CN117286161B - 麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 - Google Patents
麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 Download PDFInfo
- Publication number
- CN117286161B CN117286161B CN202311584788.0A CN202311584788A CN117286161B CN 117286161 B CN117286161 B CN 117286161B CN 202311584788 A CN202311584788 A CN 202311584788A CN 117286161 B CN117286161 B CN 117286161B
- Authority
- CN
- China
- Prior art keywords
- csadc
- dicamba
- salicylic acid
- decarboxylase
- decarboxylase gene
- 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.)
- Active
Links
- FKIKPQHMWFZFEB-UHFFFAOYSA-N 3,6-dichloro-2-hydroxybenzoic acid Chemical compound OC(=O)C1=C(O)C(Cl)=CC=C1Cl FKIKPQHMWFZFEB-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 25
- 230000015556 catabolic process Effects 0.000 title claims abstract description 24
- 239000005504 Dicamba Substances 0.000 title abstract description 36
- IWEDIXLBFLAXBO-UHFFFAOYSA-N dicamba Chemical compound COC1=C(Cl)C=CC(Cl)=C1C(O)=O IWEDIXLBFLAXBO-UHFFFAOYSA-N 0.000 title abstract description 36
- 101000821475 Cutaneotrichosporon moniliiforme Salicylate decarboxylase Proteins 0.000 title abstract description 13
- 239000013067 intermediate product Substances 0.000 title abstract description 7
- 239000002689 soil Substances 0.000 claims abstract description 8
- 230000000593 degrading effect Effects 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 108090000489 Carboxy-Lyases Proteins 0.000 claims description 24
- 102000004031 Carboxy-Lyases Human genes 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 238000003259 recombinant expression Methods 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 8
- 238000006114 decarboxylation reaction Methods 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000002744 homologous recombination Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002363 herbicidal effect Effects 0.000 abstract description 13
- 239000004009 herbicide Substances 0.000 abstract description 13
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 4
- 230000037353 metabolic pathway Effects 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 abstract 2
- 229920001184 polypeptide Polymers 0.000 abstract 2
- 239000005711 Benzoic acid Substances 0.000 abstract 1
- 235000010233 benzoic acid Nutrition 0.000 abstract 1
