CN116836902A - Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof - Google Patents
Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof Download PDFInfo
- Publication number
- CN116836902A CN116836902A CN202310759784.5A CN202310759784A CN116836902A CN 116836902 A CN116836902 A CN 116836902A CN 202310759784 A CN202310759784 A CN 202310759784A CN 116836902 A CN116836902 A CN 116836902A
- Authority
- CN
- China
- Prior art keywords
- sigk
- hyd1
- gene
- engineering bacteria
- gene fragment
- 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.)
- Pending
Links
- 241000193388 Bacillus thuringiensis Species 0.000 title claims abstract description 36
- 229940097012 bacillus thuringiensis Drugs 0.000 title claims abstract description 36
- 241000894006 Bacteria Species 0.000 title claims abstract description 33
- 238000010276 construction Methods 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
- 230000001105 regulatory effect Effects 0.000 claims abstract description 35
- 238000013518 transcription Methods 0.000 claims abstract description 33
- 230000035897 transcription Effects 0.000 claims abstract description 32
- 239000012634 fragment Substances 0.000 claims abstract description 30
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract description 22
- 210000004027 cell Anatomy 0.000 claims abstract description 21
- 101710097941 N-acetylmuramoyl-L-alanine amidase CwlA Proteins 0.000 claims abstract description 14
- 230000035772 mutation Effects 0.000 claims abstract description 14
- 238000005516 engineering process Methods 0.000 claims abstract description 9
- 230000006801 homologous recombination Effects 0.000 claims abstract description 4
- 238000002744 homologous recombination Methods 0.000 claims abstract description 4
- 239000013612 plasmid Substances 0.000 claims description 23
- 238000011144 upstream manufacturing Methods 0.000 claims description 13
- 241000255925 Diptera Species 0.000 claims description 11
- 238000012217 deletion Methods 0.000 claims description 9
- 241000256173 Aedes albopictus Species 0.000 claims description 8
- 241000256054 Culex <genus> Species 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 230000017858 demethylation Effects 0.000 claims description 6
- 238000010520 demethylation reaction Methods 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 101150025376 sigK gene Proteins 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 2
- 101710151559 Crystal protein Proteins 0.000 description 15
- 230000000749 insecticidal effect Effects 0.000 description 15
- 210000002421 cell wall Anatomy 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 8
- 230000009089 cytolysis Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 210000003969 blast cell Anatomy 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 239000012154 double-distilled water Substances 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 229960003276 erythromycin Drugs 0.000 description 4
- 230000002045 lasting effect Effects 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 101000894568 Catharanthus roseus Catharanthine synthase Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 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 2
- 230000002934 lysing effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 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
- 101100497595 Bacillus subtilis (strain 168) cwlC gene Proteins 0.000 description 1
- 101100219198 Bacillus thuringiensis subsp. israelensis cry11Aa gene Proteins 0.000 description 1
- 101100329321 Bacillus thuringiensis subsp. israelensis cry4Ba gene Proteins 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- -1 Cry4Aa Proteins 0.000 description 1
- 102000014824 Crystallins Human genes 0.000 description 1
- 108010064003 Crystallins Proteins 0.000 description 1
- 241000256113 Culicidae Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
- A01N63/23—B. thuringiensis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- 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
- C12N9/14—Hydrolases (3)
-
- 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
- C12R2001/07—Bacillus
- C12R2001/075—Bacillus thuringiensis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Agronomy & Crop Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Dentistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Insects & Arthropods (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganism engineering bacteria. Double mutation engineering bacteria of Bacillus thuringiensis subspecies israeli, which is strain Bt59 peptidoglycan hydrolase genehyd1And transcription regulatory factor genesigKDouble mutation engineering bacteria Bt59 (delta)hyd1‑sigK) Deposit number: CGMCC No.27191. The construction method of the engineering bacteria uses Bt59 geneThe cell wall hydrolase genes are obtained by using the group as a template and utilizing the overlapping PCR technologyhyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is then subjected to homologous recombination technology to obtain the cell wall hydrolase genehyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is constructed into a gene operation vector pMAD, is transformed into Bti competent cells by electric shock, and is screened at high temperature to obtain double mutation engineering bacteria Bt59 (delta)hyd1‑sigK). The engineering bacteria are applied. The invention is used for solving the technical problem of short duration of engineering bacteria of the israeli subspecies of the bacillus thuringiensis.
Description
Technical Field
The invention relates to the technical field of microorganism engineering bacteria, in particular to a double-mutation engineering bacteria of the israeli subspecies of bacillus thuringiensis, a construction method of the engineering bacteria and application of the engineering bacteria.
Background
Bacillus thuringiensis @Bacillus thuringiensisBt) is a gram-positive bacterium widely existing in soil, can synthesize insecticidal crystal protein with specific insecticidal activity while forming spores, and is widely applied to the prevention and control of agricultural, forest and sanitary pests. Bt israel subspecies @B. thuringiensissubsp.israelensisBti) is the first subspecies of Bt that proved to be toxic to dipteran larvae, and can be producedVarious insecticidal crystal proteins such as Cry4Aa, cry4Ba, cry11Aa and Cyt1Aa have toxicity activity to more than 500 kinds of mosquitoes, and flies. Crystallins are produced during phase II of spore formation and have three different types of crystal composition, located outside the outer membrane of the spore, and shed during growth with release of the spore. After the crystal protein is eaten by larvae, toxic peptide is dissociated under the action of alkaline protease in insect midgut, normal functions of intestinal tracts are destroyed, and the insect larvae die. Thanks to its broad-spectrum, efficient mosquito killing activity, safety to the environment and non-target organisms, and its remarkable economic, social and ecological benefits, bti has been successfully and widely applied to the biological control of disease-borne mosquitoes.
