[go: up one dir, main page]

WO2009018739A1 - Souche de bacillus thuringiensis, gènes cry8h, protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations - Google Patents

Souche de bacillus thuringiensis, gènes cry8h, protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations Download PDF

Info

Publication number
WO2009018739A1
WO2009018739A1 PCT/CN2008/071513 CN2008071513W WO2009018739A1 WO 2009018739 A1 WO2009018739 A1 WO 2009018739A1 CN 2008071513 W CN2008071513 W CN 2008071513W WO 2009018739 A1 WO2009018739 A1 WO 2009018739A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
protein
plant
seq
strain
Prior art date
Application number
PCT/CN2008/071513
Other languages
English (en)
Chinese (zh)
Inventor
Changlong Shu
Fuping Song
Rongyan Wang
Jie Zhang
Shuliang Feng
Dafang Huang
Zhihong Lang
Original Assignee
Institute Of Plant Protection, Chinese Academy Of Agricultural Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute Of Plant Protection, Chinese Academy Of Agricultural Sciences filed Critical Institute Of Plant Protection, Chinese Academy Of Agricultural Sciences
Publication of WO2009018739A1 publication Critical patent/WO2009018739A1/fr

Links

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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of biological control technology, and relates to a nucleotide sequence of a crySH gene highly virulent to a coleopteran pest, and relates to an amino acid sequence of a protein highly virulent to a coleopteran pest, involving, artificially designed and synthesized in a plant
  • the nucleotide sequence of the crySH gene expressed in the nucleic acid sequence encoding the nucleotide sequence of the artificially designed crySH gene, the recombinant strain containing the crySH gene, the use of the gene to construct an expression vector, and the use of the above gene
  • a method of performing plant transformation in a sequence A method of performing plant transformation in a sequence.
  • the scarab belongs to the genus Scarabaeidae, and its larvae (commonly known as ⁇ , hereinafter referred to as " ⁇ " in the present invention) are an important class of underground pests that can harm food, cotton, oil crops, vegetables, and sugar. Crops, tobacco, pasture, flowers, turfgrass, fruit trees and many other plants. A large number of investigations have shown that the hazards of cockroaches in underground pests are the highest, mainly larvae of the genus scorpionidae and larvae, accounting for 70-80% of the total number of underground pests. According to statistics, the annual occurrence of plutonium is about 100 million mu, and the serious years have reached 320 million mu. The loss of production is as high as 20% or more.
  • Baci llus thuringiensis is an extremely widely distributed Gram-positive bacterium. It forms a protein-like parasporal crystal while forming spores, for Lepidoptera, Diptera, Coleoptera, Hymenoptera, and the same wing.
  • Various insects such as Homoptera, Orthoptera, Mallophaga, and specific insecticidal activities of nematodes, mites and protozoa (Schnepf, EN et al, Microbiol. And Molecular Biology Review, 1998, 62 : 3 775-806).
  • ICPs Insecticidal Crystal Proteins
  • delta-endotoxins are harmless to humans and animals and do not pollute the environment. Therefore, Bt has been widely used in the biological control of pests.
  • cry8 genes At present, 11 kinds of cry8 genes have been found, and the encoded protein is composed of 1160-1210 amino acids with a molecular weight of 128-137 kDa.
  • Table 1 Ansano, S., Yamanaka, S. and Takeuchi, K., Protein having insecticidal activity, DNA encoding the protein, and Controll ing agent and controll ing method of noxious organi sms, 2002, JP 2002045186 - A and JP 2002045186- A/2) ).
  • Cry8Aal and Cry8Bal isolated from Mycogen of the United States have significant insecticidal activity against various pests of the family Caragidae (Tracy E.
  • cry8Bbl and cry8Bcl were isolated from the Bt strain in the United States and found to have significant insecticidal effects on Western corn rootworm and have been used in the development of transgenic insect-resistant maize (Abad, Andre, R. , Duck Nicholas, B., Feng, Xiang, Flannagan Ronald, D., Kahn, Theodore, W., Sims, Lynne, E. Genes encoding novel proteins with pesticidal activity against coleopterans, 2002, W0 02/34774 A2).