- 239000000047 product Substances 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 230000000813 microbial effect Effects 0.000 description 9
- 239000010802 sludge Substances 0.000 description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 7
- 235000011130 ammonium sulphate Nutrition 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005067 remediation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- HCJMNOSIAGSZBM-UHFFFAOYSA-N 6-methylsalicylic acid Chemical compound CC1=CC=CC(O)=C1C(O)=O HCJMNOSIAGSZBM-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001335 demethylating effect Effects 0.000 description 2
- 230000017858 demethylation Effects 0.000 description 2
- 238000010520 demethylation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- RANCECPPZPIPNO-UHFFFAOYSA-N 2,5-dichlorophenol Chemical group OC1=CC(Cl)=CC=C1Cl RANCECPPZPIPNO-UHFFFAOYSA-N 0.000 description 1
- QCEPIUWMXRQPIF-UHFFFAOYSA-N 2-chloro-6-hydroxybenzoic acid Chemical compound OC(=O)C1=C(O)C=CC=C1Cl QCEPIUWMXRQPIF-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101710091614 Decarboxylase yanB Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001558 benzoic acid derivatives Chemical class 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- 230000000911 decarboxylating effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 108010056846 dicamba O-demethylase Proteins 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004725 rapid separation liquid chromatography Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/36—Organic compounds containing halogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Medicinal Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了麦草畏厌氧降解中间产物3,6‑二氯水杨酸脱羧酶CsaDC及其应用。csaDC其核苷酸序列为SEQ ID NO.1,全长1035 bp,其氨基酸序列为SEQ ID NO.2,编码346个氨基酸。CsaDC是首次发现参与苯甲酸类除草剂麦草畏厌氧降解下游代谢途径的脱羧酶,能够有效降解麦草畏中间产物3,6‑二氯水杨酸。脱羧酶CsaDC可用于降解土壤和水体中麦草畏的残留,具有非常重要的理论和应用价值。
Description
技术领域
本发明属于环境和农业生物技术领域,涉及麦草畏脱甲基中间产物3,6-二氯水杨酸(3,6-DCSA) 脱羧酶的基因csaDC及其应用。
背景技术
化学农药作为现代农业必不可缺少的重要的生产资料,为保护农业生产、保障我国乃至世界粮食安全发挥了至关重要的作用。除草剂作为农药的重要种类,其使用能有效减轻农业劳动强度、保证农业正常生产,但是随着除草剂的大量使用,其残留对土壤造成的危害越来越严重。微生物修复技术是一种原位生物修复技术,效果好,费用低,无二次污染,适合大面积面源污染修复,是土壤有机污染物修复技术的主流和发展方向。抗除草剂转基因是解决除草剂药害的有效途径,而抗除草机的基因一般均来自微生物降解基因。