Since the crystal protein is released through the explanation of the blast cell rupture in the later stage of the spore formation and is not embedded in the cell wall, the crystal protein exposed in the environment is easy to be inactivated by the influence of natural factors such as solar radiation, temperature and the like, so that the insecticidal persistence of Bti is obviously reduced, and Ultraviolet (UV) radiation is considered as the most critical influencing factor. In order to overcome the influence of environmental adverse factors on the crystal protein, the traditional approach is mainly to add a UV protective agent or embed the crystal protein by adopting a physical material so as to improve the tolerance of Bt protein to UV and enhance the lasting stability of toxin protein. Cry1C/Ab chimeric crystal protein is expressed in the parent cell non-lysing strain Bt 407-, and the non-lysing cell wall is utilized to protect the crystal protein from being inactivated by UV damage, so that the duration of insecticidal activity of the strain is prolonged. In Bt Coulossie subspecies @B. thuringiensissubsp.kurstakiBtk) HD73 strain by knocking out key peptidoglycan hydrolase genescwlCThe parent cell is constructed to not lyse the strain and maintain the original virulence of the strain. Therefore, by knocking out the key peptidoglycan hydrolase gene, the blast cell is blocked from being cracked, and the insecticidal crystal protein is embedded by utilizing the cell wall, so that the insecticidal activity of the strain is maintained, and the lasting stability of the protein is obviously enhanced.
Among the peptidoglycan hydrolases involved in the lysis of the mother cell of the Bti strain Bt59 are CwlB, cwlC. Unlike the function in HD73, knockoutcwlCOnly delayed but not completely blocked Bt59 blast lysis. In the later growth period of the Bti spores,cwlCandcwlBare all regulated by a transcription regulating factor SigK, knocked outsigKCan completely inhibitcwlBAndcwlCbut still does not completely block Bti blast lysis.
The applicant has conducted intensive studies on the above technical problems, knockoutsigKCan completely inhibitcwlBAndcwlCbut still not completely block the cause of Bti blast lysis: the strain Bti Bt59 is involved in the lysis of blast cells and the peptidoglycan Hydrolase other than CwlB and CwlC is also Hydrolase1 (Hyd 1), and Hyd1 is specifically expressed in Bti.
Disclosure of Invention
Aiming at the problems existing in the prior art, the first object of the invention is to provide a double-mutation engineering bacterium of the israeli subspecies of the bacillus thuringiensis, which is used for solving the technical problem of short duration of the israeli subspecies engineering bacterium of the bacillus thuringiensis.
In order to solve the technical problems, the invention adopts the following technical proposal that the engineering bacteria are double-mutation engineering bacteria of the subspecies israeli, and is characterized in that the engineering bacteria are strain Bt59 peptidoglycan hydrolase geneshyd1And transcription regulatory factor genesigKDouble mutation engineering bacteria Bt59 (delta)hyd1-sigK) Deposit number: CGMCC No.27191, preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection); deposit unit address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; preservation date: 2023, 4, 23; classification naming: bacillus thuringiensis var.
The invention identifies a hydrolase gene specifically expressed in the subspecies israeli of bacillus thuringiensis through bioinformatics and in vitro activity analysishyd1Recombinant hydrolase Hyd1 has activity of hydrolyzing the cell wall of Bti and plays a key role in the cell lysis of Bti. The insecticidal crystal protein of the Bti is easily deactivated under the influence of factors such as ultraviolet rays in the environment, so that the actual use effect of the Bti is seriously influenced, and the cell wall can protect the crystal protein from the influence of the external environment, so that the lasting effect of the crystal protein is enhanced.
Applicants found that Bti strain Bt59 was involved in maternal cell lysisThe peptidoglycan Hydrolase has, in addition to CwlB and CwlC, hydrolase1 (Hyd 1), and Hyd1 is specifically expressed in Bti. Unlike the function in HD73, knockoutcwlCOnly delayed but not completely blocked Bt59 blast lysis. In the later growth period of the Bti spores,cwlCandcwlBare all regulated by a transcription regulating factor SigK, knocked outsigKCan completely inhibitcwlBAndcwlCbut does not completely block Bti blast lysis.hyd1Is regulated by a transcription regulating factor SigE,sigKthe absence of (2) does not affecthyd1Is expressed by (a). Thus, the peptidoglycan hydrolase gene is knocked out simultaneouslyhyd1And transcription regulatory factorsigKThe mother cell can be constructed without cracking the Bti strain, and the lasting period of the Bti strain is prolonged.
The second purpose of the invention is to provide a construction method of the double mutation engineering bacteria of the israeli subspecies of the bacillus thuringiensis, which is used for solving the technical problem of short duration of the existing israeli subspecies production strains of the bacillus thuringiensis.