  • the present invention provides a Bacillus thuringiensis crySH pattern gene sequence which is highly toxic to coleopteran pests such as the Great Black Stork, and is used for transforming microorganisms and plants to exhibit toxicity to related pests, and overcomes and delays The resistance of pests to engineered bacteria and transgenic plants.
  • Bacillus thuringiensis strain BT-SU4 the accession number is CGMCC2071.
  • the C. thuringiensis crySHal gene which is highly efficient against Coleoptera pests has a nucleotide sequence as shown in SEQ ID NO: 1.
  • the Bacillus thuringiensis crySHal protein which is highly efficient against Coleoptera pests is encoded by the above-described crySHal gene, and its amino acid sequence is shown in SEQ ID NO: 2.
  • crySHal protein in the preparation of a medicament for killing coleopteran pests.
  • a protein having a same function as the above-described protein and having an amino acid sequence having a homology to the sequence of SEQ ID NO: 02 or SEQ ID NO: not less than 83.5%.
  • a plant expression vector P3300U8H characterized in that the plant expression vector consists of a mcrySHal gene sequence, a constitutive expression promoter or a root-specific promoter, a terminator and a binary element capable of shuttle between Escherichia coli and Agrobacterium tumefaciens Constructed by the vector.
  • the application is to transform a plant expression vector p3300U8H containing the mcrySHal gene into a plant or microorganism to produce toxicity against a coleopteran pest.
  • the plant is turfgrass.
  • the application is to prepare a protein expressed by the mcrySHal gene into a medicament for killing coleopteran pests.
  • a transgenic plant characterized by the transformation of a foreign gene into a plant, the protein expressed by the foreign gene having the same function as any of the proteins described above.
  • a biological preparation characterized by containing any of the above proteins.
  • the present invention isolates the strain BT-SU4 from Hebei soil, and its storage number is CGMCC2071. Its biological characteristics are that spores can be produced during the growth cycle, and at the same time, corpus callosum crystals are produced.
  • a pair of universal primers were designed based on the conserved region of the cry8 gene:
  • the BT-SU4 strain was identified by PCR amplification, and the amplification result (see Figure 1) showed that the band was different from the known cry8 gene (see Table 3), indicating that the strain BT-SU4 may contain new cry8 insecticide. gene.
  • cry8H5/cry8H3 A pair of full-length gene primers cry8H5/cry8H3 was designed to amplify the full-length gene. And BamHI/Sail was introduced for cloning and expression, and the sequence of primer pair cry8H5/cry8H3 was as follows:
  • cry8H5 gg aat tcg atg agt ccg aat aat cag aat
  • cry8H3 cgc gtc gac tta cat ttc ttc tac aat caa ttc
  • the total DNA of the strain BT-SU4 was used as a template, and PCR amplification was carried out using pfu DNA polymerase.
  • the result showed that a band of about 3.5 Kb was amplified and ligated with the vector pET21b to transform Escherichia coli JM110.
  • Recombinant plasmid pSASSM (Fig. 2). Sequencing analysis of the inserted fragment revealed that the sequence SEQ ID NO 1 was a BamHI/Sail double-cut fragment of pSASSU4, and the full length of the sequence was 3672 bps. The analysis indicated that it contained an open reading frame, the position of 0RF1 was 1-3672, and the GC content was 38.
  • % a protein encoding 1223 amino acids.
  • the amino acid sequence thereof was determined to be represented by SEQ ID NO 2. Homology analysis indicated that the protein has high homology with Cry8 protein, and Table 4 is the homology data. Since the amino acid homology with the known Cry8 protein is less than 78%, the highest is only 58.2% (Cry8Bbl), which is named Cry8Hal by the Bt insecticidal crystal protein nomenclature committee.
  • the primers cry8H5/cry8H3 were introduced into the BamHI and Sail sites respectively, and the full-length gene was amplified by using the plasmid BT-SU4 plasmid DNA as a template.
  • the recombinant plasmid pSK08H was obtained by inserting the Bt expression vector pSTK (see Figure 3), and transformed into Escherichia coli SCS. 110, extraction of plasmid, electroporation into Bt crystalless mutant HD-73- (this mutant was obtained from the Biotechnology Laboratory of Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and can be provided to the public, see Li Haitao et al., Journal of Agricultural Biotechnology 2005 Vol. 13 No. 6 P. 787-791 ) , obtained the engineering strain BioT8H.