麦草畏是苯甲酸系列的激素型除草剂,具有广谱、高效和低毒的特点,对一年生和多年生阔叶杂草则有明显的防除效果,是目前世界上使用量仅次于草甘膦的除草剂。麦草畏在环境中的降解目前主要为微生物降解,目前已筛选到多种麦草畏的降解菌株,克隆到多个麦草畏降解基因,目前关于麦草畏的微生物降解基本上第一步都是脱甲基生成无除草活性的3,6-DCSA,见图1。其中麦草畏O-脱甲基酶基因 dmo (专利 US7105724B2) 转入大豆成功构建抗麦草畏转基因大豆,商标定为 Roundup Ready 2 XtendTM,该品种 2013 年在美国进行大田试验。麦草畏杂草抗性产生慢是非常理想的抗除草剂转基因的靶标除草剂。随着麦草畏混剂的开发和抗麦草畏的转基因作物技术研究的不断深入,麦草畏在世界范围内的需求量必将大幅增加。但是,到目前为止,微生物降解麦草畏微生物降解的第一步脱甲基产物 3,6-DCSA 的代谢途径及其分子机制还不清楚,这严重制约了对麦草畏的环境行为和生态安全方面的研究。
获得麦草畏脱甲基中间产物3,6-二氯水杨酸(3,6-DCSA)的降解菌株和降解基因在治理农药残留中主要具有以下作用,(一)通过现代微生物发酵技术将麦草畏降解菌株和基因制成降解菌剂或酶制剂实现土壤原位修复。(二)通过现代生物技术将降解基因导入作物构建相应的除草剂抗性转基因作物。综上所述,开展麦草畏脱甲基产物 3,6-DCSA 微生物降解代谢机制的研究具有非常重要的理论和实际应用价值。
发明内容
本发明的目的是针对现有麦草畏微生物降解途径的研究的缺乏,从能够高效降解麦草畏的厌氧活性污泥中克隆到一个脱羧酶基因csaDC,该基因是首次发现的能够催化3,6-DCSA脱羧,该基因编码的蛋白不仅能将3,6-DCSA的羧基脱除,同时能催化6-氯水杨酸和6-甲基水杨酸等多种带羧基结构的底物脱羧,在含羧基化合物生物降解和转化中具有重要的应用价值。
本发明的又一目的是提供该基因的应用。
本发明的目的通过以下技术方案实现:
一个3,6-二氯水杨酸脱羧酶基因csaDC,其核苷酸序列为SEQ ID NO.1。通过序列分析和基因比对的方法来寻找目的基因。前期通过硫酸铵分级沉淀的方法获得能够使3,6-二氯水杨酸脱羧的组分,将获得的组分进行预处理过后通过LTQ-Orbitrap XL质谱仪测定该组分所含的蛋白,使用MaxQuant(版本:1.6.17.0)的默认集分析测定蛋白质的原始数据,将测得的蛋白序列和污泥测得的宏基因组序列与NCBI数据库进行比对,找到负责3,6-二氯水杨酸脱羧的蛋白序列。所述的脱羧酶基因csaDC编码的蛋白CsaDC,其氨基酸序列为SEQID NO.2。
含有所述的3,6-二氯水杨酸脱羧酶基因csaDC的重组表达载体pET29a-csadc。
所述的重组表达载体,优选将3,6-二氯水杨酸脱羧酶基因csaDC插入pET-29a(+)所得,所述的3,6-二氯水杨酸脱羧酶基因csaDC是以合成的csaDC为DNA模板,由SEQ IDNO.3 和SEQ ID NO.4所示的引物进行PCR扩增得到。
含有所述的3,6-二氯水杨酸脱羧酶基因csaDC的高效表达菌株的构建,所述的表达菌株优选E coli. BL21 (DE3)。
所述脱羧酶基因csaDC、所述的脱羧酶基因csaDC的重组表达载体或所述的基因工程菌株在3,6-二氯水杨酸脱羧反应中的应用。
所述脱羧酶基因csaDC、所述的脱羧酶基因csaDC的重组表达载体或所述的基因工程菌株在制备降解3,6-二氯水杨酸的试剂中的应用。
所述3,6-二氯水杨酸脱羧酶CsaDC在降解3,6-二氯水杨酸中的应用。
所述3,6-二氯水杨酸脱羧酶CsaDC在去除土壤和水体3,6-二氯水杨酸中的应用。
本发明的有益效果如下:
本发明首次公开了除草剂麦草畏的厌氧微生物代谢中间产物 3,6-二氯水杨酸脱羧酶CsaDC及其应用。3,6-二氯水杨酸脱羧酶基因csaDC核苷酸及氨基酸序列分别为:SEQID NO.1,SEQ ID NO.2,基因全长为 1035 bp,编 码 346 个氨基酸。csaDC能高效快速降3,6-二氯水杨酸,因此CsaDC在厌氧降解麦草畏的过程中起着重要的作用,对去除环境中的麦草畏具有非常重要的理论和应用价值。
附图说明
图1、驯化的厌氧活性污泥降解麦草畏的代谢途径推测;
图2、 3,6-二氯水杨酸脱羧酶CsaDC纯化的SDS-PAGE电泳图。1:蛋白Marker;2:反应器中污泥破碎的粗酶;3:硫酸铵分级沉淀0-20%饱和度;4:硫酸铵分级沉淀20-40%饱和度;5:硫酸铵分级沉淀40-60%饱和度;5:硫酸铵分级沉淀60-80%饱和度;6:硫酸铵分级沉淀80-100%饱和度;
图3、 重组蛋白pET29a-csadc SDS-PAGE电泳图。1:蛋白Marker;2:重组表达菌株E.coli/BL21 (DE3)-csadc的粗酶液;3:150 mM咪唑洗脱液;
图4 、脱羧酶降解3,6-二氯水杨酸的紫外扫描图;
图5 、脱羧酶CsaDC催化3,6-二氯水杨酸的能力检测。A:脱羧酶CsaDC催化3,6-二氯水杨酸的液相检测图;B/C:脱羧酶CsaDC催化3,6-二氯水杨酸的产物LC-QTOF-MS图谱。
具体实施方式
实施例1 3,6-二氯水杨酸脱羧酶基因csaDC的查找:
用来驯化的污泥采自长期施用麦草畏且处于30-40 cm 深度的稻田土,将10 kg采集的稻田土与2 L液体基础盐培养基和0.17 mM麦草畏混合均匀,加入厌氧反应器中。每隔1-2天从反应器中取样,使用高效液相色谱(HPLC)检测麦草畏浓度,当90%麦草畏降解之后,在反应器中加入0.33 mM麦草畏,按照以上方法经过五轮富集之后,反应器中的菌群可在7天降解0.83 mM麦草畏。取富集的厌氧污泥加入到一个高1.5 cm直径200 mm的上流式厌氧反应器中进一步富集,利用蠕动泵把含2635 µM麦草畏的废水泵入上流式反应器。在维持反应器对麦草畏降解率保持在90%以上条件下,不断加大反应器进水量,直到反应器进水达到4升每天,相应的水力停留时间为3天左右。
