CN88102798A - 膦苏菌素抗性基因 - Google Patents
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Abstract
选择非真菌样细菌的膦苏菌素(PTC)，得到PTC-抗性选择体。通过构建基因库和筛选化学修饰的PTC，从这些选择体的完整DNA中得到携带抗性基因的DNA片段。可将抗性基因定位到长为2Kb的片段上，并选择PTC抗性，该基因适用于生产PTC抗性植物及其繁殖物质，也可用作标志物。获得该PTC抗性基因的微生物可用于污水处理厂。
Description
膦苏菌素（Phosphinothricin）（PTC，2-氨基-4-甲膦基丁酸）是一种谷氨酰胺合成酶抑制剂。PTC是抗生素膦基甲苏氨酰-丙氨酰-丙氨酸的“结构单位”。该三肽（PTT）具有抗革兰氏阳性和阴性菌活性，同时具有抗真菌灰绿葡萄孢的活性（Bayer等，Helv.Chim.Acta55，224，1972）。PTT是由绿色产色链霉菌Tü494（DSM40736）菌株产生的。
欧洲专利申请（EPA）0173327号描述了PTT的生物合成。其中图7涉及由吸水链霉菌FERM BP-130（ATCC21705）产生的抗性基因，但並没有详细描述其特征。
1987年2月4日出版的Franfurter Allgemeine Zeitung的报告的第29页（自然与科学增刊，左手栏上方）述及有可能从土壤链霉菌中分离到负责破坏PTC的基因。
未在先公开的欧洲专利申请（EPA）0，257，542号中提到了来源于上述PTT生产菌绿色产色链霉菌DSM60736菌株的PTC抗性基因。
关于PTC和PTT的生产以及这些化合物的抗性，迄今为止只有链霉菌已被描述并提出。
细菌中，真菌样链霉菌作为一个大菌属，在许多方面占有重要地位：链霉菌是抗生素的最重要的生产菌，它们形成即使在老化菌落中仍可保留的菌丝体，並常常伸出高度发育的气生菌丝体;另外，与遗传操作有关的一个重要特征是，在其DNA中A和T对G和C的比例约为30比70。
现已发现，其他细菌如非真菌样细菌，特别是革兰氏阴性菌也表现有PTC抗性。
德国专利2，717，440号述及PTC具有非选择性的除莠剂作用。可根据其在土壤中的短生物学半寿期来鉴别之，即这种除莠剂在土壤中很容易被微生物迅速修饰或破坏，从而失去除莠作用。已有可能从来自PTC处理过的耕地的土壤样品中分离出修饰PTC的微生物。也有可能在用诱变剂处理后，由污水淤浆中分离出有PTC抗性的细菌。
本发明涉及膦苏菌素（PTC）抗性基因，该基因可经选择有PTC抗性的非真菌样细菌，提取DNA、构建基因库、分离PTC抗性克隆、並由这些克隆中分离PTC抗性基因而制得。
本发明特别涉及由非真菌样细菌中制得的新的PTC抗性基因、含有该基因的细胞特别是植物细胞、以及含该基因的抗PTC的植物。本发明的另一个方面涉及基因在细菌和植物细胞中作为标志物的应用。本发明的其他方面和优选实施方案将在下文中详细解释。
图1显示了本发明之基因的限制性酶切图。
图2显示了载体pOCAM12，在构建基因库后，其可用于分离图1所示的基因。
选择非真菌类的抗PTC菌，除未确定特征的微生物外，最好是不产孢子的革兰氏阴性杆菌，特别是假单胞菌属的需氧微生物和产碱杆菌属与土壤杆菌属的假单胞菌样细菌、肠杆菌属和沙雷氏杆菌属的兼性厌氧肠道细菌、以及Cedecea菌属的细菌。
为本发明之目的，没有必要对PTC抗性细菌进行精确定性。只要证明其可在富含PTC的选择培养基中正常生长便足够了。
一旦确定了适当的种和属特性，也就有可能很好地确定所说种和属（其可由寄存单位得到）之菌株的特性，並从中选择PTC抗性菌株。
已对粪产碱菌和真养产碱菌、Pseudomonas paucimobilis、Enterobacter agglomerans、Serratia plymuthica、根瘤土壤杆菌和Cedecea Gr.V.的菌株进行了详尽的特性分析。基于这一知识，便有可能去利用这些细菌，如得自Deutsche Sammlung Von Mikroorganismen（德国微生物保藏中心）的产碱杆菌菌株（寄存号为DSM975或30030）。
亦可在污水处理厂中使用这些类型的PTC抗性细菌或已通过引入本发明的基因而提供了抗PTC抗性的微生物，以破坏生产残留物或污水中的PTC及其衍生物，或者使用污水处理厂中有用的微生物菌群使其破坏改变之。
按照EP-A0，257，542中所述，负责失活PTC的酶已鉴定为谷氨酸N-乙酰基转移酶。已分离出了负责这一活性的基因，並已确定具有图1所示的限制性酶切图和表1所示的DNA序列。当然，也可以用某种已知方法如亚磷酸盐法化学合成该DNA序列，並可能按照已知方法来制备经过修饰的基因。
当与适当的宿主特异性调节片段融合后，该基因可使其他对PTC敏感的生物体获得抗PTC抗性。这一抗性可被用作选择标志或制得有用的PTC抗性植物。
在用PTC处理这些有用植物后，不仅能使不需要的植物生长受到抑制，还可减少有用植物上不需要的微生物。
除特别指出者外，下列实施例中的百分数据均为重量百分比。
实施例1：
在基本培养基上筛选及修饰检测法
使用LB培养基（10g Difco bacto胰酶解胨、5g Difco bacto酵母提取物和10g氯化钠/升）由土壤样品中提取细菌，先培养过夜，再用缓冲液（10mMNa2HPO4/10mM NaCl）洗涤並铺敷在具有下列成分的选择培养基上：0.4g NaCl、0.8gKH2PO4、1.6g Na2HPO4、1.6gD，L-PTC（NH4），4ml葡萄糖溶液（10%）、0.8ml 1MMgSO4，加水至800ml;加入1.4%琼脂制成固态培养基。
