CN1207388C - 草甘膦降解酶基因和含该基因的载体 - Google Patents
草甘膦降解酶基因和含该基因的载体 Download PDFInfo
- Publication number
- CN1207388C CN1207388C CNB011148136A CN01114813A CN1207388C CN 1207388 C CN1207388 C CN 1207388C CN B011148136 A CNB011148136 A CN B011148136A CN 01114813 A CN01114813 A CN 01114813A CN 1207388 C CN1207388 C CN 1207388C
- Authority
- CN
- China
- Prior art keywords
- glyphosate
- gene
- pcrgld
- dna
- xli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000005562 Glyphosate Substances 0.000 title claims abstract description 60
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 229940097068 glyphosate Drugs 0.000 title claims abstract description 60
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 17
- 230000000593 degrading effect Effects 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 title abstract description 22
- 239000013612 plasmid Substances 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 2
- 230000037431 insertion Effects 0.000 claims description 2
- 239000013600 plasmid vector Substances 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 22
- 239000011574 phosphorus Substances 0.000 abstract description 22
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 22
- 101150082542 Gld gene Proteins 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 241000589196 Sinorhizobium meliloti Species 0.000 abstract description 4
- 108010053446 Carbon-phosphorus lyase Proteins 0.000 abstract description 3
- 230000001925 catabolic effect Effects 0.000 abstract 2
- 241000894006 Bacteria Species 0.000 description 20
- 230000015556 catabolic process Effects 0.000 description 17
- 238000006731 degradation reaction Methods 0.000 description 17
- 239000007788 liquid Substances 0.000 description 14
- 241000589180 Rhizobium Species 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000012533 medium component Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- MGRVRXRGTBOSHW-UHFFFAOYSA-N (aminomethyl)phosphonic acid Chemical class NCP(O)(O)=O MGRVRXRGTBOSHW-UHFFFAOYSA-N 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 239000007993 MOPS buffer Substances 0.000 description 3
- 241000219823 Medicago Species 0.000 description 3
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 3
