CN87105764A - 2-氨基-4-甲膦酰丁酸抗性基因及其应用 - Google Patents
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Abstract
通过在绿色产色链霉菌DSM4112中选择2-氨基-4-甲膦酰-丙氨酰-丙氨酸(PTT)抗性而产生PTT抗性的选择体。用BamHI酶切这些选择体的总DNA得到携有Phosphinothricin(PTC)抗性基因的DNA片段，克隆4.0Kb大小的片段并选择PTT抗性。此基因适用于生产PTC抗性的植物，以及作为抗性标记用于L型外消旋PTC的选择性N-乙酰化作用。
Description
Phosphinothricin（PTC，2-氨基-4-甲膦酰丁酸）是谷氨酰胺合成酶的抑制剂。PTC是抗生素2-氨基-4-甲膦酰-丙氨酰-丙氨酸的“结构单位”。此三肽（PTT）具有抗革兰氏阳性和革兰氏阴性细菌以及抗真菌灰葡萄孢（Botrytis cinerea）的活性（Bayer等人，Helv.Chim.Acta 55∶224，1972）。PTT是由绿色产色链霉菌（Streptomyces viridochromogenes）Tu 494菌株（DSM40736，DSM4112）产生的。
德国专利2，717，440号公开了PTC可起全除莠剂（total herbicide）的作用。已公开的PCT申请WO86/02097号描述了植物对PTC的抗性是归因于谷氨酰胺合成酶的过量生成。这种类型的过量生成（如从基因扩增所导致的）易遭受不稳定的风险。因此，这样一种不稳定性将带来谷氨酰胺合成酶过量生成的下降，将致使重新出现PTC的竞争性抑制作用。
与之相反，在权利要求中所限定的本发明涉及一种PTC抗性基因及其在生产PTC抗性植物中的应用。此外，该基因还可作为抗性标记使用。而且，该基因可适于L型外消旋PTC的选择性N-乙酰化作用。
本发明的PTC抗性基因能由下述步骤得到：用BamHⅠ酶切以PTT抗性选择出来的绿色产色链霉菌DSM4112菌株的总DNA，克隆大小为4.0Kb的片段，然后选择PTT抗性。限制性图谱（图1）详细描绘了该4.0Kb片段的特征。
对该4Kb片段部分进行克隆实验，以更精确地确定编码区的位置。此实验结果表明，抗性基因位于1.6Kb的SstⅡ-SstⅠ片段上（图1中位置0.55至2.15）。用BglⅡ消化产生0.8Kb大小的片段，此片段掺入质粒中并转化变铅青链霉菌（S.lividans）后赋予PTT抗性。此抗性是由PTC的N-乙酰化产生的。
对此0.8Kb片段进行Maxam和Gilbert序列分析得到DAN序列Ⅰ（见附页）。抗性基因的位置可从这一序列的开放读码（open reading frame）确定（从位置258起）。此基因读码终点位于图示的倒数第二个核苷酸（位置806），即最后一个核苷酸（位置807）是终止密码子的第一个核苷酸。
用下加线条强调标明了DNA序列Ⅰ中的Shine-Dalgarno序列，作为起始密码子的GTG也用线条标出。而且，顶部线条标出了确定的读码。
DNA序列Ⅱ显示已确定了序列的基因中的限制性切点。没有标出切断此序列六次以上的酶。
抗生素PTT由细菌摄取并被降解为PTC。后者也抑制细菌中的谷氨酰胺合成酶，致使细菌因缺乏谷氨酰胺而死亡。因此，产生PTT的细菌应当具有保护自身不受PTT影响的机理，也就是说，防止再度摄取已生产的PTT，或者对已降解的产物PTC进行修饰。但令人吃惊的是，PTT生产菌绿色产色链霉菌DSM4112对其自身的抗生素敏感。但出人意料地证实，通过选择PTT抗性，有可能以10-5的极高频率发现抗PTT的选择体，而且，此选择体还能抑制相邻菌落的本底生长。
通过分离此DNA、用BamHⅠ切断并连接到链霉菌载体中建立了这些选择体DNA的基因库。将连接混合物转化到市售的变铅青链霉菌TK23菌株中，每1μg连接混合物产生大约5000至10000个含有大约1至5Kb插入段的转化体。在这些转化体中有PTT抗性变铅青链霉菌株。有可能通过质粒分离并重新转化到变铅青链霉菌中，证明该抗性是由质粒编码的。负责此抗性的基因位于4Kb BamHⅠ片段上（图1）。编码区位于0.8Kb BglⅡ片段上。BamHⅠ片段中不含下列酶的切点：ClaⅠ、EcoRⅠ、EcoRⅤ、HindⅢ、HpaⅠ、KpnⅠ、PvuⅠ、PvuⅡ和XhoⅠ。
将抗性基因的限制性图谱（还没有详细确定其特征）与吸水链霉菌（S.hygroscopicus）FERM BP-130/ATCC21705（欧洲专利申请，公开号0，173，327，图7）相比较表明，本发明的抗性基因不同于已知基因，其为寻找PTT生物合成基因过程中发现的。
一方面使绿色产色链霉菌DSM4112和变铅青链霉菌TK23的细胞提取物与PTC和乙酰辅酶A保温，另一方面使PTT抗性的绿色产色链霉菌选择体和携有质粒的变铅青链霉菌转化体与PTC和乙酰辅酶A保温，有可能表明后面的细胞将具有乙酰化活性。层析分析表明乙酰化发生在氨基上。
由于大肠杆菌中也已发现了PTT抗性，而且抗性机理也在革兰氏阴性细菌中起作用，因此，有可能基于运送现象排除抗性。这样，与植物启动子偶联并使用合适的载体后，本发明的抗性基因可转化到植物中，从而可产生PTC抗性植物。
PTC的N-乙酰化还可用于合成的D，L-PTC的外消旋体拆开，因为只有L型发生选择性乙酰化作用。
因此，本发明还涉及使用抗性基因选择性地使外消旋PTC的L型发生N-乙酰化。
因此，本发明的抗性基因编码的PTC乙酰转移酶可用于分离外消旋PTC，例如，使外消旋体经受此酶的乙酰化作用。可使由德国专利2，717，440的方法所得到的外消旋PTC成为光学对映体，因为此酶只选择性地攻击L型，而D型则保持不变。然后，根据二者性质的差别，通过已知方法可将这样得到的混合物分离开。
已公开了这样一种方法：使N-酰基-D，L-氨基酸与固定在适当载体上的酰基转移酶接触，选择性地释放L-氨基酸，它可以在酸化以后用不与水混溶的溶剂从与N-酰基-D-氨基酸的混合物中萃取出来（见英国专利1，369，462号）。已经公开了N-酰基-D，L-PTC的相应的分离方法，如见于德国专利公开2，939，269号或美国专利4，226，941号。