采集反应器中已获得高效厌氧降解麦草畏能力的污泥送往美吉生物公司进行宏基因组测定,从而获得反应器中菌群的宏基因组序列。
上述基础盐培养基为:0.53 g NH4Cl,0.35 g K2HPO4,0.27 g KH2PO4,0.2 gFeSO4·7H2O,0.1 g MgCl2·6H2O, 0.1 g丙酮酸钠,0.1 g抗坏血酸,0.073 g CaCl2·2H2O,1.0 mg刃天青,30 mg半胱氨酸盐酸盐,1.0 mL微量元素复合液,1.0 mL维生素复合液,加去离子水定容至1L。
取上流式反应器中的污泥2 L,使用超声波细胞破碎仪(UH-650B,40%强度)进行超声破碎,将得到的粗酶液通过硫酸铵分级沉淀,获得60-80%饱和度的组分可使3,6-二氯水杨酸脱羧(图2)。对该组分进行前处理后,通过离心收集消化肽,并使用NanoDrop分光光度计进行定量。使用Ultimate 3000 RSLC纳米系统(Thermo Fisher Scientific)进行在线分离,之后使用配备有纳米电喷雾离子源的LTQ Orbitrap XL质谱仪(Thermo FisherScientific)进行数据依赖性MS/MS分析。蛋白质鉴定的原始数据由MaxQuant(版本:1.6.17.0)采用默认集进行分析,最终获得522个肽段序列。为了筛选出这522个序列中的脱羧酶序列,将这些序列分别在NCBI使用BLASTp进行搜索比对,结果显示其中一个序列(SEQID NO.2)与来自Aspergillus niger ATCC 1015的脱羧酶yanB的氨基酸序列相似性为37.6%,且该蛋白序列对应的核酸序列同时来自宏基因组测定的结果中,由此获得了3,6-二氯水杨酸降解基因,命名该基因为csaDC。
实施例2 3,6-二氯水杨酸脱羧酶基因csaDC的异源表达和功能验证:
2.1csaDC基因的克隆
以正向引物:5’-gaaggagatatacatatgGAAGTCAAGAAGAAGACATCCAAC-3’ (SeQ IDNO.3);反向引物:5’-tctcagtggtggtggtggtggtgCCGCTTTTCGGCGAGGCGT-3’ (SeQ IDNO.4),通过PCR以合成的DNA序列(SeQ ID NO.1)为模板扩增出脱羧酶csaDC基因。
特异性扩增体系(50 µL):
反应程序如下:
2.2 重组表达菌株的构建和验证
2.2.1 质粒线性化
以正向引物:5’-Catatgtatatctccttc-3’ (SeQ ID NO.5);反向引物:5’-caccaccaccaccaccactgagatccggctgctaacaaagcc-3’ (SeQ ID NO.6) 将质粒pET-29a (+) 进行线性化,并用Dpn I 消除模版污染。PCR产物使用凝胶纯化回收试剂盒,具体方法参考试剂盒说明书。0.75% 的琼脂糖核酸电泳检测PCR产物。
2.2.2重组表达菌株的构建
于冰水浴中配制如下反应体系 (10 µL) :
混匀后,在37 ℃水浴30 min,反应完成后立即置于冰水浴中冷却5 min,将同源重组产物转入大肠杆菌表达菌株E.coli BL21 (DE3) 中。挑取转化子至50 mg/L Km的3 mLLB试管中,在37 ℃、180 rpm摇床培养,提取质粒获得阳性转化子,分别进行PCR验证和送往上海生工生物技术科技有限公司进行测序,验证插入质粒pET-29a (+)的DNA片段序列是否正确,将获得的含有SeQ ID NO.1所示的-csaDC基因的阳性克隆含有pET29a-csaDC的E.coli BL21 (DE3)表达菌株命名为E.coli/ BL21 (DE3)-csaDC。
2.3CsaDC 的诱导表达与纯化
重组表达菌株E.coli/ BL21 (DE3)-csaDC在100 mL LB液体培养基中,37 ℃,180rpm培养至OD600为0.4-0.6时,加入0.10 mM IPTG, 16 ℃诱导培养8 h; 4 ℃、12000 rpm离心5 min,收集菌体,用15 mL 50 mM PBS (pH 7.0) 缓冲液重悬菌体,超声破碎5-10 min,12000 rpm离心30 min, 收集上清,用0.22 µm 的水相过滤器过滤去除菌体与破碎残渣,即获得重组表达菌株E.coli/BL21 (DE3)-csaDC的粗酶液,然后用镍离子亲和层析柱对CsaDC进行纯化。收集洗脱液,在4 ℃、50 mM PBS (pH 7.0) 缓冲液中用透析袋 (截留分子量10kDa) 透析过夜。SDS-PAGE 蛋白电泳检测纯化效果,条带大小和理论预测的大小 (39.3kDa) 相一致,见图3。
2.4CsaDC 活力测定
酶活反应体系 (1 mL):加有PBS (50 mM, pH 7.0),0.1 mM 3,6-二氯水杨酸,反应酶量 (2.3 中纯化所得) 100 µL,30 ℃ 反应。每个反应以加入3,6-二氯水杨酸开始计时,通过紫外扫描进行定时测定。3,6-二氯水杨酸的紫外吸收峰最高是319 nm,经过反应后产生脱羧产物,因此通过紫外扫描 (200-400 nm) 可以看到在319 nm 处紫外峰明显降低,275 nm 处有升高,降解情况见图4。将酶反应液通过煮沸,过滤,进行HPLC检测3,6-二氯水杨酸的降解情况。实验结果表明纯化后的脱羧酶CsaDC能够降解3,6-二氯水杨酸。酶学实验表明纯化后的CsaDC降解3,6-二氯水杨酸的比酶活为0.8 U/mg。
