CN104151410A - Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications - Google Patents
Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications Download PDFInfo
- Publication number
- CN104151410A CN104151410A CN201410392302.8A CN201410392302A CN104151410A CN 104151410 A CN104151410 A CN 104151410A CN 201410392302 A CN201410392302 A CN 201410392302A CN 104151410 A CN104151410 A CN 104151410A
- Authority
- CN
- China
- Prior art keywords
- gene
- vip3afaa
- bacillus thuringiensis
- insecticidal protein
- vegetative 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 81
- 108700031685 Bacillus thuringiensis Vip3A Proteins 0.000 title claims abstract description 15
- 241000238631 Hexapoda Species 0.000 claims abstract description 26
- 230000009261 transgenic effect Effects 0.000 claims abstract description 25
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 23
- 241000196324 Embryophyta Species 0.000 claims abstract description 21
- 244000299507 Gossypium hirsutum Species 0.000 claims abstract description 15
- 229920000742 Cotton Polymers 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 241001124076 Aphididae Species 0.000 claims abstract description 9
- 240000008042 Zea mays Species 0.000 claims abstract description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 5
- 235000005822 corn Nutrition 0.000 claims abstract description 5
- 244000068988 Glycine max Species 0.000 claims abstract description 4
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 4
- 235000018102 proteins Nutrition 0.000 claims description 13
- 241000193388 Bacillus thuringiensis Species 0.000 claims description 7
- 229940097012 bacillus thuringiensis Drugs 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 6
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 abstract 1
- 235000006008 Brassica napus var napus Nutrition 0.000 abstract 1
- 240000000385 Brassica napus var. napus Species 0.000 abstract 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 abstract 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 abstract 1
- 241000255777 Lepidoptera Species 0.000 abstract 1
- 239000002917 insecticide Substances 0.000 abstract 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 11
- 241000209094 Oryza Species 0.000 description 9
- 241000985245 Spodoptera litura Species 0.000 description 9
- 241000589158 Agrobacterium Species 0.000 description 8
- 241000607479 Yersinia pestis Species 0.000 description 7
- 235000007164 Oryza sativa Nutrition 0.000 description 6
- 235000009566 rice Nutrition 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000005562 Glyphosate Substances 0.000 description 5
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 5
- 229940097068 glyphosate Drugs 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 108700010070 Codon Usage Proteins 0.000 description 4
- 108700019146 Transgenes Proteins 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 241001233957 eudicotyledons Species 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 229920002148 Gellan gum Polymers 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000003905 agrochemical Substances 0.000 description 3
- 229920001940 conductive polymer Polymers 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 244000037666 field crops Species 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 238000009616 inductively coupled plasma Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000244202 Caenorhabditis Species 0.000 description 2
- 241000254173 Coleoptera Species 0.000 description 2
- 101710151559 Crystal protein Proteins 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000258937 Hemiptera Species 0.000 description 2
- 108700001097 Insect Genes Proteins 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 244000037671 genetically modified crops Species 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 241000218475 Agrotis segetum Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000009429 Gossypium barbadense Nutrition 0.000 description 1
- 241000255967 Helicoverpa zea Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001147398 Ostrinia nubilalis Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101150014068 PPIP5K1 gene Proteins 0.000 description 1
- 241000255969 Pieris brassicae Species 0.000 description 1
- -1 Pro tocols Chemical class 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 101150086784 cry gene Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000266 injurious effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000018322 upland cotton Nutrition 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009105 vegetative growth Effects 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Insects & Arthropods (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and a coding gene thereof, and their applications. The bacillus thuringiensis vegetative insecticidal protein has an amino acid sequence represented by SEQ ID No.1. A base sequence of the coding gene is represented by SEQ ID No.2. The applications are an application of the bacillus thuringiensis vegetative insecticidal protein in preparing insecticides, and an application of the coding gene in cultivating an insect-resistant transgenic plant. Compared with prior arts, according to the invention, Vip3Af2 gene and Vip3Aa1 gene are adopted as models, and a chimeric gene Vip3AfAa is designed and synthesized. The Vip3AfAa protein obtained by chimeric gene Vip3AfAa expression has high insecticidal activities upon lepidoptera insects and aphids. The chimeric gene Vip3AfAa can be highly efficiently expressed in plants such as cotton, corn, canola, soy-bean, and the like, and can be used for cultivating insect-resistant transgenic plants.