根据本发明保持不变的D-PTC可通过已知方法外消旋（见公开号为EP-A-0，137，371的欧洲专利申请，实例8），然后回到流程中。
有可能但并不一定要分离出酶，这里及下文中所说的“酶”总是意指具有酶促活性的部分。如果酶被分离出来，它可以以游离形式或在载体上的固定化形式使用。合适载体的例子已在欧洲专利-A0，141，223号中描述了。但方便的作法是不分离酶，而是使用任何预期可表达本发明之酶的PTC抗性细胞。因此，有可能方便地使用绿色产色链霉菌DSM4112的PTT抗性选择体。而且，有可能有利地使用任何用本发明之基因转化的预期细胞，并使之能够表达PTC乙酰转移酶。就此而论，本发明的基因（也是指其活性部分）可以以质粒整合形式或利用其它常规的基因操作方法，例如通过转染引入到宿主细胞中。例如，掺入大肠杆菌的表达质粒中并用这样的质粒转化大肠杆菌是方便可行的，例如可通过欧洲专利-A0，163，249号和0，171，024号中介绍的已知方法进行。
对于外消旋体中的L-PTC的N-乙酰化，根据本发明，可以游离或固定化形式使用表达PTC乙酰转移酶的细胞，使用常规的固定化技术进行固定（例如德国专利公开3，237，341及其中引用的文献）。
本发明中L-PTC的酶促乙酰化以常规的酶促反应方式进行，方法中的条件由所用生物的性质来确定。从原则上讲，适用于此的方法与上述选择性脱酰作用的方法相同。
下述实例进一步阐明了本发明。除非特别指出者外，其中的份数和百分数数据均是指重量。
实例1 PTT抗性选择体
在基本培养基中（Hopwood等人，《链霉菌的基因操作：实验室手册》，The John Innes Foundation Norwieh，England，p.233，1985）培养绿色产色链霉菌DSM4112菌株，加入渐增浓度的PTT。在100μg/ml的浓度下，大约在每10个菌落中发现一个抗性菌落。
实例2载体的制备
用BglⅡ切割质粒pSVHⅠ（欧洲专利0，070，522号，美国专利4，673，642号），分离大小约7.1Kb的片段并与1.1Kb具有硫链丝菌素抗性的BglⅠ片段连接（欧洲专利申请，公开号0，158，201）。得到大小约为8.15Kb的质粒pEB2（图2）。
实例3抗性基因的分离
从实例1得到的选择体中分离总DNA，并用BamHⅠ酶切。同样用BamHⅠ打开质粒pEB2，使两种混合物结合并连接。将连接混合物转化到并铅青链霉菌TK23（可从The John Innes Foundation得到）中，每1微克连接混合物得到5000至10000个具有大约1～5Kb插入段的转化体。选择PTT抗性产生两个具抗性的变铅青链霉菌菌落。从抗性菌落中分离出质粒并用BamHⅠ酶切。发现一个携有负责抗性的基因的4Kb BamHⅠ片段。此质粒被称为pPRⅠ（图3）。
将其再转化到变铅青链霉菌TK23中，证明PTT抗性是由质粒编码的，因为转化体能在含有100μg/mlPTT的基体培养上生长。
实例4证明N-乙酰化使PTC失活
检查下列菌株以证实克隆片段的乙酰化活性：绿色产色链霉菌DSM40736，绿色产色链霉菌（PTT抗性突变株）、变铅青链霉菌TK23和变铅青链霉菌TK23（pPRⅠ）。
这需将菌株接种到溶解培养基A（欧洲专利申请，公开号0，158，872第6页）中，并在轨道式震荡培养器中于30℃保温2天。收获细胞后，取1mg菌丝体在合适的缓冲液（如RS缓冲液：C.J.Thompson等人，J.Bacteriol 151∶678-685，1982）中用超声波破碎。测定PTC降解的典型试验程序如下：
在250μl粗提物中，加入100μl PTC溶液（250μg/ml）和50μl乙酰辅酶A（4mg/ml），将混合物于30℃下保温2小时。此时间过后，用HPLC测定仍然存在的PTC量。此实验基结果如下：
未反应的PTC
菌株
加入的PTC
变铅青链霉菌TK23 100%
绿色产色链霉菌（DSM40736） 72%
绿色产色链霉菌选择体 7%
变铅青链霉菌TK23（pPRⅠ） 31%
在薄层层析上（没有茚三酮显色）与参考物质相比表明，已经发生了PTC的N-乙酰化。
Claims (2)
1、一种获得Phosphinothricin(PTC)抗性基因的方法，该方法包括选择抗2-氨基-4-甲膦酰-丙氨酰-丙氨酸(PTT)的绿色产色链霉菌(Streptomyces viridochromogenes)D SM4112，从抗性菌株中分离总DNA并用BamHI酶切之，分离大小为4.0Kb的片段，以含有抗性基因的完整或亚片段形式克隆此片段并选择PTT抗性。
2、根据权利要求1的方法，其中被克隆的是位于4.0Kb BamHⅠ片段中的0.8Kb BglⅡ片段。
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863628747 DE3628747A1 (de) | 1986-08-23 | 1986-08-23 | Resistenzgen gegen phosphinothricin und seine verwendung |
| DEP3628747.4 | 1986-08-23 | ||
| DEP3637307.9 | 1986-11-03 | ||
| DE3637307 | 1986-11-03 | ||
| DE19863642829 DE3642829A1 (de) | 1986-08-23 | 1986-12-16 | Resistenzgen gegen phosphinothricin |
| DEP3642829.9 | 1986-12-16 | ||
| DEP3700313.5 | 1987-01-08 | ||
| DE19873700313 DE3700313A1 (de) | 1986-08-23 | 1987-01-08 | Verwendung eines resistenzgens gegen phosphinothricin |
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| CN87105764A true CN87105764A (zh) | 1988-11-30 |