- 241001516577 Phylloxera Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007320 rich medium Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 244000000000 soil microbiome Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 244000037671 genetically modified crops Species 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 101150112970 up gene Proteins 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种从苜蓿根瘤菌M010中克隆到的草甘膦降解酶基因(Gld)以及含有该基因的载体和转化子。含有该基因的序列总长为2670bp。含有Gld序列的转化子XLI-pCRGld的蛋白粗提物,具有降解草甘膦释放无机磷的活性,表明本发明克隆到的草甘膦降解酶,属于C-P裂解酶。
Description
本发明属于分子遗传学技术领域,特别是应用现代分子生物学方法和DNA重组技术从土壤细菌中分离获得一种草甘膦降解酶基因。
化学除草已成为现代化农业生产的重要组成部分。全世界除草剂的总用量、施用面积及费用均已超过杀虫剂和杀菌剂的总和。在众多的除草剂中,草甘膦(N-phosphonomethylglycine)是目前世界上使用最为广泛的一种。它的优点是对人畜无毒,降解快,对环境污染非常小,而且价钱便宜,因而深受欢迎。仅美国每年销售量就达几百万美元。草甘膦是一种广谱、内吸性的除草剂,它可无选择地抑制所有植物的5-烯醇式丙酮酰莽草酸-3-磷酸合成酶(下面简称:EPSPS)活性,阻断植物芳香族氨基酸的合成,因此它也抑制农作物的生长。应用基因工程技术培育对草甘膦具有抗性的农作物,对于安全使用草甘膦,无疑具有重要的意义。
目前,获得抗草甘膦转基因农作物研究途径主要有:一、导入过量表达的EPSP合成酶基因,以此抵御草甘膦的竞争性抑制;二、通过定点突变,获得对草甘膦低亲和性或无亲和性的EPSP合成酶,从而消除草甘膦的抑制作用。抗草甘膦转基因大豆是目前世界上产业化规模最大的转基因植物(2000年总面积约为1000万公顷)。然而除了大豆以外,其他转基因作物均未能商品化生产,这可能是由于植物代谢中存在着草甘膦作用的第二个靶位点。
在土壤细菌中,广泛存在降解草甘膦的酶,它们能把有机磷分解为无机磷,作为细菌的磷源加以吸收。现已证实草甘膦降解途径有二条。其一、裂解草甘膦分子为氨甲基磷酸盐(AMPA),AMPA进一步被未知的随后步骤裂解出无机磷;其二,通过C-P裂解酶活性初始裂解C-P键,得到肌氨酸。用以草甘膦作为唯一磷源的培养基,培养根瘤菌(Flavobacterium spp.)菌株及农杆菌(Agrobacteriumspp.),发现这些菌株能将草甘膦裂解成肌氨酸和无机磷。迄今,对草甘膦抗药性代谢机理方面的研究报道还很少,更没有把降解草甘膦基因导入植物并在转基因植物中表达的报道。
本发明的目的是提供一种草甘膦降解酶基因及含该基因的载体。
发明人在研究中发现苜蓿根瘤菌(Rhizobium meliloti)M010具有高效降解草甘膦的能力,提示其含有草甘膦降解酶基因。
本发明是根据基因互补的原理,利用现代分子生物学和DNA重组技术,选择具有降解草甘膦能力的微生物,从中分离和克隆草甘膦降解酶基因。本发明以苜蓿根瘤菌(Rhizobium meliloti)M010菌株为材料建立基因文库,然后转化到没有降解草甘膦能力的大肠杆菌中,通过基因互补获得含有草甘膦降解酶基因的DNA序列。
本发明所用的苜蓿根瘤菌(Rhizobium meliloti)M010购自广东省微生物研究所菌种保藏选育实验室(广东省微生物研究所菌种保藏目录1996)。
本发明的含草甘膦降解酶基因的DNA序列如序列表1所示。
下面通过实施例和附图进一步详细叙述本发明:
图1是野生型苜蓿根瘤菌M010在含有不同浓度草甘膦的无磷液体培养基中的生长曲线;
图2是含有草甘膦降解酶基因的质粒pCRGld的结构图;
图3是XLI-pCRGld以及XLI-pCR2.1转化子在含有不同浓度草甘膦的无磷液体培养基中的生长曲线;
图4是野生型苜蓿根瘤菌M010,XLI-pCRGld转化子以及XLI-pCR2.1转化子的体外降解草甘膦的活性比较。
实施例1.检测苜蓿根瘤菌M010降解草甘膦能力
将苜蓿根瘤菌M010接种在含有0,2,4,6,8和10mM草甘膦的液体根瘤菌无磷培养基(培养基成分见表一)中,测定其生长曲线。从曲线上可见,在含2.0mM草甘膦的液体无磷培养基中,该菌的生长速度最快。但是随着草甘膦浓度的提高,其生长也逐渐受到抑制(生长曲线见图1)。说明苜蓿根瘤菌M010具有较强的降解草甘膦的能力。
表一、液体根瘤菌无磷培养基成分:
MOPS/KOH pH7.4 25mM, MgSO4 2mM,
柠檬酸铁 100μM, CaCl2 1.2mM,
NH4Cl 33mM, 维生素B1 0.5mg/L,
烟酸 0.1mg/l, 生物素 0.1mg/L,
泛酸 0.5mg/L, 丁二酸钠 50mM,
微量元素:
CoCl2 0.8μM, CuSO4 40μM,
H3BO4 14μM, MnCl2 0.1μM,
Na2MoO4 10μM, ZnSO4 1.5μM.