  • the above-mentioned engineering strain BioTSH was cultured at 30 ° C in beef extract medium (peptone 5 g, beef extract 3 g, glucose 10 g, water 1000 mL, 121, 20 minutes autoclaving), and the protein was extracted for SDS-PAGE electrophoresis.
  • beef extract medium peptone 5 g, beef extract 3 g, glucose 10 g, water 1000 mL, 121, 20 minutes autoclaving
  • the results see Figure 4
  • the results showed that the cry8Hal gene in the engineered organism BioT8H was expressed, and the molecular weight of the expressed product was about 130 kDa.
  • the activity assay of Cry8Hal protein showed that the expressed Cry8Hal had the activity of killing the larvae of the black cockroach and the dark cockroach larvae.
  • the l_2010bp sequence of the crySHal gene was optimized based on differences in codon preferences between microorganisms and plants.
  • the present invention performs whole-gene synthesis according to the artificially engineered sequence of the crySHal gene, and the new gene is shown in the nucleotide sequence shown in SEQ ID NO: 3.
  • the corresponding protein sequence is shown in SEQ ID NO 4.
  • the nucleotide sequence homology of the cry8Hal gene and the mcry8Hal (modified cry8Hal) gene was only 86. 88%, and the G+C content was also increased from 37.6% of the original cry8Hal to 50.1%.
  • the frequency of amino acid codon usage of the Cry8Hal protein was adjusted to make the amino acid codon usage frequency of the mCry8Hal protein close to the frequency of use in plants, and the artificially engineered sequences of the cry8Ha gene were introduced into the BamHI and Sacl sites (see SEQ ID NO 3).
  • this vector is a common vector, GenBank accession number is Y14837
  • the recombinant plasmid is named pUC57-mcry8H.
  • the plasmid pUC57_m C ry8H was used to recover the 2.0 kb fragment, and the plasmid PCAMBIA3300 was digested with the same endonuclease (this vector is a commonly used vector, plant of the Chinese Academy of Agricultural Sciences).
  • This vector is a commonly used vector, plant of the Chinese Academy of Agricultural Sciences.
  • preservation of the Biotechnology Group of the Institute of Conservation see Zhang Xiaoguo et al., Wuhan Botanical Research, 2000 Vol. 18 No. 1 P. 15-20 ), recovering the 10 kb fragment, ligating the two fragments and transforming E.
  • the plasmid contains a constitutively expressed promoter Ubiquitin (ie, a DNA sequence that drives the ligated gene fragment for transcription and translation into a protein.
  • the constitutively expressed promoter regulates the gene at any stage of growth and development and any tissue. There are expressions), the mcrySHa gene and the NOS terminator (a DNA sequence containing a termination signal for gene expression).
  • the plasmid construction map is shown in Figure 5, and the plasmid can be transformed into a plant to obtain a transgenic plant. .
  • the artificially engineered gene mcrySHal Agrobacterium was transformed to produce a positive clone, which was then transformed into turfgrass.
  • the biological activity of the transgenic turfgrass showed that the transgenic plants showed good resistance to the black turtle (Holotrichia obl ita) ⁇ patina Performance of Anomala diverenta and Holotrichia parallels.
  • the performance of the transgenic turfgrass against the Holotrichia obl ita, Anomala diverenta and Holotrichia parallela is due to the promoter in the plant to initiate transcription of the artificially engineered mcry8Hal gene.
  • the expression cassette in the binary vector-constitutive promoter, the artificially modified mcrySHal and the terminator can only be expressed in the genome of turfgrass to express the foreign gene, so this binary vector can be used to transform any The plants in which the Agrobacterium transformation method was established, the transgenic plants obtained were all resistant to the performance of Holotrichia obl ita; Anomala diverenta and Holotrichia parallela.
  • microorganism scientific name of genus, species: Bacillus sp. Bacillus thuringiensis Depository: General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee
  • Figure 1 PCR-RFLP map of strain BT-SU4. among them:
  • FIG. 1 Recombinant plasmid pSASSM digestion map. among them:
  • FIG. 3 Recombinant plasmid pSK08H restriction map. among them:
  • Figure 6 Molecular detection of transformed turfgrass, where:
  • Table 2 shows the homologous sequences of these genes and primers.
  • Table 3 shows the size of the cleaved fragment of the cry8 gene amplification product predicted by the pair of primers. These genes can be identified by this PCR-RFLP method.
  • crySFa 2182 1293, 889 Cry8Ga 2163 1267, 570, 326 Bt strain 185 was identified with the following PCR reaction system (50 ⁇ : lOxPCR buffer 5 ⁇
  • Amplification cycle Denaturation at 94 °C for 1 minute, annealing at 54 °C for 1 minute, extension at 72 °C for 4 minutes, 25 cycles, and finally extension at 72 °C for 10 minutes.
  • a pair of full-length gene primers cry8H5/cry8H3 was designed to amplify the full-length gene. And BamHI/ Sai l was introduced for cloning and expression, and the sequence of the primer pair cry8H5/cry8H3 was as follows:
  • cry8H5 gg aat tcg atg agt ccg aat aat cag aat
  • cry8H3 cgc gtc gac tta cat ttc ttc tac aat caa ttc
  • PCR amplification was carried out using pfu DNA polymerase using the following system.
  • Amplification cycle Denaturation at 94 °C for 1 minute, annealing at 54 °C for 1 minute, extension at 72 °C for 4 minutes, 25 cycles, and finally extension at 72 °C for 10 minutes.
  • the result shows that a band of 3.5 Kb was amplified and ligated with the vector pET21b to transform Escherichia coli JM110 to obtain a positive transformant pSASSU4.
  • the inserted fragment was sequenced and analyzed, and the sequence SEQ ID NO 1 was BamHI/ Sai l double-cleaved fragment in pSASSM.
  • the full length of the sequence was 3472 bps. The analysis indicated that it contained an open reading frame.
  • the position of 0RF1 was 1-3472, and the GC content was 38. %, a protein consisting of 1157 amino acids.
  • the amino acid sequence thereof was determined to be represented by SEQ ID NO 2. Homology analysis indicated that the protein has high homology with Cry8 protein, and Table 4 is the homology data. Since the amino acid homology with the known Cry8 protein is less than 78%, the highest is only 58.2% (Cry8Bbl), and it is named Cry8Hal by the Bt insecticidal crystal protein nomenclature committee.
  • Cry8Hal 53 52 48 55 56 49 48 The present invention further analyzed the amino acid composition of the CrySHal protein (see Table 6), and found that its molecular weight was 131.56 kDa, and the isoelectric point was PH 4.735 (see Table 5), and the protein was analyzed. Biochemical indicators (see Table 5)
  • the primers cry8H5/cry8H3 were introduced into the BamHI and Sail sites respectively, and the full-length gene was amplified by using the full-length cry8Hal strain BT-SU4 plasmid DNA as a template, and inserted into the Bt expression vector pSTK (see Figure 3) to transform Escherichia coli. SCS110, plasmid was extracted and electroporated into Bt crystalless mutant HD-73- to obtain engineering strain BioT8H.
  • Bt engineering strains were inoculated on common bacterial agar medium flask for 3 days. Wild strain HD-73- was inoculated on a common bacterial agaric flask medium for 4 days. The culture was washed, diluted 2 times in a gradient, and 40 ml of the bacterial suspension was added to 200 g of fine soil (ultraviolet sterilized) having uniform thick and thin potato silk, and mixed to maintain the soil water content at 18% to 20%. 20 larvae of the chafer-infested larvae were added, and the treatment with the addition of clear water was used as a blank control, the words were inoculated at 28 °C, the number of dead insects was checked in 14 days, and the mortality was calculated.
  • fine soil ultraviolet sterilized
  • the results indicate that the engineered strains have extremely high cytotoxic activity against Holotrichia obl i ta, Anomala diverenta and Holotrichia paral lels.
  • the CrySH protein expressed has the activity of killing the black cockroach, the cockroach, and the cockroach.
  • the concentration is X mg protein per gram of soil.
  • the l_2010bp sequence of the cry8Hal gene was optimized based on differences in codon preferences between microorganisms and plants.
  • the present invention performs whole-gene synthesis according to the artificially engineered sequence of the crySHal gene, and the novel gene is shown in the nucleotide sequence shown in SEQ ID NO 3.