为完成修饰检测，将大约5ml细菌悬液减少体积到200μl並加5μg溶菌酶溶解之。加入1μCi3，4-14C-PTC后，将混合物于28℃下保温4小时。为进行分析，然后将混合物于95℃下保温10分钟，在冰上冷却並在台式离心机中离心10分钟。取10μl澄清的上清液加到纤维素薄层板（得自Merck公司，Darmstadt，纤维素F，TLC AL薄片）上进行上行层析（流动相：吡啶、正丁醇、乙酸和水，体积比为50∶75∶15∶60），然后干燥並作放射自显影。在上述条件下，PTC的Rf值约为0.31，N-乙酰基PTC的Rf值约为0.33。
为研究装配型质粒库，将最初在5ml培养基中培养的各5个克隆合并，减少体积到大约300μl並按上述方法进行检测。
实施例2
制备装配型质粒载体pOCAM12
因为装配型质粒载体pOCAM12存在其COLE1复制原点，所以在大肠杆菌中具有高拷贝数，並具有天然质粒RK2的广泛宿主及四环素抗性基因。此载体为可活动载体pRK404（G.Ditta等，Plasmid13，149-153，1985）的衍化体。在其结合了25至40Kb长的DNA片段后，即可被包装在λ噬菌体内。可按下述方法得到该载体：用HindⅢ切开载体pTJS75（T.S.Schmidhauser和D.R.Helinski，J.Bacteriology164，446-455，1985），並用DNA聚合酶Ⅰ（Klenow片段）将突出的序列填成平端。用NaeⅠ和SspⅠ切开载体pSDL12（A.Levinson等，J.Molec.Appl.Genetics2，507-517，1984），並分离出大片段。用填成平端的BglⅡ片段（其来自pHC79並含有Con区）将后者与已成线形並填成平端的质粒pTJS75相连接。合成Ti质粒的右侧边缘区（RB）（其长度为23bp）並经修整平端连接法插入之。经限制性酶切分析以确定图2所示长约8.5Kb的所需质粒pOCAM12的结构特征。
当然，亦可将用其他载体如可在市场购得的pHC79（Hohn和Collins，Gene，11，291，1980）来构建基因库。
实施例3
构建基因库
依照分离真核细胞DNA的方法（Maniatis等，Molecular Cloning，A Laboratory Manual，280-285，1982）自粪产碱菌中分离DNA並用Sau3AI切割。用BamHⅠ酶切载体pOCAM12並与大小约25-40Kb的DNA片段连接。可按照制造商推荐的方法（Amersham：in Vitro Packaging System for Lambda DNA，代号334Z号）或Maniatis等人（296-299页）指定的方法进行连接並包装到λ噬菌体中。
实施例4
感染大肠杆菌指示菌菌株DH1並筛选
首先在LBMM培养基（LB培养基加2g麦芽糖和2.5g硫酸镁七水合物/升）培养大肠杆菌DH1的细菌，达到光密度值（OD550）约为1。将200μl该悬液与多至50μl的噬菌体悬浮混合並于37℃下保温30分钟，加入1ml LBMM並继续保温50分钟。将100μl等分样品在添加了10μg四环素/ml的LB平皿上划线接种。摘取单个菌落，在5ml培养物培养后，以5个平皿为一组按上述方法检测。检测了2，700个装配型质粒克隆后，被检测的一个集群在放射自显影图谱中N-乙酰基PTC的位置上显示有预期信号。以修饰分析法检测属于该集群单个克隆，从而鉴定出负责修饰的克隆。
结果发现该克隆具有一长约25，000至30，000bp的插入物。其为大肠杆菌DH1菌株及根瘤土壤杆菌和苜蓿根瘤菌的细菌提供了抗多至50mM PTC的抗性。
经限制性酶切分析将酶学活性部位定位于插入物的约2Kb BstEⅡ-BglⅡ片段上。由限制性酶切图（图1）显示了该片段的组织特征。表1中所列出的DNA序列则进一步明确了膦苏菌素
（Phosphinothricin）抗性基因的定位和序列特征。
Claims (8)
1、一种制备膦苏菌素(Phosphinothricin，PTC)抗性基因的方法，其包括选择有抗PTC抗性的非真菌样细菌、由所选细菌中分离DNA、用该DNA构建基因库、分离抗PTC的克隆並由其DNA中制取PTC抗性基因。
2、根据权利要求1的方法，其中所说的细菌是选自假单胞菌属、产碱杆菌属、土壤杆菌属、肠杆菌属、沙雷氏杆菌属或Cedecea菌属的细菌。
3、根据权利要求2的方法，其中所说的细菌是选自Pseudomonas paucimobilis、粪产碱菌或真养产碱菌、根瘤土壤杆菌、Enterobacter agglomerans、Serratia plymuthica或Cedecea Gr.V的细菌。
4、根据权利要求1的方法，其中选择了菌种粪产碱菌的细菌、由所选细菌中提取DNA並用BstEⅡ和BglⅡ切割之，然后克隆大小约2Kb的片段並选择PTC抗性。
5、一种制备PTC抗性基因的方法，其包括化学合成表1中所示的DNA。
6、一种生产抗PTC植物细胞、植物或其部分、以及繁殖材料的方法，其包括将依照权利要求1至5所述方法制得的可表达基因插入植物细胞内。
7、一种选择植物、植物细胞或细菌的方法，其包括将依照权利要求1至5所述方法制得的可表达基因插入细胞内並选择PTC抗性。
8、一种生物降解PTC和含PTC残留物、农业用地面和污水的方法，其包括使残留物或污水与表达权利要求1至5所述基因的微生物群相接触。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873716309 DE3716309A1 (de) | 1987-05-15 | 1987-05-15 | Resistenzgen gegen phosphinothricin |
| DEP3716309.4 | 1987-05-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN88102798A true CN88102798A (zh) | 1988-11-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN198888102798A Pending CN88102798A (zh) | 1987-05-15 | 1988-05-13 | 膦苏菌素抗性基因 |