实施例2.建立苜蓿根瘤菌M010基因文库
将苜蓿根瘤菌M010单菌落接种于200ml根瘤菌丰富培养基(培养基成分见表二)中,在28℃空气浴中震荡培养48小时。离心收菌,以5ml DNA提取缓冲液(50mM Tris-Cl,10mM EDTA,1%SDS,5μg/ml溶菌酶)重悬菌体,用超声波破壁。然后用等体积的苯酚和氯仿抽提除蛋白,再通过酒精沉淀DNA。最后将DNA溶于1ml TE缓冲液(10mM Tris-HCl,1mM EDTA,pH8.0)。将DNA以限制生内切酶BamHI和EcoRI进行部分酶切。通过琼脂糖凝胶电泳将不同大小的DNA片段分开,并切下其中含有2-10kb左右片段的凝胶,用Omega公司试剂盒(Gel Extration Kit试剂盒)回收DNA。随后按载体∶插入片段=1∶3的比例,用T4连接酶将该片段连接到已用相同酶切处理的质粒pCR2.1上。将连接液转化大肠杆菌XL1-blue感受态细胞,涂布到含氨苄青霉素(Amp)50ug/ml的LB固体培养基上,37℃培养过夜。收集大约5000个转化子。
表二、根瘤菌丰富培养基成分:
酵母膏 1克 土壤浸提液 200毫升
甘露醇 10克 蒸馏水 800毫升pH7.2
[注]土壤浸提液的制法:取土壤50克,加水200毫升,在高压蒸汽锅中,以15磅压力蒸煮1小时,过滤去固体物,滤液加水补足到200毫升。
实施例3.降解草甘膦相关基因的克隆和序列分析
将上述实施例2制备的转化子菌落用大肠杆菌无磷液体培养基(培养基成份见表三)从LB固体培养基上洗脱下来,离心收集菌体,而后再用大肠杆菌无磷液体培养基重复洗涤2次,然后随机地涂布到含不同浓度草甘膦(0mM,0.05mM,0.1mM,0.25mM,0.5mM)的大肠杆菌无磷固体(培养基成份见表三)平板上,37℃培养3天。结果我们获得了一个能在含草甘膦的无磷固体培养基上生长的转化子,命名为XLI-pCRGld。只有含有完整的降解草甘膦酶基因的转化子才能利用培养基中的唯一磷源-草甘膦而生长。因此无疑XLI-pCRGld含有编码C-P裂解酶基因,我们把含有该基因的质粒命名为pCRGld。将pCRGld质粒DNA提纯并用限制性内切酶BamHI和EcoRI双酶切检测。结果表明pCRGld包含一个2.7kb插入片段(见图2)。这个克隆含有的草甘膦降解酶基因的插入片段的核酸序列(命名为Gld)已经完全测序(结果如序列表1所示)。将Gld序列与Genebank、EMBL核酸序列库比较显示,未发现具有相似功能的同源性的序列。
表三、大肠杆菌液体无磷培养基成分:
MOPS(pH7.4) 40mM, N-三甲基甘氨酸 4mM,
次氮基三乙酸 15mM, NaCl 50mM,
NH4Cl 10mM, K2SO4 0.28mM,
MgSO4 7H2O 0.02mM, MgCl2 0.6mM,
亮氨酸 30mg/L, 色氨酸 20mg/L,
葡萄糖 20g/L,
微量元素:
MnSO4 H2O 6μM mM, FeSO4 7H2O 10μM,
CaCl2 2H2O 0.6μM, CoCl2 6H2O 0.4μM,
ZnSO4 7H2O 0.4μM, H3BO3 0.3μM,
Na2MoO4 2H2O 0.04μM, CuSO4 0.04μM,
固体培养基为液体培养基中加入15g/L琼脂,12g/L柠檬酸三钠。
实施例4.检测pCRGld转化子的降解草甘膦的能力
为了更有力地证明Gld基因编码酶在草甘膦代谢中的活性,将XLI-pCRGld转化子与没有外源插入片段的XLI-pCR2.1转化子做对比。首先将转化株接种到LB培养基中,37℃过夜培养。然后离心离心收取菌体,并用大肠杆菌液体无磷培养基洗涤2次,转接到草甘膦浓度为0,0.2,0.4,1,2mM的液体无磷培养基中,37℃培养72小时。检测培养液的OD600值,比较各菌在不同草甘膦浓度下的生长情况(测定结果如图3所示)。 结果表明,XLI-pCRGld转化子在0.4mM草甘膦浓度下生长较好。而对照样品,即没有外源片段的XLI-pCR2.1转化子在浓度为0.2mM的草甘膦作用下已完全被抑制。
实施例5.测定草甘膦体外降解活性
从苜蓿根瘤菌M010,XLI-pCRGld以及XLI-pCR2.1转化子细胞中提取蛋白粗提物。