  • the nucleotide sequence homology of the cry8Hal gene and the mcry8Hal (modif ied cry8Hal) gene was only 87.08%, and the G+C content was also increased from 37.6% of the original cry8Hal to 50. 1% (Table 8).
  • the frequency of amino acid codon usage of the Cry8Hal protein was adjusted so that the frequency of amino acid codon usage of the mCrySHal protein was close to that used in plants (Table 9).
  • the artificially engineered sequences of the cry8Ha gene were introduced into the BamHI and Kpnl sites, and ligated into the pUC57 vector.
  • the recombinant plasmid was named pUC57-mcry8H.
  • the plasmid pUC57_m C ry8H (preserved by the Biotechnology Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences) was used to recover the 2.0 kb fragment, and the plasmid PCAMBIA3300 was digested with the same endonuclease (this vector is a commonly used vector, plant of the Chinese Academy of Agricultural Sciences).
  • This vector is a commonly used vector, plant of the Chinese Academy of Agricultural Sciences.
  • the Biotechnology Group of the Institute of Conservation is preserved. See Zhang Xiaoguo et al., Wuhan Botanical Research, 2000 Vol. 18 No. 1 P. 15-20).
  • the 10 kb fragment was recovered, and the two fragments were ligated to transform DH5 a to obtain a positive transformant.
  • This new construction plasmid was named p3300U8H.
  • the plasmid contains a constitutively expressed promoter Ubiquitin (ie, a DNA sequence that drives the ligated gene fragment for transcription and translation into a protein.
  • the constitutively expressed promoter regulates the gene at any stage of growth and development and any tissue. There are expressions), the mcrySHa gene and the NOS terminator (a DNA sequence containing a termination signal for gene expression).
  • the plasmid construction map is shown in Figure 5, which can express foreign genes in plants.
  • the Agrobacterium clone containing the P3300U8H plasmid was inoculated into LB liquid medium containing kanamycin 100 ⁇ g/ml, rifampicin 100 ⁇ g/ml and streptomycin 100 ⁇ g/ml, and cultured at 28 ° C with shaking.
  • kanamycin 100 ⁇ g/ml a kanamycin 100 ⁇ g/ml
  • streptomycin 100 ⁇ g/ml streptomycin 100 ⁇ g/ml
  • the reaction conditions for PCR amplification were: 94 ° C, 10 minutes, 1 cycle; 94 ° C, 1 minute, 54 ° C, 1 minute, 72 ° C, 1 minute, 30 cycles.
  • the product was electrophoresed.
  • the positive transformant amplified a fragment of 600 bp in size. 2. 6 Biological activity detection of transgenic turfgrass
  • Bioassays were performed using population planting and colony methods. In the field soil, 12 plantings were used for planting and mixing planting of transgenic plants and untransformed plants, respectively.
  • the hazard degree and hazard index of Holotrichia parallela larvae on the tested transgenic plants are as follows, see Table 10.
  • the damage rate of the P3300U8H plant was 62.5%, the hazard index was 22. 9; the non-transgenic plant hazard rate was 100%, the hazard index was 72.9.
  • the transgenic p3300U8H plants showed good anti-dark black turtle (#o 0 ir c A a parallela) characteristics.
  • the damage degree of the larvae of the North China black cockroach Holotrichia oblita) to the tested transgenic plants is as follows.
  • the P3300U8H plant has a hazard rate of 75%, a hazard index of 28.4, a non-transgenic plant hazard rate of 100%, and an untransformed plant hazard index of 75.0. It can be seen that the transgenic p3300U8H plants showed good resistance to the ⁇ o 0 ir c 'a oblita.
  • the damage degree of the larvae to the tested transgenic plants is as follows.
  • the P3300U8H plant has a hazard rate of 45%, a hazard index of 16.5, a non-transgenic plant hazard rate of 100%, and an untransformed plant hazard index of 75.0. It can be seen that the transgenic p3300U8H plants showed good characteristics against Anomala denenta.
  • the gene using a promoter that can specifically transcribe a gene at the root, the gene can be expressed at the root of the plant, and only the target pest, A ⁇ oir a oblita, can be obtained only at the root of the plant. Resistance of ⁇ Anomala diverenta) and Dark Tortoise ⁇ Holotrichia parallela). 1 /1
  • Microorganisms or other biological materials related to preservation are Microorganisms or other biological materials related to preservation