In order to solve the technical problems, the invention adopts the following technical scheme: the construction method of Bacillus thuringiensis subspecies israeli double mutation engineering bacteria uses Bt59 genome as template and utilizes overlap PCR technology to obtain cell wall hydrolase genehyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is then subjected to homologous recombination technology to obtain the cell wall hydrolase genehyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is constructed into a gene operation vector pMAD, is transformed into Bti competent cells by electric shock, and is screened at high temperature to obtain double mutation engineering bacteria Bt59 (delta)hyd1-sigK)。
The invention can quickly carry out gene modification on Bti by utilizing the gene knockout technology so as to obtain engineering bacteria with excellent properties, simultaneously the exogenous plasmid can be lost at high temperature, and the engineering bacteria do not contain exogenous genes and have no marked resistance and can be directly used for industrial production and fermentation.
The invention utilizes homologous recombination technology to successfully construct the peptidoglycan hydrolase genehyd1Deletion mutant, transcription regulatory factor genesigKDeletion mutant,hyd1AndsigKdouble deletion mutants.hyd1AndsigKthe double mutation can completely block the cell lysis of the Bti mother cell, and the cell wall can be used for embedding the crystal protein so as to enhance the environmental stability. The double-mutation engineering bacteria constructed by the invention do not produce spores, and have higher insecticidal activity and stronger ultraviolet stress resistance.
To solve the transcription regulatory factor genesigKThe technical problem of how to obtain the deleted gene fragment, namely, obtaining the transcription regulatory factor gene by utilizing the overlapping PCR technologysigKA deleted gene fragment comprising:
the Bt59 genome is used as a template, and overlapped PCR is performed by using an overlapped primer SEQ ID NO.9-10 to obtain a transcription regulatory factor genesigKDeleted gene fragment, transcription regulatory factor genesigKThe deleted gene fragment is connected to a linearized pMAD plasmid to obtain a recombinant plasmid; after demethylation of the recombinant plasmid, the recombinant plasmid is transformed into Bt59 cells by electric shock to obtainsigKDeletion mutant Bt59 (. DELTA.sigK)；
The overlapping PCR primer SEQ ID NO.9 is:
an upstream primer ATCGATGCATGCCATGGTACCCGGGATTGTACAGAGTCATCGGTAATTTC;
a downstream primer GCGTCTGCAGAAGCTTCTAGAATTCCAGAACCAGAAAGCGGACTATG.
To solve the problem of cell wall hydrolase genehyd1The technical problem of deleted Gene fragments, the use of overlapping PCR techniques, cell wall hydrolase geneshyd1A deleted gene fragment comprising:
the Bt59 genome is used as a template, and overlapped PCR is performed by using overlapped primers SEQ ID NO.11-12 to obtain the cell wall hydrolase genehyd1Deleted gene fragment, cell wall hydrolase genehyd1The deleted gene fragment is connected to a linearized pMAD plasmid to obtain a recombinant plasmid; after demethylation of the recombinant plasmid, transformation to Bt59 and Bt59 (delta) was performed by electric shock, respectivelysigK) In cells, obtainhyd1Deletion mutant Bt59 (. DELTA.hyd1) And (d) sumhyd1AndsigKdouble mutant strain Bt59 (. DELTA.hyd1-sigK)；
The overlapping PCR primer two SEQ ID NO.10-11 is:
an upstream primer CAGATCTATCGATGCATGCCATGGTACCCGAATCCATCTGTCCCGCTTAAACTG;
a downstream primer:
CATGGGAATATTCGGTGTGGGAAATTACATATCCATCGCTCCTCTTCTCTTAG。
the second object of the invention is to provide the application of the double mutant engineering bacteria of the israeli subspecies of the bacillus thuringiensis in biological control of disease-vector mosquitoes. The bacillus thuringiensis israel subspecies adopted by the invention are used as microbial pesticides, and are used for preventing and controlling disease-borne mosquitoes for a long time, and the toxins of the bacillus thuringiensis israel subspecies mainly act on the mosquitoes and are relatively safe to vertebrates.
The technical scheme of the invention is further improved, and the bacillus thuringiensis subspecies israeli double-mutation engineering bacteria are applied to biological control of disease vector mosquitoes including culex light and aedes albopictus. The invention relates to a method for preparing a peptidoglycan hydrolase gene by a gene editing technologyhyd1And transcription regulatory factor genesigKDouble mutation blocks the lysis of parent cells, and utilizes cell walls to embed crystal proteins, so that the insecticidal duration of Bti on mosquito larvae is improved.
Bacillus thuringiensis israeli subspecies peptidoglycan hydrolase genehyd1The nucleotide sequence of the peptidoglycan hydrolase gene is shown as SEQ ID NO. 1.
Transcription regulatory factor gene of bacillus thuringiensis subspecies israelisigKThe upstream sequence of the transcription regulating factor gene has the nucleotide sequence shown in SEQ ID NO. 2.
Transcription regulatory factor gene of bacillus thuringiensis subspecies israelisigKThe nucleotide sequence of the transcription regulating factor gene is shown as SEQ ID NO. 3.
Transcription regulatory factor gene of bacillus thuringiensis subspecies israelisigKAnd the downstream sequence of the transcription regulating factor gene is shown as SEQ ID NO. 4.
Bacillus thuringiensis israeli subspecies peptidoglycan hydrolase genehyd1The upstream sequence, the nucleotide sequence of the upstream of the peptidoglycan hydrolase gene is shown as SEQ ID NO. 5.