分别用5ml含有50μg/ml氨卞青霉素的LB液体培养基培养XLI-pCRGld和XLI-pCR2.1转化子。200rpm、37℃空气浴振荡培养过夜。以1%的接种量,转接到新鲜的含有50μg/ml氨卞青霉素的400ml LB液体培养基中,200rpm、37℃继续培养至OD600=1.0时,离心收取菌体。将苜蓿根瘤菌M010单菌落接种于400ml根瘤菌丰富培养基中,28℃,200rpm空气浴振荡培养至OD600=1.0,离心收取菌体。分别用40ml缓冲液A(25mM MOPS pH7.0)洗涤上述菌体,离心弃去上清,分别用5ml缓冲液A重悬各菌体。然后用Fluko公司的乳化机处理细胞悬液使其破壁。以12000rpm离心30分钟,弃去细胞碎片。上清液用于下述有关各菌体外降解草甘膦活性的测定。
用缓冲液A把草甘膦配制成490μM的浓度,调节其pH=7.0。同时将各菌上清液,也即各菌的细胞蛋白粗提物,用缓冲液A调节蛋白浓度统一为200μg/ml。分别取500μl各菌上清液与500μl490μM草甘膦(pH=7.0)混合,于室温中放置。定时取该反应液50μl,加入800μl孔雀绿溶液,精确记时60秒,立刻加入100μl 34%柠檬酸钠溶液并混匀以终止反应,静置15分钟,测定OD660,与标准曲线比较,可获知反应液中的无机磷含量。测定结果如图4所示。
从图4中可见,苜蓿根瘤菌M010细胞蛋白粗提物有较强的降解草甘膦释放无机磷的能力,XLI-pCRGld转化子也有较明显的降解草甘膦以释放无机磷的能力。并且,从曲线上可见,这两者的活性变化趋势是一致的。而对照样品XLI-pCR2.1转化子基本上没有降解草甘膦能力。
实施例6.Gld DNA序列,pCRGld质粒以及XLI-pCRGld转化子的制备
为了明确Gld基因是由苜蓿根瘤杆菌中克隆而来,我们根据序列测定结果设计引物,(1)CGC
GGATCCGCGGGTGGGTACGGGCACGT,(2)CCG
GAATTCGCCGTACATCCGTTCGTTCTC,进行PCR扩增反应,反应条件为:94℃预变性5分钟,然后94℃90秒,55℃90秒,72℃150秒,共进行30个循环,最后72℃延伸10分钟。扩增反应完毕后,琼脂糖电泳分离PCR产物,以Gel Extraction Kit(Omega公司)回收DNA,用T4连接酶连接回收DNA片段到质粒载体pCR2.1(购自美国Invitrogen公司),而后用标准氯化钙方法转化大肠杆菌XL1-blue,从而可以得到经扩增而来的Gld DNA序列,pCRGld质粒以及XLI-pCRGld转化子。对PCR扩增而来的Gld DNA序列进行序列测定,结果与我们从苜蓿根瘤杆菌直接克隆的Gld DNA序列一致。
降解草甘膦策略可成为取代EPSPS蛋白过量表达策略的一个十分有效的方法,在构建转基因农草剂的应用范围和提高农业生产力,而且还会影响新型除草剂的设计和使用。既具有基础理论研究意义,也具有巨大的市场应用前景。另外,在全球数以万计的转基因植物中,绝大多数采用抗生素抗性基因(如卡那霉素抗性基因)作为标记基因。目前人们对转基因植物的担心之一就是这类基因可能会转移到人、动物肠道微生物或环境微生物的基因组中,导致抗生素类药物或农药失效。以除草剂抗性基因替代抗生素抗性基因是发展趋势之一。
序列表1
1.序列特征:长度:2670bp;类型:核酸;链数:单链;几何结构:线性;
2.分子类型:DNA;
3.来源:克隆;苜蓿根瘤杆菌;
4.序列描述:SEQ ID NO.1
GGATCCGCGG GTGGTATCGG GCACGTCCTG GATCACCGGC GCCGACGAAC CCAACAGGCA 60
CGTGGTGGGG CTGGTCGCCG GCCGCGACTT CACCCCCGAC GGCACCATCG AGGCCGCCGA 120
TGTGCGCGAC GGCGATCCGT CGCCGGACGG CGCCGGGGTG CTCACCTCGG CCCGCGGCAT 180
CGAGATCGGC CACATCTTCC AGCTGGGCCG CAAGTACGCC GACGCGTTCG GCGCCGACGT 240
GCTCGGCGAG AACGGCAAGC CGGTGCGGCT GACCATGGGC TCCTACGGCA TCGGGGTGTC 300