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Dentistry (AREA)
  • Biomedical Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Insects & Arthropods (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une souche de Bacillus thuringiensis, des gènes CRY8H, des protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations. SEQ ID NO 1 représente une séquence nucléotidique du gène cry8H qui présente une toxicité élevée envers les nuisibles de l'ordre des coléoptères. Une autre séquence nucléotidique du gène cry8H qui est artificiellement conçu et synthétisé et qui peut être exprimé dans une plante, est représentée par SEQ ID NO 3. Une plante transgénique transformée à l'aide du gène cry8H conçu artificiellement présente une activité pesticide contre Holotrichia oblita, Anomala corpulenta et Holotrichia parallela.
PCT/CN2008/071513 2007-08-07 2008-07-01 Souche de bacillus thuringiensis, gènes cry8h, protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations WO2009018739A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200710120020.2 2007-08-07
CN2007101200202A CN101130762B (zh) 2007-08-07 2007-08-07 对鞘翅目害虫高效的苏云金芽孢杆菌cry8H基因、蛋白及其应用

Publications (1)

Publication Number Publication Date
WO2009018739A1 true WO2009018739A1 (fr) 2009-02-12

Family

ID=39128168

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2008/071513 WO2009018739A1 (fr) 2007-08-07 2008-07-01 Souche de bacillus thuringiensis, gènes cry8h, protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations

Country Status (2)

Country Link
CN (1) CN101130762B (fr)
WO (1) WO2009018739A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101113424B (zh) * 2007-07-04 2010-12-29 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8G基因、蛋白及其应用
CN101130762B (zh) * 2007-08-07 2010-08-25 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8H基因、蛋白及其应用
CN102329760B (zh) * 2011-10-19 2013-01-09 青岛农业大学 杀蛴螬类害虫的苏云金芽孢杆菌新菌株及其杀虫蛋白
CN102363760B (zh) * 2011-11-09 2013-06-05 四川农业大学 一株苏云金芽孢杆菌st8及其杀虫基因和应用
WO2014062540A1 (fr) * 2012-10-15 2014-04-24 Pioneer Hi-Bred International, Inc. Nouveau gène de bacillus thuringiensis ayant une activité contre les coléoptères
CN106916212A (zh) * 2017-04-18 2017-07-04 中国农业科学院植物保护研究所 杀虫蛋白在防治天牛科害虫中的应用
CN108124873B (zh) * 2017-11-23 2021-05-04 中国农业科学院植物保护研究所 杀虫蛋白在防治金龟子成虫中的应用
CN109776659B (zh) * 2019-03-14 2021-01-29 中国农业科学院生物技术研究所 cry2Ah-vp基因在抗黏虫中的应用
CN116649372B (zh) * 2023-07-26 2023-10-27 中国农业科学院植物保护研究所 一种微生物组合物及其在防治鞘翅目害虫中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1609191A (zh) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌菌株和基因
CN1609220A (zh) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 一种人工合成的对鞘翅目害虫高毒力的Bt cry8基因序列
WO2005063996A2 (fr) * 2003-12-23 2005-07-14 Pioneer Hi-Bred International, Inc. Activation de plantes de toxine d'insecte
CN101054589A (zh) * 2004-11-16 2007-10-17 中国农业科学院植物保护研究所 对鞘翅目害虫高效的cry8F基因、其表达蛋白及其应用
CN101113424A (zh) * 2007-07-04 2008-01-30 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8G基因、蛋白及其应用
CN101130762A (zh) * 2007-08-07 2008-02-27 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8H基因、蛋白及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063996A2 (fr) * 2003-12-23 2005-07-14 Pioneer Hi-Bred International, Inc. Activation de plantes de toxine d'insecte
CN1609191A (zh) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌菌株和基因
CN1609220A (zh) * 2004-11-16 2005-04-27 中国农业科学院植物保护研究所 一种人工合成的对鞘翅目害虫高毒力的Bt cry8基因序列
CN101054589A (zh) * 2004-11-16 2007-10-17 中国农业科学院植物保护研究所 对鞘翅目害虫高效的cry8F基因、其表达蛋白及其应用
CN101113424A (zh) * 2007-07-04 2008-01-30 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8G基因、蛋白及其应用
CN101130762A (zh) * 2007-08-07 2008-02-27 中国农业科学院植物保护研究所 对鞘翅目害虫高效的苏云金芽孢杆菌cry8H基因、蛋白及其应用

Also Published As

Publication number Publication date
CN101130762B (zh) 2010-08-25
CN101130762A (zh) 2008-02-27

Similar Documents

Publication Publication Date Title
WO2009018739A1 (fr) Souche de bacillus thuringiensis, gènes cry8h, protéines, qui présentent tous une toxicité élevée envers les insectes nuisibles de l'ordre des coléoptères, et leurs utilisations
CN101113424B (zh) 对鞘翅目害虫高效的苏云金芽孢杆菌cry8G基因、蛋白及其应用
JP3435159B2 (ja) バチルス・チュリンゲンシスの新菌株及び殺虫作用をもつそのタンパク質
UA126058C2 (uk) Інсектицидний ген і спосіб його застосування
JPH10506532A (ja) 新規な有害生物防除性タンパク質および菌株
CA2531400A1 (fr) Proteines insecticides secretees par bacillus thuringiensis et utilisations
BRPI0615649A2 (pt) método para aumentar o acúmulo de uma proteìna inseticida em uma célula hospedeira, para produzir uma célula vegetal resistente a uma praga de inseto, para controlar infestação e para proteger uma cultura, composição inseticida, produto de mercadoria, planta transgênica ou célula vegetal, sequência de nucleotìdeo, proteìna inseticida, progênie ou semente de planta, vetor, célula hospedeira e cassete de expressão
CA2117270A1 (fr) Utilisation d'isolats de bacillus thuringiensis dans la lutte contre les aphididae nuisibles
CA2775582A1 (fr) Proteines insecticides
MXPA06007005A (es) Composiciones de proteinas y genes secretados de bacillus thuringiensis y usos de las mismas.
JP2017521055A (ja) 害虫防除に有用な植物性殺虫タンパク質
CN108892721B (zh) 蝶蛹金小蜂毒液Kazal-type丝氨酸蛋白酶抑制剂PpSPI20蛋白及应用
JPH07503846A (ja) バシラスチューリンゲンシス単離体でのコガネムシ害虫抑制方法
UA124757C2 (uk) Інсектицидний поліпептид проти лускокрилого або твердокрилого шкідника та його застосування
CN111235166B (zh) 一种新型诱导表达的Cry2Ab杀虫基因及其应用
WO2006053473A1 (fr) Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres
CN111850009A (zh) 一种Cry2Ab-2杀虫基因及其应用
CN117510600A (zh) 一种杀虫蛋白的突变体及其应用
AU768073B2 (en) Insecticidal agents
CN101413007B (zh) 对鞘翅目害虫高效的苏云金芽胞杆菌cry8Ⅰ基因、蛋白及其应用
CN108822210B (zh) 蝶蛹金小蜂毒液Kazal-type丝氨酸蛋白酶抑制剂PpSPI24蛋白及应用
KR20000057484A (ko) 바실루스속 세균 및 살충성 단백질
DE60129554T2 (de) Bakterielle insektizidproteine
MXPA01012883A (es) Proteinas insecticidas a partir de paecilomices y combinaciones sinergisticas de las mismas.
CN1323169C (zh) 一种人工合成的对鞘翅目害虫高毒力的Btcry8基因序列

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08783684

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08783684

Country of ref document: EP

Kind code of ref document: A1