Bacillus thuringiensis israeli subspecies peptidoglycan hydrolase baseBecause ofhyd1And the downstream sequence of the peptidoglycan hydrolase gene is shown in SEQ ID NO. 6.
Drawings
FIG. 1 is a graph showing the hydrolytic activity of recombinant Hyd1 of the invention against Bti cell walls; in fig. 1, BSA is bovine serum albumin;
FIG. 2a shows the present inventionhyd1AndsigKcell morphology of bacillus thuringiensis subspecies israeli cultured in SSM medium for 5d after double mutation;
FIG. 2b ishyd1AndsigKcell morphology of bacillus thuringiensis subspecies israeli after double mutation in SSM medium for 6 d;
FIG. 2c ishyd1AndsigKcell morphology of Bacillus thuringiensis subspecies israeli after double mutation in SSM medium for 7 d.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The reagents and media formulations designed in the examples:
(1) LB liquid medium:
10g of tryptone, 5g of yeast extract and 5g of NaCl are added into 900ml of double distilled water, stirred and mixed uniformly, the double distilled water is used for constant volume to 1L, and the mixture is placed in an autoclave, sterilized for 20min at 121 ℃, cooled and then placed at 4 ℃ for storage.
(2) LB solid medium:
10g of tryptone, 5g of yeast extract, 5g of NaCl and 15g of agar are added into 900ml of double distilled water, stirred and mixed uniformly, the double distilled water is used for constant volume to 1L, the mixture is placed into an autoclave, the temperature is 121 ℃, sterilization is carried out for 20min, antibiotics are added to the mixture after cooling to 50 ℃, and the mixture is placed into 4 ℃ for preservation after cooling.
(3)TK Buffer：
0.05 M Tris-HCl，0.05M KCl，pH=7。
(4) SSM medium:
nutrient broth 8 g, 10% KCl 1 mL, 12% MgSO4 1 mL, 1 mol/L NaOH 1 mL, and double distilled water to 1L were placed in an autoclave and sterilized at 121℃for 20min.
(5) Fermentation medium:
2% starch, 4.5% bean cake powder, 2.0% corn steep liquor, 0.1% MgSO4,0.1% CaCO3,0.1% KH2PO4, and sterilizing at 121deg.C for 20min.
(6) Bt59 adopts the existing strain, see publication No. CN 109628362A, paragraph 0020-0022 of Chinese patent specification of publication No. 2019, 4, 16.
Example 1
Cloning of peptidoglycan hydrolase Using the Bt59 genome as template, using the upstream primer CAGCAAATGGGTCGCGGATCCGAATTCATGGATATGGTTAAAGTTTGG and the downstream primer GGTGCTCGAGTGCGGCCGCAAGCTTTTAATTTAATCTTTTTTTCCAAGhyd1The gene fragment SEQ ID NO.1, and a 6 XHis tag (CATCATCATCATCATCAC) was introduced at the C-terminus for recombinant protein purification. The fragment of interest was ligated with linearized pET21b plasmid (Novagen) according to the instructions of the ClonExpressII One Step Cloning Kit kit from Vazyme, and the recombinant plasmid was sequenced and transformed into VazymeEscherichia coliBL21 (DE 3) strain. The isopropyl-beta-D-thiogalactoside IPTG with a final concentration of 0.5. 0.5 mM was used to culture 12. 12 h at 150 rpm and 18℃to induce the expression of the target protein. After the induction, the bacterial cells were collected, the bacterial cells were broken by using a ultrasonic breaker Biosafer 900 from Sifei, and the recombinant proteins were detected by SDS-PAGE and purified by an AKTA Pure rapid protein purification system from GE.
Bt59 cells were cultured in LB medium to logarithmic phase, collected by centrifugation, and resuspended in TK buffer, and the cells were disrupted by using a Saifer ultrasonic disrupter Biosafer 900. After high-speed centrifugation of the disrupted cell fluid, the pellet was collected, resuspended in 4% (W/V) sodium dodecyl sulfate, and boiled for 5-10 min, washed twice with 1M NaCl, washed three times with deionized water, the residual SDS was removed, and the prepared cell wall was stored at-80 ℃ for later use. Suspending the prepared Bt59 cell wall with TK buffer to make the absorbance OD of the suspension 540 About 0.3, adding purified Hyd1 into suspension, incubating at 37deg.C, and collecting the mixture for absorbance measurement for analysis of recombinant cell wall hydrolase activity, and the results are shown in FIG. 1, which shows that recombinant hydrolase Hyd1 has activity in hydrolyzing Bt59 cell walls.