ACGGATGGTC GCGGTGATCG CCGAACAGCA GCATGACGAG ATCGGCTTGC GCTGGCCGGC 360
TTCGGTCGCG CCGTTCGACG TGCACGTGGT CATCGCCAAC AAGGACGCCG GTGCCCGCGA 420
GGGCGCCACC GAGTTGGCCG GTGAGCTGTC CCGGCTGGGC TTCGAGGTGC TCTTCGACGA 480
CCGCACCGCC TCACCGGGGG TGAAGTTCAA GGACGCCGAA CTGCTCGGCA TGCCGTGGAT 540
CGTGGTGGTC GGGCGGGGCT GGGCCGACGG GGTCGTGGAG CTGCGCAACC GATTCACCGG 600
CGAGGCCCGC GAGATCGCGG CCGAGGGTGC GGCCGCCGAG GTGGCCGCCC AGCTGCGGGC 660
CTAACCGGTC TTCTCTGGCC CGCCCGGGAA TGCCGTGGTG ATCTGGGGGA CTCCGAGCAC 720
CTGGTTCCAG CGGGCAGCCA GCACCGCGGC CTGCGTCATC GCGGTCACCG CGAAGCCGCG 780
GTCCTCGGTT GTCTGCGCCT GTTCCAGCAC GGCTCGCCAT GCGGTGGCGG TGCACCGCCA 840
GTTCGGCGGC ATCGCCGGGG TCGGTCACCT CCAGCGGCAA GCTCGTAACC GGCCGCCGGC 900
AGTGGCGGGG TCACCGAGTG TTCGGCCAGC ATGACCAGTG CCTCTTCGCG GCGCGCCCGG 960
TGCTGGGCCA TCGACTCGGC GACCAGGGCG TTGTCCTCGG CCGTGGAATG CGCCGAGACC 1020
ACGCCGTAGC CGTAGATCGC GCCGTGCTCG GTGGCGACCG CGTCGAACAA CGCCGCCTGG 1080
GCCGCGTCGG CCGGCCGGCC CGGATCGGTG TCAGGCTGCG GTGAGGTCAC GCTTTCTTCT 1140
CCGGATCACC CAGTGCCACT TGATAAGCCG CGGTACAGGA GGCGGCGATG GAACCCAGCA 1200
GTCCGGCCCG GTAACCGGTC ATCTCGGTGG CGGCCCGGGC GGCATCCTCG GCCGACTCGC 1260
GCAGCGCCTT GGCGACGGCG GTGACGGTCG GCTTGGCGGC CGGTGCCGCG GTGGAACTGG 1320
TCGTCGTGGA CGGCGCTGCG CTGGTGGTGG GCGCCTCACC GTCGGTGAGG CGGGTGATCT 1380
CATCGGAGAG GGCACGGGCG TGCGCGGTGC GCTCGCCGGC GATCGCGGTG AGGGTGGCCA 1440
CCATGTCGGG GGGCAGCGTC CGTCCGGGCG GCGCCGTCGC CGCCTCACCG GCGAGCAGGC 1500
TGTCGGCGTT GGCGCGGTCG AACTGGGTCC GCAGGTCGGC GAGATCGGCC GGTGGGGGTG 1560
GCGGTGAACA GGCCGTCGTC CCGGCGAGGA GCCCCACTGC CAGGGTGGCT GGGACCGCGG 1620
CGATCAACAG GCGGCGCCGG GTCAGGGTGG GTGGGGCGCT CGGCACGCCC ACATCCTGCC 1680
ATGTTCGGTT GCGCTGCTCG TTTGGTGATT GGCACGTCGC GCCTGGCGTA TCGTTTGTTA 1740
ACCGAACCCG GGTCCCGCCG ACCGGGGAAT GTGTCCGCAC AACTCAAGAT GAGGAGCTCG 1800