Description
Technical field
The present invention relates to biological prevention field, be specifically related to bacillus thuringiensis vegetative insecticidal protein Vip3A fAa and encoding gene thereof and application.
Background technology
Diseases and pests of agronomic crop is one of Main Agricultural disaster of China, and it has, and kind is many, impact is large, also often break out the feature of causing disaster, and its occurrence scope and severity often cause heavy losses to Chinese national economy, particularly agriculture production.According to statistics, China due to illness nearly 50,000,000,000 jin of pest injurious loss grain, 1,800 ten thousand tons of all kinds of cash crop every year.Past is in order to prevent and treat the various insect pests of crop, chemical pesticide is widely used, yet, on the one hand due to long-term a large amount of agricultural chemicals that use, cause Some Insects to produce certain resistance or resistance, the using dosage that causes agricultural chemicals is increasing year by year or is using brand-new agricultural chemicals; On the other hand, due to the long-term chemical pesticide that excessively uses, cause the environment that the mankind depend on for existence to be polluted in various degree.Therefore, need to find the various disease and pests of better approach prevention and control.Along with the development of biotechnology, utilizing anti insect gene to cultivate insect-resistant transgenic plants is an effective approach.
Bacillus thuringiensis (Bacillus thuringiensis, Bt) be the important donor bacterium of Plant Extrinsic Anti-insect Genes, the parasporal crystal toxin that its toxicity activity produces while being mainly derived from sporulation, be insecticidal crystal protein (insecticidal crystalline protein, ICP), comprise crystal toxin (crystalline toxin, Cry) and lysis toxin (cytolytic toxin, Cyt).ICP, by Cry gene and Cyt genes encoding, has strong toxicity to sensitive insect, and to higher animal and people's nontoxicity.
The insecticidal crystalline gene of having reported has 748 kinds, has much been widely used in insect-resistant transgenic breeding.Yet because the genetically modified crops of most of commercialization utilization are insecticidal crystal protein class, along with the expansion of these genetically modified crops cultivated areas, insect produces resistance to single insecticidal proteins has become a severe problem.Therefore finding new anti insect gene seems particularly important.
Scientist is through unremitting effort, the separated non-parasporal crystal insecticidal proteins that obtains having desinsection toxicity from the Bt bacterial strain of some vegetative growth stages, after synthetic, be secreted into outside born of the same parents, Here it is is called as the Vegetative Insecticidal Proteins (vegetative insecticidal protein, Vip) of the Tribactur of the pest-resistant albumen of the s-generation.Vips is mainly divided into Vip1, Vip2, Vip3 and Vip4 Four types totally 108 insecticidal proteins, and wherein 77 albumen belong to Vip3.The insecticidal spectrum of Vip3 is different from ICPs with insecticidal activity, and the former has toxic action to insects such as lepidopteran, Coleoptera and Homopteras, and pest-resistant spectrum is wider.At present, these genes are widely used in pest-resistant transgenic rice, corn and cotton breeding.
Yet the resource of Vip3 gene is limited after all, and there is larger difference in different intergenic resistances, the gene of bacterium is rich in AT simultaneously, this certainly will affect the expression of gene in plant, corresponding gene is carried out artificial reconstructed, can further improve the utilising efficiency of gene.
Document (Fang et al., Characterization of chimeric Bacillus thuringiensis Vip3toxins, Applied and Environmental Microbiology, 2007,73 (3): 956-961; Fang Jun, Bacillus thuringiensis Vegetative Insecticidal Proteins Vip3 gene and the application in transgenic paddy rice thereof, 2008, Ph D dissertation, Zhejiang University Library) by the N end of Vip3Ab2 or C end is chimeric with C end or the N end of Vip3Aa1, synthetic Vip3AbAa and Vip3AaAb, result shows, the insecticidal spectrum of mosaic gene is wider, and higher to the insecticidal activity of Some Insects.Therefore, by synthetic mosaic gene, be the effective way of transformation Vip3 gene.
Summary of the invention
The invention provides a kind of bacillus thuringiensis vegetative insecticidal protein Vip3A fAa, this bacillus thuringiensis Vegetative Insecticidal Proteins can be in vegetable cell high efficient expression, the insects such as lepidopteran, Coleoptera and Homoptera are had to higher insecticidal activity.
Bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A aBb, its aminoacid sequence is as shown in SEQ ID No.1.
The present invention also provides the encoding gene of described bacillus thuringiensis vegetative insecticidal protein Vip3A fAa, and its base sequence is as shown in SEQ ID No.2.This encoding gene designs and synthesizes by following thinking:
(1) obtain 400 amino acid whose 1200 base sequences of coding Vip3Af2 albumen n end in Vip3Af2 gene (GenBank Accession No:DD319822);
(2) obtain 389 amino acid whose 1167 base sequences of coding Vip3Aa1 PROTEIN C end in Vip3Aa1 gene (GenBank Accession No:L48811);
(3) two base sequences that chimeric step (1) and (2) obtain, obtain a preliminary original base sequence of transforming;
(4) get rid of the sequence that typically causes this unsettled AT of being rich in of plant gene transcription that exists in described original base sequence and conventional restriction endonuclease sites, under the prerequisite that does not change aminoacid sequence, carry out codon displacement, obtain the base sequence after improvement;
(5) normal chain of the base sequence after improving and corresponding minus strand are carried out to BLAST2 analysis, under the prerequisite that does not change aminoacid sequence, carry out codon displacement, get rid of the inverted repeats existing in sequence; Finally obtain the base sequence as shown in SEQ ID No.2.
Codon displacement in step (4) and (5) is to adopt the main preference codon of Plant Genome to replace corresponding codon in the base sequence after described original base sequence or correction.To improve the expression efficiency of described encoding gene in target plant.
In the present invention, the main preference codon of described Plant Genome is monocotyledon rice (Liu Qingpo, the codon usage of paddy rice and the impact of initial sum terminator codon flanking sequence on genetic expression, 2005, doctorate paper, Zhejiang University Library) and dicotyledons intend south (the Duret L.and Mouchiroud D.Expression pattern and that is situated between, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, the codon of the equal preference of nuclear gene group and Arabidopsis.1999.PNAS.96:4482-4487.).
Base sequence shown in the SEQ ID No.2 of acquisition is recombinated in the host cells such as intestinal bacteria and expressed, obtain the bacillus thuringiensis vegetative insecticidal protein Vip3A fAa with aminoacid sequence shown in SEQID No.1.
The G+C content of described encoding gene is 59.37%, with the highest homology of existing Vip3 gene be 88.56%; And the G+C content of described original base sequence is only 31.05%, both homologys are only 63.05%.
The highest homology that described bacillus thuringiensis vegetative insecticidal protein Vip3A fAa and existing Vip3 albumen are Vip3Aa1 albumen is 98.2%, with the homology of Vip3Af2 albumen be 90.2%.
The present invention also provides expression unit, expression vector or the transformant that contains described encoding gene.As preferably, the promotor of described expression unit is T7 promotor, lac promotor or araBAD promotor.Under the effect of these promotors, Vip3AfAa albumen can directly be realized soluble-expression in born of the same parents in e. coli host cell.The initial carrier of described expression vector can be selected pET28a (+).
The present invention also provides the application of described encoding gene in cultivating insect-resistant transgenic plants.Specifically comprise:
(1) build the plant expression vector that contains described encoding gene;
(2) described plant expression vector is passed through to agrobacterium mediation converted plant callus;
(3) plant callus is transferred on selective medium and continued to cultivate, after seedling differentiation, transplant to land for growing field crops, screening obtains insect-resistant transgenic plants.
Due to when designing described encoding gene, that the codon that adopts unifacial leaf model plant paddy rice and dicotyledonous model plant to intend the common preference of southern Jie's nuclear gene group carries out codon displacement, therefore this gene is applicable to unifacial leaf and dicotyledons, for cultivating corresponding insect-resistant transgenic plants.As preferably, described insect-resistant transgenic plants is insect-resistant transgenic cotton, corn, rape and soybean.
The present invention also provides the application of described bacillus thuringiensis vegetative insecticidal protein Vip3A fAa in preparing sterilant.The object of killing of described sterilant is preferably lepidopterous insects.Described lepidopterous insects is as prodenia litura, cabbage caterpillar, bollworm, black cutworm and Pyrausta nubilalis (Hubern). etc., more preferably prodenia litura.Described sterilant kill more preferably aphid of object.
Compared with prior art, beneficial effect of the present invention is:
It is source that Vip3Bb1 gene and Vip3Aa1 gene are take in the present invention, designs and synthesizes mosaic gene Vip3AaBb, and mosaic gene Vip3AaBb expresses the Vip3AfAa albumen obtaining lepidopterous insects, aphid are had to higher insecticidal activity; Mosaic gene Vip3AaBb can be in the vegetable cells such as cotton, corn, rape and soybean high efficient expression, can be used for cultivating corresponding insect-resistant transgenic plants.
Accompanying drawing explanation
Fig. 1 is the aminoacid sequence comparison chart of Vip3Aa1, Vip3Af2 and Vip3AfAa protein;
Fig. 2 A is the resistance result figure of transgene cotton to glyphosate;
Fig. 2 B is the resistance result figure of non-transgenic cotton to glyphosate;
Fig. 3 A is the resistance result figure of non-transgenic cotton to aphid;
Fig. 3 B is the resistance result figure of transgene cotton to aphid.
Embodiment
The molecular biology that following examples are used and biochemical method are known technology, the Molecular Cloning:A Laboratory Mannual being published by Cold Spring Harbor Laboratory Press (2001) that the Current Pro tocols in Molecular Biology being published by John WTle y and Sons company writing at Ausubel and J.Sambrook etc. write, the documents such as 3rd ED. all have detailed explanation.In following examples, experiment material used is commercially available purchase product if no special instructions.
The design of embodiment 1 mosaic gene and synthetic
Take Vip3Af2 gene and Vip3Aa1 gene is source, design mosaic gene Vip3AfAa, and concrete steps are as follows:
(1) obtain 400 amino acid whose 1200 base sequences of coding Vip3Af2 albumen n end in Vip3Af2 gene (GenBank Accession No:DD319822);
(2) obtain 389 amino acid whose 1167 base sequences of coding Vip3Aa1 PROTEIN C end in Vip3Aa1 gene (GenBank Accession No:L48811);
(3) two base sequences that chimeric step (1) and (2) obtain, obtain a preliminary original base sequence of transforming, and as shown in SEQ ID No.3, the G+C content of this sequence only has 31.05%;
(4) do not changing under the prerequisite of aminoacid sequence, the sequence and the conventional restriction endonuclease sites that typically cause this unsettled AT of being rich in of plant gene transcription that in described original base sequence, exist are replaced into monocotyledon rice (Liu Qingpo, the codon usage of paddy rice and the impact of initial sum terminator codon flanking sequence on genetic expression, 2005, doctorate paper, Zhejiang University Library) and dicotyledons intend south (the Duret L.and Mouchiroud D.Expression pattern and that is situated between, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis.1999.PNAS.96:4482-4487.) codon of the equal preference of nuclear gene group, obtain the base sequence after improving,
(5) normal chain of the base sequence after improving and corresponding minus strand are carried out to BLAST2 analysis, do not changing under the prerequisite of aminoacid sequence, the inverted repeats existing in sequence is replaced into the codon that monocotyledon rice and dicotyledons are intended the equal preference of southern Jie's nuclear gene group, obtain final chimeric gene sequence Vip3AfAa, as shown in SEQ ID No.1, the sequence homology shown in this sequence and SEQ ID No.3 only has 63.05%.Sequence shown in SEQ ID No.1 and the plasmid PUC-Vip3AfAa that contains this DNA fragmentation all entrust Sangon Biotech (Shanghai) Co., Ltd. to complete.
The expression of embodiment 2 mosaic genes
The Vip3AfAa mosaic gene that utilizes embodiment 1 to obtain is expressed cloud gold genus bacillus vegetative insecticidal protein Vip3A fAa, specifically comprises:
Vip3AfAa mosaic gene is building up to escherichia coli plasmid expression vector pET28a (+) upper, and transforms escherichia coli expression host BL21 (DE3); Inoculate single bacterium colony in 5 milliliters of LB substratum, 37 ℃ of overnight incubation, then dilute and be cultured to OD in 1:100 ratio
600for 0.4-0.6, then adding final concentration is that the IPTG of 1mM carries out abduction delivering, and induction time is 4-6 hour; Centrifugal collection thalline, adds 20mL sterilized water resuspended, liquid nitrogen multigelation 6 times, and the centrifugal thalline that goes, obtains supernatant liquor.
The aminoacid sequence of Vip3AfAa albumen is as shown in SEQ ID No.1.The aminoacid sequence of Vip3AfAa albumen is carried out to Blast2 with Vip3Aa1 albumen and Vip3Af2 albumen respectively and compare, in the homology of protein level, be respectively 98.2% and 90.2% (seeing Fig. 1).
The insecticidal activity of embodiment 3 Vip3AaBb albumen
The supernatant liquor that embodiment 2 is obtained is fed lepidopterous insects prodenia litura, and take clear water, transform and have the colibacillary fermented supernatant fluid of pET28a (+) empty carrier for contrast, the insecticidal activity of detection Vip3AfAa albumen to prodenia litura, the results are shown in Table 1.
The insecticidal activity comparison of three kinds of samples of table 1 to prodenia litura
As can be seen from Table 1, Vip3AfAa albumen has significant insecticidal activity to prodenia litura, and after feeding 24h, the average mortality of prodenia litura reaches 86.7%, and after feeding 48h, the average mortality of prodenia litura reaches 100%.And the prodenia litura of other two kinds of samples of feeding, mortality ratio is lower.
Embodiment 4: for cultivating insect-resistant transgenic cotton
(1) structure of carrier
According to the sequence of SEQ ID NO.1, synthesize respectively two primers (primer sequence entrusts extra large Sani bio tech ltd to synthesize), from plasmid PUC-Vip3AfAa, pcr amplification goes out Vip3AfAa gene, and primer sequence is as follows:
Upstream primer:
5’-CACGGGGGACTCTAGAACAATGAACAAGAACAACACTAAG-3’(SEQ?ID?NO:4)；
Downstream primer:
5’-CGGGGGATCCTCTAGTCACTTGATAGAAACGTCGTA-3’(SEQ?ID?NO:5)；
PCR reaction system is:
PCR reaction parameter:
98 ℃, 10 seconds, 55 ℃, 15 seconds, 72 ℃, 2 minutes 30 seconds, 35 circulations; 72 ℃ are extended 5 minutes.
PCR product is after PCR product purification test kit purifying, with XbaI enzyme cutting double T-DNA carrier pLM-B001, with Clontech's
hD Cloning Kit is cloned into pLM-B001 by Vip3AfAa, identifies the positive colony of acquisition check order (PE company, 377 sequenators with XbaI enzyme cutting again; Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) checking, the plasmid called after pLM-Vip3AfAa that checks order correct.
(2) Agrobacterium preparation
By electrization, carrier pLM-Vip3AfAa is imported to Agrobacterium LBA4404; Get containing the Agrobacterium of carrier pLM-Vip3AfAa and draw plate, choose single bacterium colony and cultivate in LB substratum, for Cotton Transformation is prepared Agrobacterium.
(3) acquisition of transgene cotton
1) choose the hypocotyl of Upland Cotton Ke word 312 aseptic seedling, with scalper, be cut into 0.5-0.6cm segment, be inoculated in (MS+B on inducing culture
5organism+2,4-D 0.1mg/L+KT 0.1mg/L+ glucose 30g/L+phytagel 2.5g/L) evoked callus;
Callus is at subculture medium (MS (saltpetre doubles, and ammonium nitrate reduces by half)+B
5organism+2,4-D 0.05mg/L+KT 0.1mg/L glucose 30g/L+phytagel 2.5g/L) subculture several times after, select grain of rice shape Granulous callus, proceeded to division culture medium (MS+B
5organism+glucose 30g/L+phytagel 2.5g/L+KT 0.15mg/L+IBA 0.5mg/L), in, be further divided into embryoid;
2) the glycerine pipe that takes out the agrobacterium strains of preserving in Ultralow Temperature Freezer is in thawing on ice, on LB plate, rule, 26.5 ℃ of dark 36-48hr that cultivate, treat to grow single bacterium colony clearly in ware, picking list bacterium colony, in other LB plate line, is secretly cultivated 36-48hr for 26.5 ℃, treat that in ware, growing enough bacterium colonies finishes to cultivate, media surface bacterium colony is scraped in the MGL substratum in triangular flask, and 27 ℃, 200r/min shake 2hr, and OD value can be used for infecting between 0.5-1.5;
3) callus that is divided into embryoid is proceeded to sterile petri dish from triangular flask, remove rataria, turn white and the dead callus waiting out of order, blow and within 5 minutes, make surface micro-a little dry; Activated Agrobacterium bacterium liquid is poured into wherein, Agrobacterium bacterium liquid was advisable just to cover embryoid surface, stir evenly, standing 5-10 minute, outwells bacterium liquid, with filter paper, blots remaining bacterium liquid, blow and within 5 minutes, make surface dry a little, thin layer is scattered in the common culture medium that is lined with filter paper, and the 19-21 ℃ of dark 38-42 hour that cultivates treats that small part callus surface occurs that distant bacterium colony finishes common cultivation;
4) callus through cultivating is altogether taken out and immersed containing in the sterilized water of 500mg/L Cef together with filter paper, callus is cleaned up, outwell washing lotion, be placed in the sterilized water that contains 500mg/L Cef and soak 15-20min; Between soak period, stir, removes rataria more, turns white and the dead callus waiting out of order; Outwell washing lotion, use sterile water wash three times, filter paper suck dry moisture, rickle divides to intersperse among to be selected, on substratum one, to blow and within 10 minutes, make surface dry a little; The low light level is cultivated 20 days left and right subcultures and is proceeded to the two normal illuminations cultivations of selection substratum, cultivate 20 days left and right subcultures and proceed to the three normal illuminations cultivations of selection substratum, cultivate 20-30 days, in the dead callus of black, occur that the normal callus of the less growth of light yellow particle is kanamycin-resistant callus tissue.Generally can select on substratum three again subculture once to increase callus quantity kanamycin-resistant callus tissue mono-clonal;
5) from selecting substratum, picking mono-clonal resistance callus is inoculated into respectively on division culture medium, and 20 days left and right subcultures once, as far as possible clear distinguishing between difference clone in the process of differentiation and seedling;
6) kanamycin-resistant callus tissue obtaining after screening is transferred on pre-division culture medium and (first secretly cultivated 5-7 days, illumination in then 16 hours differentiation is germinateed) 4-6 week, after resistance seedling grows up to, transfer on root media and take root, finally regeneration plant is washed away to cultivate based on greenhouse or field and cultivate, until results T1 seed;
7) by T1 for seed in land for growing field crops, with the special primer of marker gene and goal gene, identify marker gene and the goal gene in transgenic progeny, obtain insect-resistant transgenic cotton.
(4) Resistance Identification of transgene cotton
By (3) part steps 7) the insect-resistant transgenic cotton seeds kind that obtains is in land for growing field crops, with glyphosate (expression product of above-mentioned marker gene has resistance to glyphosate), smear transgenic progeny, obtain the transgenosis pure lines (as Fig. 2 A and Fig. 2 B) of stably express.From Fig. 2 A and Fig. 2 B, transgenic progeny has resistance to glyphosate.
Meanwhile, field test transgenic progeny is to the resistance of aphid (being shown in Fig. 3 A and Fig. 3 B).From Fig. 3 A and Fig. 3 B, transgenic progeny does not obviously have aphid damage, and aphid is had to significant resistance.
Claims (10)
1. bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A fAa, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.
2. the encoding gene of bacillus thuringiensis vegetative insecticidal protein Vip3A fAa as claimed in claim 1, is characterized in that, base sequence is as shown in SEQ ID No.2.
3. contain the expression unit of encoding gene as claimed in claim 2.
4. expression as claimed in claim 3 unit, is characterized in that, promotor is T7 promotor, lac promotor or araBAD promotor.
5. contain the expression vector of encoding gene as claimed in claim 2.
6. contain the transformant of encoding gene as claimed in claim 2.
7. the application of encoding gene in cultivating insect-resistant transgenic plants as claimed in claim 2.
8. application as claimed in claim 7, is characterized in that, described insect-resistant transgenic plants is insect-resistant transgenic cotton, corn, rape or soybean.
9. the application of bacillus thuringiensis vegetative insecticidal protein Vip3A fAa in preparing sterilant as claimed in claim 1.
10. application as claimed in claim 9, is characterized in that, the object of killing of described sterilant is aphid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410392302.8A CN104151410A (en) | 2013-08-15 | 2014-08-11 | Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310354718.6 | 2013-08-15 | ||
| CN2013103547186A CN103421099A (en) | 2013-08-15 | 2013-08-15 | Vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of vegetative insecticidal protein Vip3AfAa and application |
| CN201410392302.8A CN104151410A (en) | 2013-08-15 | 2014-08-11 | Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104151410A true CN104151410A (en) | 2014-11-19 |
Family
ID=49646515
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013103547186A Pending CN103421099A (en) | 2013-08-15 | 2013-08-15 | Vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of vegetative insecticidal protein Vip3AfAa and application |
| CN201410392302.8A Pending CN104151410A (en) | 2013-08-15 | 2014-08-11 | Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013103547186A Pending CN103421099A (en) | 2013-08-15 | 2013-08-15 | Vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of vegetative insecticidal protein Vip3AfAa and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN103421099A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651377A (en) * | 2013-11-25 | 2015-05-27 | 浙江大学 | Novel insecticidal protein |
| CN105017391A (en) * | 2015-06-30 | 2015-11-04 | 杭州瑞丰生物科技有限公司 | Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012006271A1 (en) * | 2010-07-07 | 2012-01-12 | Syngenta Participations Ag | Control of coleopteran insect pests |
| CN103145814A (en) * | 2013-02-25 | 2013-06-12 | 北京大北农科技集团股份有限公司 | Insecticidal protein, and coding gene and use thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101385467B (en) * | 2001-03-30 | 2014-11-05 | 辛根塔参与股份公司 | Novel pesticidal toxins |
| AR035799A1 (en) * | 2001-03-30 | 2004-07-14 | Syngenta Participations Ag | INSECTICIDE TOXINS ISOLATED FROM BACILLUS THURINGIENSIS AND ITS USES. |