Example 2
Bt59 genome is used as template and gene is regulated according to transcriptionsigKSEQ ID NO.2-4, overlapping PCR primer (ATCGATGCATGCCATGGTACCCGGGATTGTACAGAGTCATCGGTAATTTC/GCGTCTGCAGAAGCTTCTAGAATTCCAGAACCAGAAAGCGGACTATG) is designed, and the target gene is obtained through overlapping PCRsigKThe deleted gene fragment was ligated into the linearized pMAD plasmid according to the instructions of the Vazyme company ClonExpress II One Step Cloning Kit kit. After demethylation of the recombinant plasmid, the recombinant plasmid was transformed into Bt59 cells using a GenePulser Xcell electroporator from Bio-Rad, voltage 2.4. 2.4 kV, resistance 1000. OMEGA. After identification, single colony which is successfully transformed is selected, and is respectively subjected to shaking flask culture at 30 ℃ and 39 ℃ and then diluted by a certain multiple and coated on an LB plate, the single colony is selected to respectively correspond to prick spots on the plate containing erythromycin of the LB plate, and the single colony which grows on the LB but cannot grow on the plate containing erythromycin is selected for PCR identification, thus obtainingsigKDeletion mutant Bt59 (. DELTA.sigK)。
Example 3
Bt59 genome is used as a template and is used for preparing the gene of the hydrolasehyd1The sequence SEQ ID NO.1, 5-6, design overlapping PCR primer (CAGATCTATCGATGCATGCCATGGTACCCGAATCCATCTGTCCCGCTTAAACTG/CATGGGAATATTCGGTGTGGGAAATTACATATCCATCGCTCCTCTTCTCTTAG), and obtain the target gene by overlapping PCRhyd1The deleted gene fragment was ligated into the linearized pMAD plasmid according to the instructions of the Vazyme company ClonExpress II One Step Cloning Kit kit. After demethylation of the recombinant plasmid, the recombinant plasmid was transformed into Bt59 and Bt59 (. DELTA.) respectively using a GenePulser Xcell electroporator from Bio-Rad company, voltage 2.4 kV, resistance 1000. OMEGAsigK) In cells. After identification, single colony which is successfully transformed is selected, and is respectively subjected to shaking flask culture at 30 ℃ and 39 ℃ and then diluted by a certain multiple and coated on an LB plate, the single colony is selected to respectively correspond to prick spots on the plate containing erythromycin of the LB plate, and the single colony which grows on the LB but cannot grow on the plate containing erythromycin is selected for PCR identification, thus obtaininghyd1Deletion mutant Bt59 (. DELTA.hyd1) Andhyd1andsigKdouble mutant strain Bt59 (. DELTA.hyd1-sigK). Bt59 (. DELTA.hyd1-sigK) The strain was cultured in SSM medium for 5d, 6d, and 7d, and the cell morphology was observed in an optical microscope, and the results are shown in fig. 2a, 2b, and 2c. As can be seen from FIG. 2, the mutant Bt59 (. DELTA.hyd1-sigK) After 7d incubation in SSM medium, the blast cells did not lyse.
Example 4
Bt59 and Bt59 (Δhyd1-sigK) Culturing 43 h in fermentation medium, at which time Bt59 parent cells have been completely lysed, bt59 (deltahyd1-sigK) The blast cells did not lyse. The strain fermentation broth is taken to be diluted to 0.0781,0.156,0.313,0.625,1.250,2.500,5.000 mu L/L in a gradient way and is respectively used for measuring the insecticidal activity of Bt59 and Bt59 (delta hyd 1-sigK) on Aedes albopictus and culex pallidum and calculating LC by using PoloPlus software 50 Values.
Example 5
Bt59 and Bt59 (Δhyd1-sigK) Culturing 43 h in fermentation medium, collecting Bt59 and Bt59 (delta)hyd1- sigK) The fermentation broth was subjected to ultraviolet irradiation for 30 min at 365 and nm, and then was diluted to 0.156,0.313,0.625,1.250,2.500,5.000 and 10.000. Mu.L/L, respectively, for determination of Bt59 and Bt59 (. DELTA.) after ultraviolet irradiationhyd1-sigK) Insecticidal Activity against Aedes albopictus and culex pallidum and calculation of LC using PoloPlus software 50 Values.
Table 1 shows the results of toxicity measurements of Bacillus thuringiensis subspecies israeli on Aedes albopictus
Table 2 shows the results of toxicity measurements of the Bacillus thuringiensis subspecies israeli on culex light-colored mosquitoes
As can be seen from Table 1, bt59 and Bt59 (Δhyd1-sigK) LC for Aedes albopictus 50 2.066 ppm and 2.075 ppm, respectively, with no significant difference; bt59 and Bt59 (ΔA) in example 5hyd1-sigK) LC for Aedes albopictus 50 3.156 ppm and 2.333 ppm, respectively, with significant differences (P<0.05). After ultraviolet irradiation, bt59 (. DELTA.hyd1-sigK)The insecticidal activity to Aedes albopictus is 26.08% higher than Bt 59. As can be seen from Table 2, bt59 and Bt59 (Δhyd1-sigK) LC for culex light color 50 0.314 ppm and 0.364 ppm, respectively, with no significant difference; bt59 and Bt59 (ΔA) in example 5hyd1-sigK) LC for culex light color 50 0.586 ppm and 0.373 ppm, respectively, with significant differences (P<0.05). After ultraviolet irradiation, bt59 (. DELTA.hyd1-sigK) The insecticidal activity to culex light color is 36.35% higher than Bt 59.