CCGTGGCATC GGATGACCCG CTGCGGTCTG CGCGATTACC GTCGCGGGAG CAGGTGATCG 1860
AGCTCCTCGA CGAAGAGTTC AGCCGGGCGG GCTACGAAGT CGAAGACGTG GTGATCGGCG 1920
GTGGCCGTCC GCCGCGGCTC ACCGTGATCG CCGATGCCGA CAACGGCGTC GATCTGGATG 1980
CCATTGCCAC CTTGTCGAAG GTGGCCTCGG CGGCCCTGGA TGCCGCCGAA CAGGCCACCG 2040
GGGACTACGG CGCTTATGTG CTGGAGGTGA CCTCCCGCGG CGTCGACCGA CCGCTGACCA 2100
CCGAGAAGCA CTTCCGTAGG TCCCGTGGCC GCAAGGTCGA GGTCACGCTG GCCGACGGAT 2160
CGAGCCTGAC GGGCCGGATC GGGCAGACCG CCGACGGGGT GGTGCACTTC GTCGTCGCTG 2220
CGGGCAAGGA TTTCGAGATC CGACCGGTCG ATGTCACCGG AATCACCAAA GCAATTGTGC 2280
AAGTTGAATT TTCGCCTCCG AATCGGCGTG AATTGGAATT GTCCGAGCAG TCTGGAAAGG 2340
GGGACGACGC ATGAACATCG ACATGGCGGC GCTCCATGCC ATCGAGTCCG ACAAGGGAAT 2400
CTCGGTCGAT ATCGTCGTGG ACACCATCAA ATCCGCGTTG CTGACCGCGT ACCGGCACAC 2460
CGAGGGCCAC GAGGCCGACG CGCGTATCGA CGTCGACCGC AAGACCGGTG TGGTCAAGGT 2520
GATGGCCAGG GAAACCGATG GCGACGGCAA CCTGATCAAC GAATGGGACG ACACCCCAGA 2580
GGGATTCGGC CGCATCGCGG CGACCACCGC GCGCCAGGTG ATCCTGCAGC GGCTGCGCGA 2640
CGCCGAGAAC GAACGGATGTA CGGC
GAATTC 2670
Claims (3)
1.命名为Gld的含有草甘膦降解酶基因的DNA,其特征是该DNA的总长为2670bp,具体序列如序列表序列SEQ ID NO.1所示。
2.命名为pCRGld的质粒,其特征是在pCR2.1质粒载体上位于BamHI和EcoRI酶切位点间有2.7kb插入片段,该插入片段为权利要求1所述的命名为Gld的DNA。
3.命名为XLI-pCRGld的转化子,其特征是它由权利要求2所述的质粒pCRGld转化大肠杆菌XLI-Blue而获得。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011148136A CN1207388C (zh) | 2001-06-19 | 2001-06-19 | 草甘膦降解酶基因和含该基因的载体 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB011148136A CN1207388C (zh) | 2001-06-19 | 2001-06-19 | 草甘膦降解酶基因和含该基因的载体 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1392261A CN1392261A (zh) | 2003-01-22 |
CN1207388C true CN1207388C (zh) | 2005-06-22 |
Family
ID=4661424