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| Country | Link |
|---|---|
| US (1) | US5077399A (zh) |
| EP (1) | EP0290986B1 (zh) |
| JP (1) | JPS645493A (zh) |
| CN (1) | CN88102798A (zh) |
| AU (1) | AU613367B2 (zh) |
| DE (2) | DE3716309A1 (zh) |
| DK (1) | DK264288A (zh) |
| ES (1) | ES2058172T3 (zh) |
| FI (1) | FI882227A (zh) |
| HU (1) | HUT46943A (zh) |
| IL (1) | IL86378A0 (zh) |
| NZ (1) | NZ224602A (zh) |
| ZA (1) | ZA883390B (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5276268A (en) * | 1986-08-23 | 1994-01-04 | Hoechst Aktiengesellschaft | Phosphinothricin-resistance gene, and its use |
| CN87100603A (zh) * | 1987-01-21 | 1988-08-10 | 昂科公司 | 抗黑素瘤疫苗 |
| US6803499B1 (en) | 1989-08-09 | 2004-10-12 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
| US7705215B1 (en) | 1990-04-17 | 2010-04-27 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
| CA2074355C (en) | 1990-01-22 | 2008-10-28 | Ronald C. Lundquist | Method of producing fertile transgenic corn plants |
| US6025545A (en) * | 1990-01-22 | 2000-02-15 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
| US6777589B1 (en) | 1990-01-22 | 2004-08-17 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
| US6946587B1 (en) | 1990-01-22 | 2005-09-20 | Dekalb Genetics Corporation | Method for preparing fertile transgenic corn plants |
| US6329574B1 (en) | 1990-01-22 | 2001-12-11 | Dekalb Genetics Corporation | High lysine fertile transgenic corn plants |
| DE4003045A1 (de) * | 1990-02-02 | 1991-08-08 | Hoechst Ag | Virus/herbizidresistenz-gene, verfahren zu ihrer herstellung und ihre verwendung |
| US5739082A (en) * | 1990-02-02 | 1998-04-14 | Hoechst Schering Agrevo Gmbh | Method of improving the yield of herbicide-resistant crop plants |
| US6395966B1 (en) | 1990-08-09 | 2002-05-28 | Dekalb Genetics Corp. | Fertile transgenic maize plants containing a gene encoding the pat protein |
| US6118047A (en) | 1993-08-25 | 2000-09-12 | Dekalb Genetic Corporation | Anthranilate synthase gene and method of use thereof for conferring tryptophan overproduction |
| US6326527B1 (en) | 1993-08-25 | 2001-12-04 | Dekalb Genetics Corporation | Method for altering the nutritional content of plant seed |
| US6281411B1 (en) | 1993-08-25 | 2001-08-28 | Dekalb Genetics Corporation | Transgenic monocots plants with increased glycine-betaine content |