In conclusion, the invention is realized by knocking out the peptidoglycan hydrolase genehyd1And transcription regulatory factorsigKThe double mutant strain Bt59 (deltahyd1-sigK) The parent cell is not cracked, does not produce spores, does not contain resistance genes, is safe to the environment, has higher insecticidal activity and ultraviolet radiation resistance, and has higher application value. The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Bacillus thuringiensis israeli subspecies peptidoglycan hydrolase genehyd1The nucleotide sequence of (2) is SEQ ID NO.1, as follows:
atggatatggttaaagtttggttagatgctggacacggtgcacatgattcaggtgccgttgggcattccttattagaaaagaatattgtgttagaacttgctttatctacatatgaatatttaaatgaacattatgacgatatcatagtaggtatgactagatttaatgatacatttaaaactcttcaagaaagatgcaatatggccaatcggttcggcgcagatatatttgtctcattccattgtaatagtggtgctacaaacggtgttcccggtaatggatttgaaacatttcgttttcctggtactacaggtgatacactacgtttacagcaagtgacacaccattctatcttttctttttatgctaaacatggacttcgtaatcgaggaatgaaggaagcttcatttgccgttttaagaggaacaaacatgccggctgttctaacagaaaacttattcatgaatcatactgaaatattaaaatttaataatgccacttttctatatcatgttgctgaagcacatgcacatggtatcgctgaatactttggattaaagccaaaagtagataaaaataaattggtacccatttatcagattcatctcggagattttaacagtatcgaatggactgctgatactttgcgtaaagtaaaagagatttttccaggctatggagtttggacaaaatgccttgaaggtaatgcacatcgcatcatactgggagattttagcggtgaatcatgggctgacgatactttatcacaactacaaggaaattttccagactatggggcttggaaaaaaagattaaattaa
transcription regulatory factor gene of bacillus thuringiensis subspecies israelisigKThe upstream nucleotide sequence is SEQ ID NO.2 as follows
attgtacagagtcatcggtaatttcattctcgttagtttgaagttcaactaagaaagatgtactacgggaagaatctgttttgcttgtaatatgtaattcagtaggaatcatgggtatttcataaaactcattttttattatagtattgtagtcttctgaaaatttacgaacttcttttggaaaagagtcttcttcttgtgtatttaaaaaggttttatgttgctttggaaatttttattaaactttaatgtttctttaaagtaataattggatccagtcaaaaataaacaactacaaattaataaaaaattcgatacggattttgaaagtgaaagaaaaatagtatacatgtaataattccaattacatagagaataaagataagtagttctagttttgagtgaaaggaaaaaccatagttatgaaaaaatttcattccagttataatttgagctccaaaaaataaagaaattccaaattgaaaaaataataaatcaggccctcttttaataacaaaagtaatgaacgaaatgaaaagtaaaaatcccctaaataaattgttacatttaataatcgatctaatgtaatcatgaatagtgtcccctttaatagcatattcatatataaataacaatttcttttaattataatataacaataaaatattttaaatgttagaagtgttgagttttataaataaactgtacagacacaaacaacctcacgcctacatacacataataaagaccactattttggcggaggtgaaaggt
Transcription regulatory factor gene of bacillus thuringiensis subspecies israelisigKThe nucleotide sequence of (2) is SEQ ID NO. 3, as follows:
ttgagtctattcgccgcaattggatatatggttcgagaagtgtttgtctttgtttcttatgtaaagaataatgcgttcccgcagccgttatcatcagatgatgagagaaagtacttagagttaatggagcaaggtgatgctcaagcgagaaatcttttgattgaacataatttacggcttgtagctcatatcgttaaaaaatttgagaacacaggagaagatgcagaagatttaatttcaattggtacgatcgggctcattaaagcgatcgagagctattctgcaggaaagggaacaaagcttgcgacgtatgcagcacgctgtattgaaaatgaaattttgatgcatttacgtgtactaaagaaaacgaaaaaagatgtttcacttcatgatccgattgggcaagataaagagggcaatgaaatatcgcttattgatatattaaaatcagagtctgaagatgtaattgatatgattcagcttagtatggagttagaaaaaattaaagagtatatcgatattttagacgaaagagaaaaagaagtgattgtgaaacgttttggacttgggctagataaggagaaaacacagcgggaaatcgctaaggcgcttggtatttctagaagctacgtatcaaggattgaaaagcgtgctttaatgaagatgttccacgagtttgtaagggcagagaaagagaaaaaagcgaaagagtaa
transcription regulation of Bacillus thuringiensis subspecies israeliFactor-controlling genesigKThe nucleotide sequence downstream is SEQ ID NO. 4 as follows:
Agtgtattagaagcagccatattttaaaaggggctgcttttcttattgtaaataagaaaagaaaacgcgttatgctaagaagaaatacatgttcactgttgaaaatgacggattctatctgtaatattagaaggtgctaaataatttaagaataataccatacttagttttctgtatgtataataaggctgctttatatgatatgtgggaataagaaccaattttacatatgatatagaaagaaaagaagggaaaactaaagttatagactgattagggggtttcgcaatttgcgacgtacattatatcgacttatgatagaacttacaaatggtcgctttacttcttatatattacgtaaatttgcacaatctcgtttgagctctatcattattccatcgtatgcgaaagtttttcaaattaatcaagatgagatggaaaagggcttgaaggaatatagaacattgcatgaattatttacgcgtaagctaaaagaaggaaagcgtagtattgatacagatgcatcgagtatcgttagtcctgttgatggtgtttttgcagatcatggtcctattgaggatacaaaaacatttgatattaaaggaaagcgctattcaattgtggatatgctaggtaatgaagaacgtgcaacgcgatatgcaggcggtacatatatggtaatttatttgagtccaagtcactatcatcgcattcatagtccgctttctggttctg
bacillus thuringiensis israeli subspecies peptidoglycan hydrolase genehyd1The upstream nucleotide sequence is SEQ ID NO. 5 as follows:
gaatccatctgtcccgcttaaactgaattacagaatttattaacaattaaatgacaaggatttcgcatttttgaatttgtttaatgtaaaaaattttacaacagaactatttatcttaataatcggatgactttctacaactacataactttaatattgatttagtttcttctagtttgagctgtaacagcatttctttttttattactgttttgagagtatctaccgtgctctcgttagttgccaacaattgatcaagcgatacttttccctttttaaattcagtcacaactgcttgaaccttttcaccttctgcattaattacgtgggctagtcctaattcttctaatccaattgatgctaagataatattaattgcctgctcacgagttatatccaatccttctggaatatttggaattcccatttatttcacccctcatttatattttgggaccatttaaatttgacaagtagtaaacattatattcactactatgcccatatatgcaaaaatagttctttcagaagatacataagggacaaaagagctttagaacatattgatttctttgatcgtatgtgaaatataacgaatagcttaacatcctaaaaatttaagacaatatcgcttaatgcgtgaataactctaataattcaagattgactttaacatcctatccacttaaattactcctatatttttctattactttaacaaagcctttatggccaaccgctaccaatttaatatctatttttgaattattatcttataaaactctctatacatgattctcttcattaaactcttttacatgttagctaaatttcaagtactttattatatgttactacatataataaagtactaagagaagaggagcg
bacillus thuringiensis israeli subspecies peptidoglycan hydrolase genehyd1The nucleotide sequence of the downstream is SEQ ID NO6, the following steps:
tttcccacaccgaatattcccatgaaattaagaattaaaatttaaaaattctccttttactttaaaatttatattatagaacttttaaaaagatataaggtctggctacaggcagctagattatattcagctcttaataaattgccgtaatttgttatgtgcaaaaaacaggagttttaaatagcaaactcctgtttttttacatgcaataaattaaacttattttttatactaagaaatagaaggtggcgaagatgattgtattagcgatttagcttcttctaacttaaattgtaataacatttccttttttattaccgttttaagagtttgggttacactttcatttgtagctaataattggtcaaaggtaactgtttctttttcaaatccagccacaactgcttgcaccttttcgccctctgcattaattacgtgagctagccctaattcttctaatccaatagaagctaaaataatattaattgcttggtctctagtaatatctaaaccttctggaattgttggcatccccattaatatcattccttttcactttatttataatcattgtatcttgaaatattatatgtaacagctattccaaaggtgcagggggaataaatttaatccaaaatatttttatgaaaattcaattcagtacatgaaattacaagactaccagatcgtatattttaaaggcaaacatatccaacaagttattaacgttatatcatctggataatattgttattttattttttaattactttgatgtatcacaactgaaacaaactagttgttccatttcacaaaaattaatattatcggaatttataaacttttctgaaaaatagtatggaaaatacacgattctgataaaaaaacagtctcactattactatcttttctaaatagttaaaagagaatatactttatgcccatataacttattgtacagttcattatgtaaatgttccctgcgtatgtg
cloninghyd1Gene primer SEQ ID NO.7-8
Upstream primer CAGCAAATGGGTCGCGGATCCGAATTCATGGATATGGTTAAAGTTTGG
A downstream primer GGTGCTCGAGTGCGGCCGCAAGCTTTTAATTTAATCTTTTTTTCCAAG.
The overlapping PCR primer SEQ ID NO.9-10 is:
an upstream primer ATCGATGCATGCCATGGTACCCGGGATTGTACAGAGTCATCGGTAATTTC;
a downstream primer GCGTCTGCAGAAGCTTCTAGAATTCCAGAACCAGAAAGCGGACTATG.
The overlapping PCR primer two SEQ ID NOS.11-12 are:
an upstream primer CAGATCTATCGATGCATGCCATGGTACCCGAATCCATCTGTCCCGCTTAAACTG;
a downstream primer:
CATGGGAATATTCGGTGTGGGAAATTACATATCCATCGCTCCTCTTCTCTTAG。
Claims (6)
1. a double mutant engineering bacterium of the israel subspecies of Bacillus thuringiensis, which is characterized in that the engineeringBacterial strain Bt59 peptidoglycan hydrolase genehyd1And transcription regulatory factor genesigKDouble mutation engineering bacteria Bt59 (delta)hyd1-sigK) Deposit number: CGMCC No.27191.
2. The method for constructing double mutant engineering bacteria of the subspecies israeli of the Bacillus thuringiensis as set forth in claim 1, wherein the cell wall hydrolase genes are obtained by using the Bt59 genome as a template and utilizing the overlap PCR techniquehyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is then subjected to homologous recombination technology to obtain the cell wall hydrolase genehyd1Deleted gene fragment and transcription regulatory factor genesigKThe deleted gene fragment is constructed into a gene operation vector pMAD, is transformed into Bti competent cells by electric shock, and is screened at high temperature to obtain double mutation engineering bacteria Bt59 (delta)hyd1-sigK)。
3. The construction method according to claim 2, wherein the transcription regulatory factor gene is obtained by using an overlap PCR techniquesigKA deleted gene fragment comprising:
the Bt59 genome is used as a template, and overlapped PCR is performed by using an overlapped primer SEQ ID NO.9-10 to obtain a transcription regulatory factor genesigKDeleted gene fragment, transcription regulatory factor genesigKThe deleted gene fragment is connected to a linearized pMAD plasmid to obtain a recombinant plasmid; after demethylation of the recombinant plasmid, the recombinant plasmid is transformed into Bt59 cells by electric shock to obtainsigKDeletion mutant Bt59 (. DELTA.sigK)；
The overlapping PCR primer SEQ ID NO.9-10 is as follows:
an upstream primer ATCGATGCATGCCATGGTACCCGGGATTGTACAGAGTCATCGGTAATTTC;
a downstream primer GCGTCTGCAGAAGCTTCTAGAATTCCAGAACCAGAAAGCGGACTATG.