| CN1040772C CN1040772C (zh) | 1998-11-18 |
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| CN98115017A Expired - Lifetime CN1124347C (zh) | 1986-08-23 | 1998-06-19 | 2－氨基－4－甲膦酰丁酸抗性基因 |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0257542B1 (zh) |
| JP (4) | JPH0797994B2 (zh) |
| CN (2) | CN1040772C (zh) |
| AT (1) | ATE75776T1 (zh) |
| AU (1) | AU604743B2 (zh) |
| CA (1) | CA1337597C (zh) |
| DK (1) | DK175254B1 (zh) |
| ES (1) | ES2038631T3 (zh) |
| FI (1) | FI100251B (zh) |
| GR (1) | GR3005200T3 (zh) |
| HU (1) | HU217208B (zh) |
| IL (1) | IL83604A (zh) |
| NZ (1) | NZ221526A (zh) |
Families Citing this family (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3765449D1 (de) * | 1986-03-11 | 1990-11-15 | Plant Genetic Systems Nv | Durch gentechnologie erhaltene und gegen glutaminsynthetase-inhibitoren resistente pflanzenzellen. |
| CN87100603A (zh) * | 1987-01-21 | 1988-08-10 | 昂科公司 | 抗黑素瘤疫苗 |
| DE3716309A1 (de) * | 1987-05-15 | 1988-11-24 | Hoechst Ag | Resistenzgen gegen phosphinothricin |
| DE3732972A1 (de) * | 1987-07-02 | 1989-01-12 | Hoechst Ag | Resistenzgen gegen phosphinothricin und seine verwendung |
| DE4126414A1 (de) * | 1991-08-09 | 1993-02-11 | Hoechst Ag | Deacetylasegene zur erzeugung von phosphinothricin oder phosphinothricyl-alanyl-alanin, verfahren zu ihrer isolierung und ihre verwendung |
| DE4222407C1 (de) * | 1992-07-08 | 1993-10-07 | Max Planck Gesellschaft | Modulartiges Promotor-Konstrukt |
| AR008158A1 (es) * | 1996-09-05 | 1999-12-09 | Syngenta Participations Ag | Proceso para el control de malas hierbas en cultivos de plantas utiles que son resistentes a un fosfo-herbicida y una composicion herbicida para dicho uso. |
| US6586367B2 (en) | 1996-09-05 | 2003-07-01 | Syngenta Crop Protection, Inc. | Process for the control of weeds |
| PL215314B1 (pl) | 1998-08-13 | 2013-11-29 | Bayer Cropscience Ag | Zastosowanie kompozycji zawierajacej glifosat i pendimetaline oraz sposób zwalczania szkodliwych roslin w uprawach kukurydzy |
| DE19836659A1 (de) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Herbizide Mittel für tolerante oder resistente Baumwollkulturen |
| DE19836660A1 (de) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Herbizide Mittel für tolerante oder resistente Sojakulturen |
| DE19836684A1 (de) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Herbizide Mittel für tolerante oder resistente Reiskulturen |
| DE19836700A1 (de) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Herbizide Mittel für tolerante oder resistente Getreidekulturen |
| AU2003207323B2 (en) | 2002-02-26 | 2007-09-13 | Syngenta Limited | A method of selectively producing male or female sterile plants |
| US7795505B2 (en) | 2004-11-17 | 2010-09-14 | Hokko Chemical Industry Co., Ltd. | Herbicide-resistance gene and utilization thereof |
| KR100743611B1 (ko) * | 2006-03-28 | 2007-08-01 | 최광술 | 산업용 로봇의 케이블 조절장치 |
| AR074941A1 (es) | 2009-01-07 | 2011-02-23 | Bayer Cropscience Sa | Plantas transplastomicas exentas de marcador de seleccion |
| EA201290572A1 (ru) | 2009-12-23 | 2013-05-30 | Байер Интеллектуэль Проперти Гмбх | Растения, устойчивые к гербицидам - ингибиторам hppd |
| ES2659086T3 (es) | 2009-12-23 | 2018-03-13 | Bayer Intellectual Property Gmbh | Plantas tolerantes a herbicidas inhibidores de HPPD |
| ES2668198T3 (es) | 2009-12-23 | 2018-05-17 | Bayer Intellectual Property Gmbh | Plantas tolerantes a herbicidas inhibidores de HPPD |
| UY33139A (es) | 2009-12-23 | 2011-07-29 | Bayer Cropscience Ag | Plantas tolerantes a herbicidas inhibidores de las hppd |
| EP2516632B1 (en) | 2009-12-23 | 2018-02-14 | Bayer Intellectual Property GmbH | Plants tolerant to hppd inhibitor herbicides |
| BR112012029634A2 (pt) | 2010-05-21 | 2015-10-20 | Bayer Ip Gmbh | agentes herbicidas para as culturas de arroz tolerantes ou resistentes |
| US20110287933A1 (en) | 2010-05-21 | 2011-11-24 | Bayer Cropscience Ag | Herbicidal composition for tolerant or resistant oilseed rape crops |
| EP2571365A1 (de) | 2010-05-21 | 2013-03-27 | Bayer Intellectual Property GmbH | Herbizide mittel für tolerante oder resistente getreidekulturen |
| US20110294663A1 (en) | 2010-05-21 | 2011-12-01 | Bayer Cropscience Ag | Herbicidal composition for tolerant or resistant corn crops |
| WO2012004013A2 (en) | 2010-07-08 | 2012-01-12 | Bayer Bioscience N.V. | Glucosinolate transporter protein and uses thereof |
| CN103443279A (zh) | 2011-01-24 | 2013-12-11 | 拜尔作物科学公司 | rd29启动子或其片段用于棉花中转基因的胁迫诱导型表达的用途 |
| EP2524602A1 (de) | 2011-05-20 | 2012-11-21 | Bayer CropScience AG | Herbizide Mittel für tolerante oder resistente Sojakulturen |
| UA117816C2 (uk) | 2012-11-06 | 2018-10-10 | Байєр Кропсайєнс Акцієнгезелльшафт | Гербіцидна комбінація для толерантних соєвих культур |
| DE202014101590U1 (de) | 2014-04-03 | 2014-04-29 | Igus Gmbh | Führungssystem für Versorgungsleitungen und Roboter mit Führungssystem |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3765449D1 (de) * | 1986-03-11 | 1990-11-15 | Plant Genetic Systems Nv | Durch gentechnologie erhaltene und gegen glutaminsynthetase-inhibitoren resistente pflanzenzellen. |
-
1987
- 1987-08-19 EP EP87112023A patent/EP0257542B1/de not_active Expired - Lifetime