| US5717129A (en) * | 1995-02-16 | 1998-02-10 | Pioneer Hi-Bred International, Inc. | Methods for maintaining sterility in plants |
| CN102282156B (zh) * | 2008-11-19 | 2017-03-15 | 国家政治研究所高级研究中心(高级研究中心) | 能够代谢亚磷酸盐作为磷源的转基因植物和真菌 |
| US11920141B2 (en) * | 2021-09-24 | 2024-03-05 | Oms Investments, Inc. | Glufosinate resistance cassettes and plants comprising the same |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6158589A (ja) * | 1984-08-30 | 1986-03-25 | Meiji Seika Kaisha Ltd | ビアラホス生産遺伝子及び耐性遺伝子 |
| ZA871352B (en) * | 1986-02-27 | 1987-08-12 | The General Hospital Corporation | Plant cells resistant to herbicidal glutamine synthetase inhibitors |
| EP0257542B1 (de) * | 1986-08-23 | 1992-05-06 | Hoechst Aktiengesellschaft | Resistenzgen gegen Phosphinothricin und seine Verwendung |
| CN87100603A (zh) * | 1987-01-21 | 1988-08-10 | 昂科公司 | 抗黑素瘤疫苗 |
-
1987
- 1987-05-15 DE DE19873716309 patent/DE3716309A1/de not_active Withdrawn
-
1988
- 1988-05-07 EP EP88107372A patent/EP0290986B1/de not_active Expired - Lifetime
- 1988-05-07 DE DE8888107372T patent/DE3881959D1/de not_active Expired - Fee Related
- 1988-05-07 ES ES88107372T patent/ES2058172T3/es not_active Expired - Lifetime
- 1988-05-12 FI FI882227A patent/FI882227A/fi not_active Application Discontinuation
- 1988-05-13 ZA ZA883390A patent/ZA883390B/xx unknown
- 1988-05-13 AU AU16146/88A patent/AU613367B2/en not_active Ceased
- 1988-05-13 NZ NZ224602A patent/NZ224602A/en unknown
- 1988-05-13 HU HU882397A patent/HUT46943A/hu unknown
- 1988-05-13 DK DK264288A patent/DK264288A/da not_active Application Discontinuation
- 1988-05-13 CN CN198888102798A patent/CN88102798A/zh active Pending
- 1988-05-14 JP JP63118043A patent/JPS645493A/ja active Pending
- 1988-05-15 IL IL86378A patent/IL86378A0/xx unknown
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1990
- 1990-04-27 US US07/515,671 patent/US5077399A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| EP0290986A1 (de) | 1988-11-17 |
| DE3881959D1 (de) | 1993-07-29 |
| DK264288D0 (da) | 1988-05-13 |
| IL86378A0 (en) | 1988-11-15 |
| DK264288A (da) | 1988-11-16 |
| AU613367B2 (en) | 1991-08-01 |
| EP0290986B1 (de) | 1993-06-23 |
| ES2058172T3 (es) | 1994-11-01 |
| AU1614688A (en) | 1988-11-17 |
| ZA883390B (en) | 1988-11-14 |
| DE3716309A1 (de) | 1988-11-24 |
| HUT46943A (en) | 1988-12-28 |
| JPS645493A (en) | 1989-01-10 |
| US5077399A (en) | 1991-12-31 |
| FI882227A (fi) | 1988-11-16 |
| NZ224602A (en) | 1992-03-26 |
| FI882227A0 (fi) | 1988-05-12 |
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