酶促反应后的产物采用HPLC和LC-QTOF-MS技术进行检测和鉴定分析。HPLC检测分析得到在酶反应体系中加入纯化的CsaDC后有新的产物产生 (保留时间为9.89 min),LC-QTOF-MS检测结果分析得到此产物为3,6-二氯水杨酸脱掉羧基所得,即产物为2,5-二氯苯酚 (图5)。
Claims (10)
1.一种脱羧酶基因csaDC,其核苷酸序列为SEQ ID NO.1。
2.权利要求1所述的脱羧酶基因csaDC编码的蛋白CsaDC,其氨基酸序列为SEQ IDNO.2。
3.含有权利要求1所述的脱羧酶基因csaDC的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于,是将权利要求1所述的脱羧酶基因csaDC与线性化的pET-29a(+)质粒同源重组所得。
5.含有权利要求1所述的脱羧酶基因csaDC的基因工程菌株。
6.权利要求5所述的基因工程菌株,其特征在于,所述的基因工程菌株为大肠杆菌E. coli BL21 (DE3)。
7.权利要求1所述脱羧酶基因csaDC、权利要求3所述的脱羧酶基因csaDC的重组表达载体或权利要求5所述的基因工程菌株在3,6-二氯水杨酸脱羧反应中的应用。
8.权利要求1所述脱羧酶基因csaDC、权利要求3所述的脱羧酶基因csaDC的重组表达载体或权利要求5所述的基因工程菌株在制备降解3,6-二氯水杨酸的试剂中的应用。
9.权利要求2的所述蛋白CsaDC在降解3,6-二氯水杨酸中的应用。
10.权利要求2的所述的蛋白CsaDC在去除土壤或水体中3,6-二氯水杨酸的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311584788.0A CN117286161B (zh) | 2023-11-27 | 2023-11-27 | 麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311584788.0A CN117286161B (zh) | 2023-11-27 | 2023-11-27 | 麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117286161A CN117286161A (zh) | 2023-12-26 |
CN117286161B true CN117286161B (zh) | 2024-03-19 |
Family
ID=89258989
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311584788.0A Active CN117286161B (zh) | 2023-11-27 | 2023-11-27 | 麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117286161B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1379539A2 (en) * | 2001-02-28 | 2004-01-14 | Board of Regents of the University of Nebraska | Methods and materials for making and using transgenic dicamba-degrading organisms |
CN108441503A (zh) * | 2018-03-22 | 2018-08-24 | 南京农业大学 | 麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用 |
CN111118037A (zh) * | 2019-10-14 | 2020-05-08 | 中国农业科学院生物技术研究所 | 具有除草剂麦草畏降解功能的基因dicx4及其应用 |
CN116286889A (zh) * | 2022-08-02 | 2023-06-23 | 南阳师范学院 | 四氢叶酸依赖型麦草畏脱甲基酶基因dmt06及其应用 |
-
2023
- 2023-11-27 CN CN202311584788.0A patent/CN117286161B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1379539A2 (en) * | 2001-02-28 | 2004-01-14 | Board of Regents of the University of Nebraska | Methods and materials for making and using transgenic dicamba-degrading organisms |
CN108441503A (zh) * | 2018-03-22 | 2018-08-24 | 南京农业大学 | 麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用 |