| US7378493B2 (en) * | 2002-03-06 | 2008-05-27 | Syngenta Participations Ag | Vip3 toxins and methods of use |
-
2013
- 2013-08-15 CN CN2013103547186A patent/CN103421099A/en active Pending
-
2014
- 2014-08-11 CN CN201410392302.8A patent/CN104151410A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012006271A1 (en) * | 2010-07-07 | 2012-01-12 | Syngenta Participations Ag | Control of coleopteran insect pests |
| CN103145814A (en) * | 2013-02-25 | 2013-06-12 | 北京大北农科技集团股份有限公司 | Insecticidal protein, and coding gene and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 方军: "苏云金杆菌营养期杀虫蛋白Vip3基因及其在转基因水稻中的应用", 《浙江大学博士学位论文》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651377A (en) * | 2013-11-25 | 2015-05-27 | 浙江大学 | Novel insecticidal protein |
| CN105017391A (en) * | 2015-06-30 | 2015-11-04 | 杭州瑞丰生物科技有限公司 | Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103421099A (en) | 2013-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105624177A (en) | Insect-fusion-resistant gene, coding protein, carrier and application thereof | |
| CN104488945B (en) | The purposes of insecticidal proteins | |
| CN102732554A (en) | Method for raising insect resistance of plants | |
| CN110117320A (en) | Cotton GhCAL-D07 gene is promoting the application in flowering of plant | |
| CN107723303A (en) | Insect-resistant fusion gene, encoding proteins, carrier and its application | |
| CN101580843B (en) | Artificial synthesized Bt insecticidal gene for transgenic anti-insect plants | |
| CN102559554B (en) | Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene | |
| CN106832001A (en) | A kind of desinsection fusion protein, encoding gene and its application | |
| CN106399339A (en) | Anti-herbicide and anti-pest fusion gene, encoded protein thereof, and application of encoding protein | |
| CN104046636A (en) | Codon vegetalization-transformed PMI gene and applications thereof | |
| CN102776226B (en) | Method for producing cecropins by using alfalfa as bioreactor | |
| CN104531656A (en) | Phosphomannose isomerase from chlorella variabilis and application thereof | |
| CN104151410A (en) | Bacillus thuringiensis vegetative insecticidal protein Vip3AfAa and coding gene thereof, and their applications | |
| CN101926365B (en) | Use of bacillus thuringiensis cry1Ai in pest control, modified mcry1Ai gene and use of modified mcry1Ai gene | |
| CN104522033B (en) | The purposes of insecticidal proteins | |
| CN104151409B (en) | Bacillus thuringiensis vegetative insecticidal protein Vip3A aBb and encoding gene thereof and application | |
| CN109867715A (en) | A kind of chloroplast protein and ATPase enzymatic activity mutant are improving the application in stress resistance of plant | |
| CN103215290A (en) | Insect-resistant fusion gene as well as insect-resistant fusion protein and application of insect-resistant fusion gene and insect-resistant fusion protein | |
| CN104151408A (en) | Bacillus thuringiensis vegetative-period insecticidal protein Vip3DAa, and coding gene and applications thereof | |
| CN102533851A (en) | Corn gene normal position transformation method mediated by high throughput agrobacteria | |
| CN102746391A (en) | Arsenic-resistance related protein PvArrp1, and coding gene and application thereof | |
| CN101870979B (en) | Insecticidal gene cryX with high toxicity to lepidopterous pests and application thereof | |
| CN103570810B (en) | A kind of bacillus thuringiensis Vegetative Insecticidal Proteins, gene, carrier and application | |
| CN107058376B (en) | Method for preventing and treating hemipteran pests of crops | |
| CN101413007B (en) | Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses 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 |
Application publication date: 20141119 |
|
| RJ01 | Rejection of invention patent application after publication |