4. The method according to claim 3, wherein the cell wall hydrolase gene is produced by an overlap PCR techniquehyd1A deleted gene fragment comprising:
the Bt59 genome is used as a template, and overlapped PCR is performed by using overlapped primers SEQ ID NO.11-12 to obtain the cell wall hydrolase genehyd1Deleted gene fragment, cell wall hydrolase genehyd1The deleted gene fragment is connected to a linearized pMAD plasmid to obtain a recombinant plasmid; after demethylation of the recombinant plasmid, transformation to Bt59 and Bt59 (delta) was performed by electric shock, respectivelysigK) In cells, obtainhyd1Deletion mutant Bt59 (. DELTA.hyd1) And (d) sumhyd1AndsigKdouble mutant strain Bt59 (. DELTA.hyd1-sigK)；
The overlapping PCR primer two SEQ ID NO.10-11 is:
an upstream primer CAGATCTATCGATGCATGCCATGGTACCCGAATCCATCTGTCCCGCTTAAACTG;
a downstream primer:
CATGGGAATATTCGGTGTGGGAAATTACATATCCATCGCTCCTCTTCTCTTAG。
5. use of a double mutant engineering bacterium of the species israeli according to any one of claims 1-4 in the biological control of disease-mediated mosquitoes.
6. The use according to claim 5, wherein the vector mosquitoes include culex pallidum and aedes albopictus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310759784.5A CN116836902A (en) | 2023-06-26 | 2023-06-26 | Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310759784.5A CN116836902A (en) | 2023-06-26 | 2023-06-26 | Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116836902A true CN116836902A (en) | 2023-10-03 |
Family
ID=88164555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310759784.5A Pending CN116836902A (en) | 2023-06-26 | 2023-06-26 | Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116836902A (en) |
-
2023
- 2023-06-26 CN CN202310759784.5A patent/CN116836902A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TULLY et al. | Spiroplasma mirum, a new species from the rabbit tick (Haemaphysalis leporispalustris) | |
| JPS6251981A (en) | Cloning and development of bacillus sringencis toxin gene noxious to beetle | |
| NO153305B (en) | PROCEDURE FOR AA TRANSFORMER THE SPOROGEN BACILLUS SUBTILIS RUB 830 TO THE ASPOROGEN BACILLUS SUBTILIS RUB 331 APPLICABLE AS A HOST IN A VERT-VECTOR SYSTEM | |
| US20220112455A1 (en) | Auxotrophic strains of staphylococcus bacterium | |
| WO2006053473A1 (en) | A bacillus thuringiensis and genes with high larvicidal activity to coleopterans | |
| JPH05500151A (en) | New strain of Bacillus thuringiensis | |
| US5516693A (en) | Hybrid gene incorporating a DNA fragment containing a gene coding for an insecticidal protein, plasmids, transformed cyanobacteria expressing such protein and method for use as a biocontrol agent | |
| CN109797124B (en) | Bacillus thuringiensis with good insecticidal performance and safety to environment and application thereof | |
| Fulano et al. | Functional divergence of flagellar type III secretion system: A case study in a non-flagellated, predatory bacterium | |
| JPS62503074A (en) | Methods for stabilizing unstable genetic replicons | |
| KR101621155B1 (en) | Method for inhibiting or preventing formation of biofilm using Omp1X gene | |
| CN116836902A (en) | Bacillus thuringiensis subspecies israeli double-mutation engineering bacterium and construction method and application thereof | |
| Grishechkina et al. | Search for natural isolates of Bacillus thuringiensis for development of ecologically friendly biologicals | |
| CN114891806B (en) | Citrobacter welchii yqjH gene knockout mutant strain and application thereof | |
| CN116396915A (en) | Bacillus subtilis without specific resistance gene and application thereof | |
| CN112522132B (en) | Bacillus SJ110, insecticidal protein, vip3-like insecticidal gene and application | |
| CN108359673A (en) | A kind of Bt cry11 genes, coding albumen and its application efficiently killing edible mushroom eye fungus gnat | |
| CN109825531B (en) | Method for removing pXO2 plasmid in Bacillus anthracis | |
| CN109825530B (en) | Method for removing pXO1 plasmid in Bacillus anthracis | |
| CN108410884A (en) | The application of bacteriophage Vp670 perforin genes holA | |
| US20120015404A1 (en) | Gene cluster for thuringiensin synthesis | |
| Yari et al. | Effects of Protoplast Fusion on δ-endotoxin Production in Bacillus thuringiensis Spp.(H14) | |
| CN101812424A (en) | Thymine auxotrophic bacillus anthracis and application thereof | |
| Borovsky et al. | Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes | |
| WO2022095295A1 (en) | New bacillus strain hsy204, and insecticidal genes and use thereof |
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 |