CN111118037A (zh) * | 2019-10-14 | 2020-05-08 | 中国农业科学院生物技术研究所 | 具有除草剂麦草畏降解功能的基因dicx4及其应用 |
WO2021073220A1 (zh) * | 2019-10-14 | 2021-04-22 | 中国农业科学院生物技术研究所 | 具有除草剂麦草畏降解功能的基因dicX4及其应用 |
CN116286889A (zh) * | 2022-08-02 | 2023-06-23 | 南阳师范学院 | 四氢叶酸依赖型麦草畏脱甲基酶基因dmt06及其应用 |
Non-Patent Citations (3)
Title |
---|
Anaerobic degradation of dicamba and metribuzin in riparian wetland soils;Edelgard W. Pavel 等;《Water Research》;19990131;第33卷(第1期);摘要,第87-94页 * |
麦草畏降解菌株Ochrobactrum sp. 3-3的分离鉴定及其降解特性;于林鹭 等;《应用与环境生物学报》;20170425;第23卷(第2期);第283-288页 * |
麦草畏降解菌株Sphingobium sp.Dca-5的分离、鉴定及降解特性;李娜 等;《应用与环境生物学报》;20190825;第25卷(第4期);第943-949页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117286161A (zh) | 2023-12-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220220516A1 (en) | Method for asymmetrically preparing l-phosphinothricin by oxidation-reduction reaction through biological multi-enzyme coupling | |
CN107794271B (zh) | 一个龙胆酸双加氧酶及其编码基因和应用 | |
US20240101974A1 (en) | D-amino acid oxidase mutants and uses thereof in preparing l-glufosinate | |
CN109971773B (zh) | 一种编码可降解3-氯龙胆酸的龙胆酸双加氧酶DsmI的基因dsmI及其应用 | |
US10787489B2 (en) | Biocatalyst comprising photoautotrophic organisms producing recombinant enzyme for degradation of harmful algal bloom toxins | |
CN114525268B (zh) | 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用 | |
CN109762800B (zh) | 一种啶虫脒酰胺酶基因aceAB及其编码蛋白质及其应用 | |
CN117286161B (zh) | 麦草畏厌氧降解中间产物3,6-二氯水杨酸脱羧酶CsaDC及其应用 | |
CN112522228B (zh) | 一种来源于氨氧化假诺卡氏单胞菌的r-转氨酶及其合成方法 | |
CN110055268B (zh) | 水解酶基因ameH及其编码的蛋白和应用 | |
CN111118037B (zh) | 具有除草剂麦草畏降解功能的基因dicx4及其应用 | |
CN109456986B (zh) | 对芳氧苯氧丙酸类除草剂中间体具有手性选择性的双加氧酶Snpd及其编码基因和应用 | |
CN108441503B (zh) | 麦草畏中间产物3,6-二氯龙胆酸脱氯酶DsmH2及其编码基因的应用 | |
CN113735282B (zh) | 一种老黄酶oye2蛋白及其在铬污染中的应用 | |
CN113930401B (zh) | 提高漆酶催化活性的方法及突变体Lcc9-M1、基因和应用 | |
WO2016015668A1 (zh) | 氨基甲酸酯类农药降解酶CFH与其编码基因cfd以及二者的应用 | |
CN109439573B (zh) | 对s-敌草胺具有专一转化功能的菌株、酰胺水解酶、编码基因及其应用 | |
CN108624570B (zh) | 六价铬还原酶及其在治理水体铬污染中的应用 | |
CN110872591B (zh) | 除草剂麦草畏降解基因dicX1及其应用 | |
CN110951664A (zh) | 一种重组谷氨酸棒杆菌及其在生产2-吡咯烷酮中的应用 | |
CN114438065B (zh) | 喹啉酸中间产物6-羟基喹啉酸脱羧酶QuiB及其应用 | |
CN111139249B (zh) | 运动金球菌麦草畏降解基因dicM的用途 | |
CN113388627B (zh) | 还原酶lx05基因,含有该基因的基因工程菌及其应用 | |
CN111139238B (zh) | 除草剂麦草畏的降解基因dicX3及其应用 | |
CN111139248B (zh) | dicX5基因及其对除草剂的降解功能 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |