbaI片段。这种结构然后通过EcoRI消化切下并克隆到pUC1813的EcoRI位点上。此pUC1813克隆再用XbaI消化，并将由35S启动子、壳蛋白编码区和云扁豆蛋白多腺苷化信号组成的片段连接到pGA482的XbaI位点上。然后如前述加入葡糖苷酸酶基因以产生质粒pGA482/CP19A-/411GUS(图4)。通过Maxam-Gilbert测序法证实质粒的结构。
通过将由pUC1813(见上)得到的云扁豆蛋白多腺苷化信号置于pDH51/CP19的XbaI位点构建出另一种质粒(用于试验A-残基片段对表达的影响)。通过EcoRI消化，从pDH51/CP19上切下一个含有35S启动子、壳蛋白编码序列和云扁豆蛋白及35S多腺苷化信号的片段，并连接到pUC1813中。然后通过部分XbaI消化从该质粒上切下包含35S启动子、壳蛋白编码序列(包括人工合成的A-残基片段)以及云扁豆多腺苷化信号的片段，并连接到pGA482的XbaI位点上。随后如前述插入葡糖苷酸酶基因，得到的质粒称为pGA482/CP19/411/GUS(图5)，通过Maxam-Gilbert定序法证实其结构。
将这些质粒或它们的衍生物转移到农杆菌株A208、C58、LBA4404、C58Z707、A4RS、A4RS(pRi278b)以及其他菌株中，而它们又被用于通过一份美国专利申请中描述的方法将CMV-C壳蛋白基因插入到植物细胞中，该专利申请是在同一天提交的，标题为“发芽植物种子的农杆菌中介的转化”，并且作为本文的证据A(ExhibitA)。
Claims (6)
1.一种制备具有下列顺序的黄瓜花叶病毒株C(CMV-C)壳蛋白基因的方法，1 GAATTCCCGC CGCAATCGGG AGTTCTTCCG CGTCCCGCTC CGAAGCCTTC51 AGACCGCAGG TGGTTAACGG TCTTTAGTCA CTTTGGTGCG TATTAGTATA101 TAAGTATTTG TGAGTCTGTA CATAATACTA TATCTATAGA GTCCTGTGTG151 AGTTGATACA GTAGACATCT GTGACGCGAT GCCGTGTTGA GAAGGGATCA201 CATCTGGTTT TAGTAAGCCT ACATCATAGT TTTGAGGTTC AATTCCTCTT251 ACTCCCTGTT GAGTACCTTA CTTTCTCATG GATGCTTCTC CGACGAGATT301 GTCGTTATTG TCTACTGACT ATATAGAGAG TGTGTGTGCT GTGTTTTCTC351 TTTTGTGTCG TAGAATTGAG TCGAGTCATG GACAAATCTG AATCAACCAG401 TGCTGGTCGT AACCATCGAC GTCGTCCGCG TCGTGGTTCC CGCTCCGCCC451 CCTCCTCCGC GGATGCTAAC TTTAGAGTCT TGTCGCAGCA GCTTTCGCGA501 CTTAATAAGA CGTTAGCAGC TGGTCGTCCA ACTATTAACC ACCCAACCTT551 TGTAGGGAGT GAACGCTGTA GACCTAGGTA CACGTTCACA TCTATTACCC601 TAAAGCCACC AAAAATAGAC CGTGAGTCTT ATTACGGTAA AAGGTTGTTA651 CTACCTGATT CAGTCACGGA ATATGATAAG AAGCTTGTTT CGCGCATTCA701 AATTCGAGTT AATCCTTTGC CGAAATTTGA TTCTACCGTG TGGGTGACAG751 TCCGTAAAGT TCCTGCCTCC TCGGACTTAT CCGTTGCCGC CATCTCTGCT801 ATGTTCGCGG ACGGAGCCTC ACCGGTACTG GTTTATCAGT ATGCCGCATC851 TGGAGTCCAA GCCAACAACA AACTGTTGTT TGATCTTTCG GCGATGCGCG901 CTGATATAGG TGACATGAGA AAGTACGCCG TCCTCGTGTA TTCAAAAGAC951 GATGCGCTCG AGACGGACGA GCTAGTACTT CATGTTGACA TCGAGCACCA1001 ACGCATTCCC ACATCTGGAG TGCTCCCAGT CTGATTCCGT GTTCCCAGAA1051 CCCTCCCTCC GATCTCTGTG GCGGGAGCTG AGTTGGCAGT TCTACTACAA1101 ACTGTCTGGA GTCACTAAAC GTTTTACGGT GAACGGGTTG TCCATCCAGC1151 TTACGGCTAA AATGGTCAGT CGTGGAGAAA TCCACGCCAG CAGATTTACA1201 AATCTCTGAG GCGCCTTTGA AACCATCTCC TAGGTTTCTT CGGAAGGGCT1251 TCGGTCCGTG TACCTCTAGC GCAACGTGCT AGTTTCAGGG TACGGGTGCC1301 CCCCCACTTT CGTGGGGGCC TCCAAAAGGA GACCAAAAAA AAAAAAAAAA1351 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA1401 AAAAAAAAAAA AAAAAAAAA GAATTC该方法包括如下步骤：
选择已被CMV-C感染的植株；从CMV-C中分离RNA；构建CMV-C cDNA文库；合成一个其顺序与上述顺序互补或同源的寡核苷酸探针；通过与该寡核苷酸探针杂交而选择编码CMV-C壳蛋白基因的cDNA。
2.制备含CMV-C壳蛋白基因的植物转化载体的方法，该方法包括如下步骤：选择具有权利要求1中所示顺序的CMV-C壳蛋白基团；将该CMV-C壳蛋白基因亚克隆到一个微T DNA细菌质粒中，该质粒具有至少一个启动子、一个多腺苷化信号和一个植物可表达的NPTII基因，从而使CMV-C壳蛋白基因位于启动子的3＇端和多聚腺苷化信号的5＇端。
3.制备含植物转化载体的细菌细胞的方法，该方法包括如下步骤：按照权利要求2制备一个植物转化载体；将该植物转化载体转移到农杆菌中。
4.制备含CMV-C壳蛋白基因的转化植物细胞的方法，该方法包括如下步骤：按照权利要求3的方法制备含植物转化载体的细菌细胞；将该植物转化载体转移到植物细胞中。
5.一种制备含CMV-C壳蛋白基因的转基因植物的方法，该方法包括如下步骤：按照权利要求4制备含CMV-C壳蛋白基因的转化植物细胞；由该转化植物细胞再生植物。
6.一种制备病毒抗性植物的方法，该方法包括如下步骤：按照权利要求5的方法制备转基因植物；繁殖该转基因植物。
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|---|---|---|---|---|
| FR2651504B1 (fr) * | 1989-08-11 | 1993-09-10 | Biosem | Plantes transgeniques appartenant a l'espece cucumis melo. |
| EP0480310A3 (en) * | 1990-10-09 | 1992-11-19 | Takeda Chemical Industries, Ltd. | Virus-resistant tomato plants |
| JP3452372B2 (ja) * | 1992-10-21 | 2003-09-29 | 日本たばこ産業株式会社 | 組換えベクター及びそれを用いてジャガイモにｐｖｙ−ｔに対する免疫性を付与する方法並びにｐｖｙ−ｔ免疫性ジャガイモ |
| US6160201A (en) * | 1993-07-09 | 2000-12-12 | Seminis Vegetable Seeds, Inc. | Lettuce infectious yellows virus genes |
| WO1996021018A1 (en) * | 1994-12-30 | 1996-07-11 | Asgrow Seed Company | Plants resistant to c strains of cucumber mosaic virus |
| CN1086420C (zh) * | 1995-03-29 | 2002-06-19 | 北京大学 | 一种培育抗黄瓜花叶病毒病甜椒的方法 |
| IT1283631B1 (it) * | 1996-05-09 | 1998-04-23 | Metapontum Agrobios Srl | Metodo per la preparazione di piante transgeniche resistenti ad in- fezioni virali e piante cosi' ottenute |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4069157A (en) * | 1975-11-20 | 1978-01-17 | E. I. Du Pont De Nemours And Company | Ultrafiltration device |
| US6608241B1 (en) * | 1985-10-29 | 2003-08-19 | Monsanto Technology Llc | Protection of plants against viral infection |
| GB8601680D0 (en) * | 1986-01-23 | 1986-02-26 | Agricultural Genetics Co | Modification of plant viruses |
| AU616635B2 (en) * | 1986-04-02 | 1991-11-07 | Pioneer Hi-Bred International, Inc. | Virus resistant plants having coat protein |
| ES2076143T3 (es) * | 1986-04-02 | 1995-11-01 | Pioneer Hi Bred Int | Plantas resistentes a virus que tienen arn antisentido. |
| ES2026581T3 (es) * | 1987-02-20 | 1992-05-01 | Takeda Chemical Industries, Ltd. | Plantas resistentes a infeccion por virus de mosaico de pepino. |
-
1988
- 1988-12-08 DE DE3850261T patent/DE3850261T2/de not_active Expired - Fee Related
- 1988-12-08 AT AT89901429T patent/ATE107361T1/de not_active IP Right Cessation
- 1988-12-08 AU AU29276/89A patent/AU2927689A/en active Granted
- 1988-12-08 WO PCT/US1988/004321 patent/WO1989005858A1/en active IP Right Grant
- 1988-12-08 EP EP89901429A patent/EP0391972B1/en not_active Expired - Lifetime
- 1988-12-08 JP JP1501329A patent/JP3055787B2/ja not_active Expired - Lifetime
- 1988-12-08 AU AU29276/89A patent/AU621336B2/en not_active Ceased
- 1988-12-21 CN CN88109272A patent/CN1034426C/zh not_active Expired - Fee Related
-
1989
- 1989-08-21 KR KR1019890701559A patent/KR900700604A/ko not_active Application Discontinuation
-
1990
- 1990-05-22 DK DK126590A patent/DK126590A/da not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0391972A1 (en) | 1990-10-17 |
| KR900700604A (ko) | 1990-08-16 |
| JPH03501680A (ja) | 1991-04-18 |
| EP0391972B1 (en) | 1994-06-15 |
| AU621336B2 (en) | 1992-03-12 |
| DE3850261D1 (de) | 1994-07-21 |
| DK126590D0 (da) | 1990-05-22 |
| DE3850261T2 (de) | 1994-10-27 |
| JP3055787B2 (ja) | 2000-06-26 |
| WO1989005858A1 (en) | 1989-06-29 |
| CN1042182A (zh) | 1990-05-16 |
| AU2927689A (en) | 1989-07-19 |
| DK126590A (da) | 1990-05-22 |
| ATE107361T1 (de) | 1994-07-15 |
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