CN106011013A - Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein - Google Patents
Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein Download PDFInfo
- Publication number
- CN106011013A CN106011013A CN201610472022.7A CN201610472022A CN106011013A CN 106011013 A CN106011013 A CN 106011013A CN 201610472022 A CN201610472022 A CN 201610472022A CN 106011013 A CN106011013 A CN 106011013A
- Authority
- CN
- China
- Prior art keywords
- cry8ax
- gene
- bacillus thuringiensis
- killing
- insecticidal
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 70
- 230000000749 insecticidal effect Effects 0.000 title claims abstract description 20
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 18
- 241000193388 Bacillus thuringiensis Species 0.000 title claims abstract description 15
- 229940097012 bacillus thuringiensis Drugs 0.000 title claims abstract description 15
- 230000014509 gene expression Effects 0.000 title claims abstract description 11
- 241000607479 Yersinia pestis Species 0.000 claims abstract description 25
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 239000002917 insecticide Substances 0.000 claims description 11
- 235000018102 proteins Nutrition 0.000 claims description 11
- 230000000813 microbial effect Effects 0.000 claims description 7
- 241000254173 Coleoptera Species 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 4
- 230000001988 toxicity Effects 0.000 abstract description 4
- 206010059866 Drug resistance Diseases 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 231100000614 poison Toxicity 0.000 abstract description 2
- 239000002574 poison Substances 0.000 abstract description 2
- 230000001934 delay Effects 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- 241000238631 Hexapoda Species 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 13
- 244000144987 brood Species 0.000 description 11
- 244000037671 genetically modified crops Species 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000009261 transgenic effect Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 230000001018 virulence Effects 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 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 5
- 238000002156 mixing Methods 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 101710151559 Crystal protein Proteins 0.000 description 3
- 241001520019 Holotrichia oblita Species 0.000 description 3
- 241000346653 Holotrichia parallela Species 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 235000004252 protein component Nutrition 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 description 2
- 101100497222 Bacillus thuringiensis cry1Af gene Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 101150086784 cry gene Proteins 0.000 description 2
- 101150041868 cry1Aa gene Proteins 0.000 description 2
- 101150065438 cry1Ab gene Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101100497219 Bacillus thuringiensis subsp. kurstaki cry1Ac gene Proteins 0.000 description 1
- 241000825627 Colaphellus bowringi Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000654838 Exosporium Species 0.000 description 1
- 108700001097 Insect Genes Proteins 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 241000193157 Paraclostridium bifermentans Species 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 230000002016 colloidosmotic effect Effects 0.000 description 1
- 229920001940 conductive polymer Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 101150092571 cry1Ac gene Proteins 0.000 description 1
- 101150102059 cry3Aa gene Proteins 0.000 description 1
- 101150060198 cyt gene Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000019371 dormancy process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000005357 flat glass Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 230000000361 pesticidal effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000000352 supercritical drying Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
-
- 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
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- 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
-
- 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)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Pest Control & Pesticides (AREA)
- Medicinal Chemistry (AREA)
- Dentistry (AREA)
- Virology (AREA)
- Environmental Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to bacillus thuringiensis 3-1-a, an insecticidal gene cry8Ax, an expression protein and applications of the bacillus thuringiensis 3-1-a, the insecticidal gene cry8Ax and the expression protein and belongs to the technical field of biological control. The preservation number of the bacillus thuringiensis 3-1-a is CGMCC No.12440. The insecticidal protein separated from the strain 3-1-a has an amino acid sequence represented as SEQ ID NO:2, and preferably, a nucleotide sequence of the gene for encoding the insecticidal protein is represented as SEQ ID NO:1. The gene has certain poison activity on agricultural pests so as to be applied to transformation of microorganisms and plants, is enabled to show toxicity on related pests, and overcomes and delays drug resistance of the pests to engineering bacteria and genetically modified plants.
Description
Technical field
The present invention relates to technical field of biological control, particularly the present invention relates to that coleoptera agricultural pests are had high virulence
Bt killing gene and by the protein of this coded by said gene.
Background technology
Thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of widely distributed gram sun
Property antibacterial, be a kind of to insect virulence strong and entomopathogen avirulent to natural enemy, to higher mammal and people's avirulence.
It is to study the most deep, the most widely used microbial insecticide at present, active to 16 mesh 3000 various pests.Bt
Insecticidal crystal protein (Insecticidal CrystalProteins, ICPs), also referred to as δ-endogenous toxin can be formed in the sporulation phase
Element (delta-endotoxin), its shape, structure and size all with its virulence close relation [Schnepf.E,
Crickmore.N,Van Rie.J.,Lereclus.D,Baum.J,Feitelson.J,Zeigler.D.R.,
Dean.D.H.Bacillus thuringiensis and its pesticidal crystal
proteins.Microbiol.Mol.Biol.Rev,1998,62(3):775-806.].Clone from Schnepf in 1981 etc.
First ICPs gene of Bt, and in 1985 have delivered its DNA base sequence and the aminoacid sequence of encoding proteins thereof,
(in April, 2014) totally 776, wherein cry gene 738, cry pattern gene 289 at present；Cyt gene 38, cyt pattern
Gene 11.Now, use and spray chemical pesticide control means and no doubt can alleviate insect crops are caused harm, but chemistry agriculture
Medicine causes environmental pollution, for a long time, sprays chemical insecticide in a large number, not only can strengthen the Drug resistance of insect, make beneficial insect and
Its ecosystem wrecks, and serious environment pollution, improve production cost, destroy ecological balance.Thuringiensis
Insecticidal crystal protein is widely used in pest control because of its good disinsection effect, the advantage such as safe efficient.Su Yun gold brood cell
Bacillus is in addition to directly as biological pesticide, and the example transgenic anti-insect plants that beats the world for 1996 is approved application in the U.S.,
The gene that it uses is from Bt cry1Ac.In ensuing several years, turn the pest-resistant Semen Maydis of cry1Ab gene, turn cry3Aa base
The pest-resistant Rhizoma Solani tuber osis of cause etc. are at a distance of coming out.In China, started formal popularization from 1998 and contain the pest-resistant of cry1Ac/cry1Ab gene
Since cotton, it is widely planted.In genetically modified crops business-like first 12 years (1996-2007), owing to obtaining
Continual and steady income, peasant planting genetically modified crops amount increases year by year.Since nineteen ninety-six, genetically modified crops within 2015, it have been
The 20th year of successful commercialization plantation.According to statistics, the cultivated area of global genetically modified crops in 2015 about 1.797 hundred million hectares, with
2014 1.815 hundred million hectares compare minimizing 1%.In these 20 years, it is public that plantation genetically modified crops area about 2,000,000,000 has been accumulated in the whole world
Hectare, being equivalent to the twice of the total land surface of the U.S., conservative estimation peasant benefits to exceed about 150,000,000,000 dollars.By the end of 2015 12
Month, genetically modified crops are successfully planted by existing 28 countries, and wherein 20 is developing country, and the kind of genetically modified crops, model
Enclose and cultivated area also increases at the development along with transgenic technology and continuation is kept growth trend.China 750
The smallholder of ten thousand scarcity of resources has planted the Bt Cotton Gossypii of 4,200,000 hectares, and employing rate is 90%, and average each household peasant planting 0.5 is public
Bt Cotton Gossypii just.Genetically modified crops commercialization all brings economy and Environmental Effect to the peasant of industrialized country and developing country
Benefit.Thuringiensis and Gene mining thereof have become important topic in Agricultural Sustainable Development.
Owing to the anti insect gene kind of the insect-resistant transgenic crops of current commercialization is more single, such spread kind
Plant and there is the risk that insect refuge reduces and pest resistance to insecticide rises.It is thus desirable to constantly separate high virulence or new base
Because combining the risk avoiding pest resistance to insecticide to rise.Therefore, screening and separating clones new, the Bt killing gene of high virulence, can
With abundant killing gene resource, provide new gene source for genetically modified crops and engineered strain, improve Bt transgenic product
Insect resistant effect, and the insect resistance risk to Bt toxalbumin can be reduced, it is to avoid new ecocatastrophe comes, and has important
Economy, society and ecological benefits.
Summary of the invention
The present invention provides a kind of to coleopteran pest Colcaphellusbowringi, the thuringiensis 3-of colorado potato bug isoreactivity
1-a, and parasite killing new gene gene cry8Ax and its crystal insecticidal proteins, to be applied to microbial and plant, be allowed to table
Reveal the toxicity to relevant insect, and overcome, delay insect that the Drug resistance of engineering bacteria and transgenic plant is produced.
Bacillus thuringiensis bacterial strain 3-1-a, its deposit number is: CGMCC No.12440.
Bacillus thuringiensis bacterial strain 3-1-a is killing coleoptera Colcaphellusbowringi, answering in colorado potato bug agricultural pests
With.
Insecticidal proteins Cry8Ax, its aminoacid sequence is as shown in SEQ ID NO:2.
Killing gene cry8Ax, encoding insecticidal proteins Cry8Ax.
Killing gene cry8Ax, its nucleotide sequence is as shown in SEQ ID NO:1.
A kind of expression vector, is characterized in that containing cry8Ax gene.
Described expression vector is pEB-cry8Ax, and its skeleton carrier is pEB, and its structure is as shown in Figure 4.
A kind of microbial transformant, is characterized in that containing cry8Ax gene.
Killing gene cry8Ax is killing coleoptera agricultural pests Colcaphellusbowringi, the application in colorado potato bug.
Described application is to be transformed in plant by killing gene cry8Ax, makes plant express the resistance to agricultural pests, or
Person is that the albumen expressed by killing gene cry8Ax kills agricultural pests as the effective ingredient of biological insecticides.
The present inventor isolated one thuringiensis strain bacterial strain of bacillus 3-1-soil near Heilongjiang Province's Anda City
A, its deposit number is CGMCC No.12440, this Biological Characteristics of Strain for brood cell can be produced in growth cycle, and
There is the parasporal crystal of poisoning coleoptera agricultural pests Colcaphellusbowringi insecticidal activity effect simultaneously；One is obtained from this bacterial strain
The positive colony of new gene, i.e. pEB-cry8Ax (see Fig. 4), it is carried out sequencing analysis, BLAST is applied also in NCBI website
The biosoftwares such as application DNAMAN are analyzed, and analysis result proves that the gene cloned is cry8Ax gene, this gene code
Frame is to be 2217bp by size, encodes 738 amino acid residues, is 80.2% with the amino acid similarity degree of Cry8Ab, so should
Gene should belong to the new gene of the tertiary gradient.
Cry8Ax gene can be shown relevant coleoptera evil by the conventional method microbial of biotechnology, plant
Insect poison.
Said gene is converted bacterial strain, expresses the albumen obtained and can make biological pesticide for killing coleopteran pest.
Meanwhile, plant can be proceeded to and build insect-resistant transgenic plants, for the preventing and treating of insect.
The Bt cry8Ax gene order of separating clone of the present invention and gene expression product thereof can ape leaves big to agricultural pests
First has certain poisoning power, and cry8Ax can expand coleopteran pest Colcaphellusbowringi, the insecticidal spectrum of colorado potato bug.By application
In microbial and plant, make them show the toxicity to relevant insect, can overcome or delay insecticide to engineering bacteria and to turn
The drug-fast generation of gene plant.
Bacillus thuringiensis bacterial strain 3-1-a, preservation information:
Bacterium classification is named: thuringiensis (Bacillus thuringiensis)
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on May 13rd, 2016
Deposit number: CGMCC No.12440
Accompanying drawing explanation
The form of Fig. 1 optical microphotograph Microscopic observation bacterial strain 3-1-a thalline,
Fig. 2 cry8Ax full length gene PCR result,
Fig. 3 cry8Ax gene at the SDS-PAGE of expression in escherichia coli albumen,
Wherein: 1.Bt 3-1-a protein component M: high-molecular-weight protein Marker；2:Cry8Ax protein component, 3:
Cry8Ea protein component 4:pEB empty carrier component
The structure chart of Fig. 4 recombinant vector pEB-cry8Ax,
Fig. 5 sequence homology analysis.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail
Embodiment 1, isolated Bacillus thuringiensis bacterial strain 3-1-a
What the laboratory worker of the applicant separated from Heilongjiang Province's Anda City soil obtains a thuringiensis strain bud
Born of the same parents bacillus, brood cell's ecto-entad of thuringiensis is followed successively by Exosporium, Ya Baoyi, cortex, brood cell's inwall, protoplasm
Film and protoplast.The main component of its mediopellis is Peptidoglycan, and the polysaccharide teichoic acid without trophocyte, it remains bud
The dewatering state of born of the same parents and thermostability, on the other hand, during sporulation, can produce a large amount of DPA-Ca sequestration thing so that brood cell
In biomacromolecule form heat-resisting gel, heat treatment 20min at 80 DEG C, thuringiensis brood cell also will not death also
And the brood cell of dormancy processes 15min under the sub-lethal temperature of 75 DEG C, activation effect is best, not only promotees its fast-germination, also may be used
Improve the survival rate (explaining sub-cattle 1990) of brood cell.According to this characteristic, temperature screening (Knowles B H, Ellar D can be implemented
J.Colloid-osmotic lysis is a general feature of the mechanism of action of
Bacillus thuringiensis d-endotoxins with different specificity[J].Biochimica
et biophysica acta,1987,924:509-518.；Dai Lianyun, Wang Xue engage. eight nature reserve area sylvogenic soils of China
The distribution [J] of Su Yun gold brood cell's bar in earth. microorganism journal, 1994,30 (2) 117-121).
1, the separation of 1 bacterial strain
1) soil sample taking subpackage joins in the big centrifuge tube of 50ml, to conical pipe taper.
2) add aquesterilisa at 15ml, put into bead 5～10.
3) with agitator, soil sample is smashed.
4) 80 DEG C are put in water-bath, 20 minutes.
5) take the EP pipe of 1.5ml, each pipe adds 1ml aquesterilisa, then from 50ml pipe, take 10 microlitre bacterium solution join EP
Pipe mixes.
6) from EP pipe, take 100 microlitres to be sprayed onto in 1/2LB culture medium, smoothen.
7) put in 30 DEG C of incubators and cultivate 2～3 days.
8) microscopy is observed.
Crystal Observation
Optical microscope:
By born of the same parents' crystalline substance mixing drop on microscope slide, smear uniformly, dry fixing, carbolfuchsin dye liquor dyeing 3min, clearly
Water rinse, 100x oil mirror carries out microscopy, carbolfuchsin dye liquor preparation method see document (Baroy F, Lecadet M M,
Deleluse A.Cloning and sequencing of three new putative toxin genes from
Clostridium bifermentans[J].Gene,1998,211:293-295).As shown in Figure 1.In 1/2LB culture medium
Forming single bacterium colony after cultivating 48h, observe that bacterial strain 3-1-a thalline is elongated rod shape under optical microscope, brood cell is oval bar-shaped, brilliant
Body is biconial.
Electronic Speculum microexamination:
SEM sample preparation: spore crystalline substance mixing drop on sheet glass, be dried, fix through osmic acid, and after through ethanol gradient take off
Water, critical point drying, ion sputtering metal spraying (2nm is thick), New Bio-TEM H-7500 scanning electron microscopic observation is taken pictures.Such as Figure 1B institute
Show.
Biological characteristis shows, to the primary dcreening operation of Colcaphellusbowringi raw survey result be corrected mortality be 80%.
By this bacterial strain preservation, its deposit number CGMCC No.12440.
The crystal habit that Bt bacterial strain 3-1-a produces, as shown in arrow under optical microscope in Fig. 1, is from left to right followed successively by
Sphaerocrystal and brood cell；
Embodiment 2. obtains new gene
By genome sequencing, find on bacterial strain Bt3-1-a genome high with cry8Ab1 similarity containing one
Cry gene, design total length primer 8AF (5'ATGAATAGTGTATTGAATA3') and
8AR (5'ATAAAGTGGTGAAAGATTAGTTGG3'),
Use rapid clon method that the new cry8Ax gene in this bacterial strain is carried out separating clone.
Use pfuDNA polymerase, carry out PCR amplification by following system.
Ultra-pure water is mended to 50 μ L, and mixing is centrifugal.
Amplification cycles: 94 DEG C of degeneration 1 minute, anneals 1 minute for 54 DEG C, and 72 DEG C extend 1 minute, 25 circulations, last 72 DEG C
Extend 10 minutes.As shown in Figure 2.
2.2 connection scheme
Supply volume to 10 μ L with ultra-pure water, fully mix, 16 DEG C connect 4h or 4 DEG C connect overnight.
The total length primer of design cry8Ax genoid, amplification obtains full-length gene, by it with carrier pEB (open carrier, this
Institute's laboratory has preservation, can be with external disclosure granting) carrier is attached, is transformed in competence JM109, through resistance screening,
PCR identification and analysis, filters out the positive recombiant plasmid containing cry8Ax gene and sees Fig. 4.Fig. 3 PCR qualification result.By purification sheet
Section is connected conversion e. coli jm109 with carrier pEB, obtains positive transformant.Carry out sequencing analysis to inserting segment, obtain sequence
Row SEQ ID NO 1, its aminoacid sequence is shown in SEQ ID NO 2.The purpose bar that size is correct has been obtained by PCR amplification
Band, is purified purpose band and checks order.Sequencing result shows, the cry8Ax gene size in 3-1-a bacterial strain is 2217bp,
Encode 738 amino acid residues, be 80.2% with the amino acid similarity degree of cry8Ab1.See Fig. 5
2.3 convert scheme
2.3.1 escherichia coli convert
1. picking list bacterium colony shakes overnight incubation in 5ml LB；
2. being inoculated in LB fluid medium by 1% inoculum concentration, 37 DEG C, 230rpm cultivates 2-2.5hr, (OD600=0.5-
0.6)；
3.4 DEG C, 4,000rpm are centrifuged 10min；
4. abandon supernatant, add the 0.1M CaCl of pre-cooling250ml suspension cell, is placed in more than 30min on ice；
5.4 DEG C, 4,000rpm are centrifuged 10min, reclaim cell；
6. with the 0.1M CaCl of 2-4ml ice pre-cooling2Re-suspended cell, is distributed in 200 μ l/0.5mL centrifuge tubes, in 4 DEG C of guarantors
Deposit (one week can be preserved).
7. take 200 μ l competent cells and 5 μ L connect product and fully mix, ice bath 30min.
8.42 DEG C of heat shock 1.5min, ice bath 3min.
9. add 800 μ l LB culture medium 37 DEG C and cultivate 45min.
10. take 200 μ l coated plates, add corresponding antibiotic, and IPTG, X-gal, 37 DEG C of cultivation.
Embodiment 3, gene expression and determination of activity
3.1.1 extract plasmid DNA above-mentioned clone, proceed in recipient bacterium Rosetta (DE3), it is thus achieved that expression strain.
After IPTG abduction delivering, carry out SDS-PAGE protein electrophoresis detection.
Abduction delivering process is as follows:
1) activated spawn (37 DEG C, 12hr)；
2) 10% (37 DEG C, 2hr) it is inoculated in LB culture medium；
3) inducer IPTG, 150rpm, 18-22 DEG C of low temperature induction 4-20h are added；
4) centrifugal thalline of collecting, addition 10mM Tris Cl (pH 8.0) suspends；
5) broken thalline (ultrasonic disruption is complete)；
Centrifugal 12,000rpm 10min 4 DEG C；
Collect supernatant and precipitate each 10-15 μ L, respectively electrophoresis detection.
Polyacrylamide gel configuration is as follows.
Loading: loading 10-15 μ l, electrophoresis: 130-150V constant voltage.
Dyeing and decolouring: taking out gel after electrophoresis, after distilled water flushing, put in dyeing liquor, 60rpm vibrates dyeing
About 1hr, decolour in destaining solution about 2hr, and decolouring is to gel background transparent, and it is clear to rinse in clear water to protein band.
By recombiant plasmid pEB-cry8Ax, (see Fig. 4) is transformed in E.coli Rosetta (DE3), IPTG abduction delivering,
SDS-PAGE (12%) gel electrophoresis.Result shows, it is efficient in escherichia coli that cry8Ax gene can be transferred through expression vector pEB
Express and be about 80kDa albumen, and the pEB empty carrier proceeding to Rosetta (DE3) induced through IPTG does not has special purpose bar
Band produces (Fig. 3).
3.2Bt bacterial strain 3-1-A and the insecticidal activity assay of cry8Ax gene coded protein
By Bt bacterial strain 3-1-A and cry8Ax gene expression albumen, it is diluted with water to variable concentrations, the parasite killing to Colcaphellusbowringi
Activity, concrete grammar is as follows, and the testing sample measuring 10-20mL joins in sterilizing culture dish, then chooses fresh green vegetable
Blade, puts in culture dish and immerses testing sample solution, 10s equably.Prior layer overlay preservative film on testing stand,
The blade soaking sample is placed in room temperature on preservative film dry, it is ensured that blade two sides is all dried, and is respectively placed in culture dish.With
Brush pen accesses second instar larvae lightly, connects 30 cephalonts, often processes in triplicate, build lid after having connect insect in each culture dish
Son, and fix with rubber band, prevent larva escape.Being placed in illumination box by culture dish, periodicity of illumination is 14h, 10h, training
Support and case is placed basin regulate humidity, and need every day and check whether blade has phenomenon that is withered or that rot, if having
The phenomenon of steam coagulation.Being 27 DEG C to the condition of culture for examination insecticide, relative humidity is more than 80%, to big ape leaf after 96h
First carries out investigation borer population dead, alive, and calculates mortality rate and LC50., Holotrichia oblita, the insecticidal activity of Holotrichia parallela, specifically side
Method uses feedstuff mixing method to carry out insecticidal bioactivity mensuration as follows.The expressing protein sample of variable concentrations gradient will be prepared also
It is sub-packed in the culture dish of sterilization, respectively with feedstuff stirring and evenly mixing, selects active newly hatched larvae and be connected on feedstuff, each
Process is repeated 3 times, each be repeated as 30 examination worms.Negative control is that 10mmol/L Tris-Cl solution is made.The raising bar of examination worm
Part be relative humidity be 70%-80%, temperature be that the illumination box of 27 DEG C is cultivated, raise that 48h " Invest, Then Investigate " is dead, borer population of living
Amount, calculates mortality rate.
With coleopteron Colcaphellusbowringi (Colaphellus bowringi) newly hatched larvae for test insecticide, utilize induction
The Cry8Ax albumen expressed carries out biological activity determination primary dcreening operation to Colcaphellusbowringi.Biological activity determination result shows: work as Cry8Ax
When the concentration of albumen is 100 μ g/mL, mortality rate is 62.5%, and compared with matched group, corrected mortality is 56.89%.To big ape
Chrysomelid LC50 is 83.329 μ g/mL (54.418-124.408) μ g/mL.The parasite killing of Holotrichia oblita, Holotrichia parallela is lived
When property is than relatively low 200 μ g/mL, respectively mortality rate is 24,36%.
Beneficial effects of the present invention: the Bt cry8Ax gene order of separating clone of the present invention and gene expression product energy thereof
Enough to Lepidoptera generation virulence, there is certain toxic action especially for Colcaphellusbowringi, can expand coleopteran pest insecticide big
The chrysomelid Holotrichia oblita of ape, the insecticidal spectrum of Holotrichia parallela, by being applied to microbial and plant, make them show right
The toxicity of relevant insect, can overcome or delay insecticide generation drug-fast to engineering bacteria and transgenic plant.
Claims (10)
1. Bacillus thuringiensis bacterial strain 3-1-a, its deposit number is: CGMCC No.12440.
2. the application in killing agricultural pests of the Bacillus thuringiensis bacterial strain 3-1-a described in claim 1.
3. insecticidal proteins Cry8Ax, its aminoacid sequence is as shown in SEQ ID NO:2.
4. killing gene cry8Ax, coding insecticidal proteins Cry8Ax described in claim 3.
5. the killing gene cry8Ax described in claim 4, its nucleotide sequence is as shown in SEQ ID NO:1.
6. an expression vector, is characterized in that containing the Cry8Ax gene described in claim 4 or 5.
The most according to claim 6, expression vector, for pEB-cry8Ax, its skeleton carrier is pEB, and its structure is as shown in Figure 4.
8. a microbial transformant, is characterized in that containing the cry8Ax gene described in claim 4 or 5.
9. the application in killing coleoptera agricultural pests of the killing gene cry8Ax described in claim 4 or 5.
Application the most according to claim 9, is that the killing gene cry8Ax described in claim 4 or 5 is transformed into plant
In, make plant express the resistance to agricultural pests, or by the killing gene cry8Ax expression described in claim 4 or 5
Albumen kills agricultural pests as the effective ingredient of biological insecticides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610472022.7A CN106011013A (en) | 2016-06-24 | 2016-06-24 | Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610472022.7A CN106011013A (en) | 2016-06-24 | 2016-06-24 | Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106011013A true CN106011013A (en) | 2016-10-12 |
Family
ID=57084313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610472022.7A Pending CN106011013A (en) | 2016-06-24 | 2016-06-24 | Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106011013A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106928329A (en) * | 2017-03-06 | 2017-07-07 | 中国农业科学院植物保护研究所 | A kind of new insecticidal proteins and its nucleotide sequence |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695449A (en) * | 2016-04-05 | 2016-06-22 | 中国农业科学院植物保护研究所 | New gene screening method |
-
2016
- 2016-06-24 CN CN201610472022.7A patent/CN106011013A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695449A (en) * | 2016-04-05 | 2016-06-22 | 中国农业科学院植物保护研究所 | New gene screening method |
Non-Patent Citations (4)
| Title |
|---|
| CHANGLONG SHU,ET AL: "Characterization of Two Novel cry8 Genes from Bacillus thuringiensis Strain BT185", 《CURRENT MICROBIOLOGY》 * |
| NCBI: "hypothetical protein [Bacillus thuringiensis] NCBI Reference Sequence: WP_061885066.1", 《NCBI》 * |
| 孙钰航等: "辽宁千山地区含cry8 类基因的苏云金芽孢杆菌的分离、克隆与表达", 《江苏农业科学》 * |
| 杜立新: "苏云金芽孢杆菌cry8E基因转录调控", 《中国博士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106928329A (en) * | 2017-03-06 | 2017-07-07 | 中国农业科学院植物保护研究所 | A kind of new insecticidal proteins and its nucleotide sequence |
| CN106928329B (en) * | 2017-03-06 | 2020-09-22 | 中国农业科学院植物保护研究所 | Novel insecticidal protein and nucleotide sequence thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102703338B (en) | Bacillus thuringiensis YBT-008 for killing ditylenchus destructor and application thereof | |
| CN102559554B (en) | Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene | |
| CN103160449A (en) | Bacillus thuringiensis MB-15 strain and preparation method of wettable powder thereof | |
| CN101531980A (en) | Bacillus thuringiensis HS18-1 and application thereof | |
| CN114214220B (en) | Bacillus thuringiensis and application thereof in promoting plant growth | |
| CN101984045B (en) | The Cry8Na1 gene of bacillus thuringiensis, expression protein and application thereof | |
| CN102154171A (en) | Bacillus thuringiensis with high efficiency on mosquito larvae | |
| CN104388349B (en) | Thuringiensis secretes killing gene sip1A, expressing protein and its application | |
| CN104611260B (en) | Thuringiensis LTS290, killing gene cry57Ab, expressing protein and its application | |
| CN110093301B (en) | Bacillus thuringiensis and application thereof in preventing and treating lepidoptera pests | |
| CN104673706B (en) | Thuringiensis FH21, killing gene, expressing protein and its application | |
| CN103627660A (en) | Bacillus thuringiensis (Bt) with high activity for field anti-Bt diamondback moth and application thereof | |
| CN103952418B (en) | Kill novel vip3-like gene and the application thereof of lepidopterous insects | |
| CN103695362B (en) | The activated bacillus thuringiensis HLJ-66 of one strain Bt-resistant plutella xylostella and application thereof | |
| CN101717437B (en) | Bacillus thuringiensis Cry9E gene, protein and applications thereof | |
| CN108486008B (en) | Bacillus thuringiensis YN108 with high toxicity to lepidoptera pests, and culture method and application thereof | |
| CN106011013A (en) | Bacillus thuringiensis 3-1-a, insecticidal gene cry8Ax expression protein and applications of bacillus thuringiensis 3-1-a and insecticidal gene cry8Ax expression protein | |
| CN103333230B (en) | Bacillus thuringiensis Genes cry1Da3 and application thereof | |
| CN103570811B (en) | Bacillus thuringiensis gene cry1Ah3 and application thereof | |
| CN114276945B (en) | Bacillus thuringiensis and application thereof | |
| CN105368733A (en) | New strain of bacillus thuringiensis and application thereof | |
| CN104211790B (en) | A kind of efficient Bt PROTEIN Cs ry21NJ, encoding gene and its application for killing homoptera pest | |
| CN105367636B (en) | A kind of Bt PROTEIN C ry1Dd1, its encoding gene and application | |
| CN103555614B (en) | One strain suppresses bacillus thuringiensis and the application thereof that coconut stem rushes down blood disease, kills lepidoptera pest | |
| CN103396977B (en) | Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161012 |