- 1987-08-19 AT AT87112023T patent/ATE75776T1/de not_active IP Right Cessation
- 1987-08-19 ES ES198787112023T patent/ES2038631T3/es not_active Expired - Lifetime
- 1987-08-20 FI FI873610A patent/FI100251B/fi not_active IP Right Cessation
- 1987-08-21 CA CA000545037A patent/CA1337597C/en not_active Expired - Lifetime
- 1987-08-21 DK DK198704378A patent/DK175254B1/da not_active IP Right Cessation
- 1987-08-21 AU AU77318/87A patent/AU604743B2/en not_active Expired
- 1987-08-21 IL IL8360487A patent/IL83604A/en not_active IP Right Cessation
- 1987-08-21 NZ NZ221526A patent/NZ221526A/xx unknown
- 1987-08-22 JP JP62209123A patent/JPH0797994B2/ja not_active Expired - Lifetime
- 1987-08-22 CN CN87105764A patent/CN1040772C/zh not_active Expired - Lifetime
- 1987-08-23 HU HU727/87A patent/HU217208B/hu unknown
-
1990
- 1990-02-28 JP JP2049140A patent/JP2749424B2/ja not_active Expired - Lifetime
-
1992
- 1992-07-16 GR GR920401371T patent/GR3005200T3/el unknown
-
1994
- 1994-10-14 JP JP6249627A patent/JPH07147985A/ja active Pending
-
1996
- 1996-10-09 JP JP8268910A patent/JP2815847B2/ja not_active Expired - Lifetime
-
1998
- 1998-06-19 CN CN98115017A patent/CN1124347C/zh not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| FI100251B (fi) | 1997-10-31 |
| AU7731887A (en) | 1988-05-19 |
| CN1040772C (zh) | 1998-11-18 |
| JPH03103193A (ja) | 1991-04-30 |
| ES2038631T3 (es) | 1993-08-01 |
| JPH07147985A (ja) | 1995-06-13 |
| CA1337597C (en) | 1995-11-21 |
| ATE75776T1 (de) | 1992-05-15 |
| HUT44621A (en) | 1988-03-28 |
| DK437887D0 (da) | 1987-08-21 |
| EP0257542A2 (de) | 1988-03-02 |
| IL83604A0 (en) | 1988-01-31 |
| CN1225393A (zh) | 1999-08-11 |
| GR3005200T3 (zh) | 1993-05-24 |
| FI873610A0 (fi) | 1987-08-20 |
| EP0257542A3 (en) | 1990-03-07 |
| JPH0797994B2 (ja) | 1995-10-25 |
| JPH09107981A (ja) | 1997-04-28 |
| FI873610L (fi) | 1988-02-24 |
| HU217208B (hu) | 1999-12-28 |
| JPS6371183A (ja) | 1988-03-31 |
| JP2815847B2 (ja) | 1998-10-27 |
| CN1124347C (zh) | 2003-10-15 |
| NZ221526A (en) | 1989-03-29 |
| JP2749424B2 (ja) | 1998-05-13 |
| IL83604A (en) | 2004-02-19 |
| DK175254B1 (da) | 2004-07-26 |
| AU604743B2 (en) | 1991-01-03 |
| DK437887A (da) | 1988-02-24 |
| EP0257542B1 (de) | 1992-05-06 |
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| C14 | Grant of patent or utility model | ||
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| C15 | Extension of patent right duration from 15 to 20 years for appl. with date before 31.12.1992 and still valid on 11.12.2001 (patent law change 1993) | ||
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