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB011148136A Expired - Fee Related CN1207388C (zh) | 2001-06-19 | 2001-06-19 | 草甘膦降解酶基因和含该基因的载体 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1207388C (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1313614C (zh) * | 2005-10-17 | 2007-05-02 | 中国农业科学院生物技术研究所 | 草甘膦乙酰转移酶基因及其应用 |
-
2001
- 2001-06-19 CN CNB011148136A patent/CN1207388C/zh not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN1392261A (zh) | 2003-01-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102268391B (zh) | 氢氧化细菌wmq-7及其分离方法和应用 | |
CN117701479B (zh) | 一株能够降解苯腈类除草剂的菌株及其微生物菌剂和应用 | |
CN104845926B (zh) | 一种有利于重组蛋白胞外分泌的基因敲除大肠杆菌及其应用 | |
CN101165172B (zh) | 一种重组甲基营养杆菌及其应用 | |
CN110699288B (zh) | 防治马铃薯黑痣病的解淀粉芽孢杆菌菌株及菌剂和应用 | |
CN1207388C (zh) | 草甘膦降解酶基因和含该基因的载体 | |
CN107858364A (zh) | 一种适于甲醇酵母表达的耐高温高比活细菌植酸酶基因 | |
CA3147940A1 (en) | Novel xanthomonas strains and related methods | |
CN101701214B (zh) | 一种宽pH适用性的木聚糖酶XYNA4及其基因和应用 | |
CN102234624B (zh) | 一种表达产生枯草芽孢杆菌精氨酸酶的基因工程菌及构建方法 | |
KR100702728B1 (ko) | 환상 뎁시펩티드 합성효소 및 그의 유전자 및 환상뎁시펩티드의 대량생산계 | |
CN110055268A (zh) | 水解酶基因ameH及其编码的蛋白和应用 | |
CN110352961B (zh) | 一种经基因工程改造的大肠杆菌菌株及其应用 | |
KR101654793B1 (ko) | 토양 개선 및 환경 정화용 질소 고정력을 갖는 바실러스 서브틸리스 bk418 균주 및 이의 배양 방법 | |
CN1238503C (zh) | 草甘膦抗性基因及含该基因的载体和转化子 | |
CN106811427B (zh) | 角度双加氧酶基因dpeA1A2及其应用 | |
CN105062906A (zh) | 一种优化有机磷水解酶酵母工程菌及其酶的生产方法 | |
CN102757972B (zh) | 酰胺酶基因cmeH及其编码蛋白质及其应用 | |
CN112300975B (zh) | 一株广藿香青枯病菌的低致病力突变株及其应用 | |
CN1182242C (zh) | 一种生态安全型根瘤菌制剂生产工艺与方法 | |
CN116240227A (zh) | 一种黏细菌来源的β-1,3-葡聚糖酶及其组合物在植物疫霉病生物防治中的应用 | |
CN111411115A (zh) | 基因和改良根瘤菌的方法 | |
CN116925196A (zh) | 小分子热休克蛋白rsp_1572在提高宿主菌环境耐受性中的应用 | |
CN102559711B (zh) | 鱼腥蓝细菌2-酮基-肌醇脱氢酶基因及其应用 | |
CN114304192A (zh) | 一种耐硼赖氨酸芽孢杆菌细菌素类似物的制备方法及在防治木薯细菌性枯萎病中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C19 | Lapse of patent right due to non-payment of the annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |