WO2006053473A1 - Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres - Google Patents

Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres Download PDF

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WO2006053473A1
WO2006053473A1 PCT/CN2005/001133 CN2005001133W WO2006053473A1 WO 2006053473 A1 WO2006053473 A1 WO 2006053473A1 CN 2005001133 W CN2005001133 W CN 2005001133W WO 2006053473 A1 WO2006053473 A1 WO 2006053473A1
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gene
protein
amino acid
genes
strain
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Chinese (zh)
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Fuping Song
Jie Zhang
Shuliang Feng
Dafang Huang
Rongyan Wang
Zhihong Lang
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Biotechnology Research Institute of CAAS
Institute of Plant Protection of CAAS
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Biotechnology Research Institute of CAAS
Institute of Plant Protection of CAAS
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    • 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
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • the invention belongs to the technical field of biological control.
  • the present invention relates to a strain of Bacillus thuringiensis which is active against a coleopteran pest, and relates to a nucleotide sequence of a highly virulence CA ⁇ £ and ctySF gene of a coleopteran pest and an amino acid sequence of a protein encoded thereby, involving synergy
  • the cry8E and cr?F gene expression products were used in combination.
  • the scarab belongs to the genus Scarabaeidae, and its larvae (commonly known as ⁇ , hereinafter referred to as " ⁇ ") are an important class of underground pests that can harm food, cotton, oil crops, vegetables, Sugar crops, tobacco, pasture, flowers, turfgrass, fruit trees and many other plants.
  • ⁇ larvae
  • a large number of investigations have shown that the hazards of cockroaches in underground pests are the highest, among which larvae are mainly larvae and larvae, accounting for the total amount of underground pests.
  • the annual occurrence of cockroaches in the country is about 100 million mu. In severe years, it has reached 300 thousand ounces of 'mu, and the loss of production is as high as 20%. Some plots are even out of production.
  • the area with the largest area and the largest amount of occurrence is the Huanghuaihai area, which mainly harms crops such as grain and oilseeds.
  • the damage in other areas is also very serious, such as the damage to sugarcane, which is common in Guangdong, Guangxi, Yunnan, Sichuan, Fujian and other places.
  • BaciU thw'ingie is, referred to as Bt), is an extremely widely distributed Gram-positive bacterium. It forms a protein-like parasporal crystal while forming spores, for Lepidoptera, Diptera, Coleoptei'a, Hymenoptera. , Homoptera, Orthoptera, various insects such as Mallophaga, and specific insecticidal activities of nematodes, mites and protozoa (Schnepf, EN et al, Microbiol. And Molecular Biology Review, 1998, 62:3 775-806). Insecticidal Crystal Proteins (ICPs), also known as 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.
  • ICPs Insecticidal Crystal Proteins
  • the object of the present invention is to provide a Bacillus thuringiensis strain having high virulence to important coleopteran pests such as Diptera, Coleoptera, and two novel " ⁇ E and a' y 8F pattern gene sequences, and a pair of Diablo
  • the combination of the cr ⁇ E and cr y 8F genes with significant synergistic effects, and the microorganisms and plants transformed with the genes a'y8E, crySF and their genes, which show toxicity to related pests and overcome and delay pests Resistance to engineered bacteria and transgenic plants is produced.
  • the present invention also provides a separate ⁇
  • the proteins encoded by the genes, Cry8Eal and Cry8Fal have an amino acid sequence homology of not less than 83.5%, and a biological preparation containing Cry8Eal protein or/and Cry8Fal protein, which expands the application range of the protein.
  • the present invention isolates Bt strain 1 85 by itself.
  • the soil was collected from the apple orchard in Shunping County, Baoding City, Hebei province.
  • 0.1-0.2 g of soil sample was placed in a test tube containing 10 ml of sterilized water and glass beads, shaken on a vortex shaker for 3 minutes, the soil particles were broken, and then placed on a 200 rp m shaker for 10 minutes, 75 In a water bath of °C water bath for 1 7 minutes, fully kill the non-spore bacteria.
  • the coating was uniform, and cultured at 37 ° C for three days, picking up a Bt colony smear microscopy.
  • a strain of Bt containing a spherical body was found (see Figure 1).
  • the culture condition of the strain was 30, ordinary LB medium, pH 7.0.
  • the strain is of the genus Bacillus (fi"c/7/wy) and Bacillus thuringiensis, and has been preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee. The retention period is January 1, 2004, and the preservation number is For CGMCC NO. I 242.
  • the information about this strain has been identified as follows: It can form buds and form spherical parasporal crystals.
  • SDS-PAGE electrophoresis indicates that the insecticidal crystal protein is about 130kDa (see Figure 2); its crystal protein is expressed at 14 hours, and the growth curve indicates that it has entered a stagnation period of 14 hours (see Figure 3), indicating that the crystal protein promoter may be spore-dependent;
  • the test showed that the strain had obvious insecticidal effect on the underground pest of the genus Diptera, and the TF mortality rate was 90% in 7 days, and it also had certain insecticidal activity against the leaf beetle (see Table 2).
  • S3un8 5-' CTGACTGATTTCCACCATCACG-3.
  • Table 3 shows the homologous sequences of these genes and primers.
  • Table 4 shows the amplification products of the ⁇ . ⁇ gene predicted by the pair of primers, and the size of the fragment, and these genes can be identified by this PCR-RFLP method.
  • crySAa cggcaaacttagtGgaatgc 2101-2120 cgtgatggtggaaatcaAtcag 3273-3294 crySBa cggcCaacttagtGgaatgc 2089-2108 cgtgatggtggaaatcaAtcag 3261-3282 crySBb cggcaaacttagtGgaatgc 2104-2123 cgtgatggtggaaacaAAcag 3276-3297 crySBc cggcaaacttagtGgaatgc 2116-2135 cgtgatggtggaaatcaAAcag 3288-3309 crySCa cggcaaacttaAtagaatgc 2092-2111 cgtgatggtgcaaatcagAcag 3282-3303 crySDo TAAAaaacttagtagaatgc 2044-2063 cgtgatggtgCGaatcagTcag 3234-3255 Note: "N
  • crySDa 1212 197, 92, 310, 613 Bt strain 185 was identified using the following PCR reaction system (50 ⁇ ):
  • Amplification cycle 94 ⁇ denaturation for 1 minute, annealing at 54 ° C for 1 minute, 72 ° C for 4 minutes, 25 cycles, most Extend at 72 ° C for 10 minutes.
  • results show a different map from the known c'S gene, indicating that strain 185 may contain a new o insecticidal gene.
  • the total DNA of Bt strain was digested with Pstl and Kpnl, and two DNA fragment libraries were constructed with vector pBluescript SK(+). Then, two DA libraries were detected by primer S5un8/S3un8 (see Table 3) and PCR method to obtain two DNA libraries.
  • the positive clones pSS.3612 and pSS162 were inserted into the lpkb fragment of llkb and the Kpnl fragment of 2. Okb, respectively (see Figure 5).
  • the pSS3612 was digested with restriction enzymes, and the 7 kb Pstl and Kpnl double-cleavage fragments contained the full-length gene of cry8Eal.
  • the fragment was subcloned with pBluescript SK(+) to obtain pSS3612-7, and the 4 kb Kpnl fragment was subcloned to obtain PSS3612- 4 (see Figure 6).
  • the inserts of pSS36l2 4 and pSS3612-7 were sequenced. The sequence SEQ ID NO 1 was obtained.
  • sequence SEQ ID NO 1 is Pst and l ( p n double-cut fragment in pSS3612, the full length of the sequence is 7276bps, and the analysis indicates that it contains two large open reading frames, the position of 0RF1 is 3658-7152, and the position of 0RF2 is 2799 — 3377.
  • the position S of 0RF1 is 3658-7152, (; C content is 38.03%, encoding a protein consisting of 1164 amino acids.
  • the amino acid sequence is determined as shown in SEQ ID NO 2. Homology analysis indicates that the protein has a Cry8 protein Higher homology, Table 5 is its homology data. The amino acid homology with the known Cry8 protein is less than 78%, and the highest is only 58.2% (Cry8Bbl), which is named by the Bt insecticidal crystal protein nomenclature committee. Cry8EaL.
  • the present invention further analyzed the amino acid composition of the CrySEal protein (see Table 6, Figure 7), and found that its molecular weight was 131.56 kDa and the isoelectric point was pH 4.735 (see Figure 8).
  • PSS162 plasmid The inserted sequence of PSS162 plasmid was analyzed.
  • the fragment was 2.3 kb in length and had a complete 3' end sequence. It was completely homologous to the C ⁇ 3 ⁇ 43 ⁇ 47 sequence, but the 5' end was quite different, and the complete reading frame was lacking.
  • Okb fragment produced by Clal digestion contains a 5' reading frame (see attached Figure 10), the fragment was subcloned with pBluescript SK (+), the positive clone was named pSS266-3, the fragment was sequenced, and the resulting sequence was spliced with the inserted sequence in PSS162 to obtain a 3.9 kb fragment.
  • the above 3.9 kb nucleic acid fragment was sequenced, and the obtained nucleotide sequence was as shown in SEQ [D NO 3 ]. Analysis of this sequence revealed that the sequence contains a large open reading frame 357-3878: GC content of 36.88%: a protein encoding 1174 amino acids (the amino acid sequence of the encoded protein is set forth in SEQ ID NO: 4 Show). Further homology analysis indicated that the protein has high homology with Cry8 proteins (see Table 5 above for homology data). Since the amino acid homology with the known C.ry8 protein is less than 78% and the highest is only 64.8% (Cry8Eal), the protein is named Cry8Fal by the Bt insecticidal crystal protein nomenclature committee.
  • the amino acid composition of the Cry8Fal protein was further analyzed by the bioanalytical software Bioedit, and the results are shown in Table 1 and Figure 11. The results showed that the protein had a molecular weight of 133.08 kDa and an isoelectric point of pH 4.565 (see Figure 12).
  • the present invention designed a primer for expressing two genes, and the sequence is as follows:
  • 8F1 CGCGGATCC (Ba HI) GATGAGTCCAAATAATCAAAATG 8F4: CCGCTCGAG (Xlw I) CTCTACGTCAACAATCAATCAATTC
  • Primers 8E1 and 8E2 were introduced into the B-Hi and Sal ⁇ loci, respectively.
  • the full-length gene was amplified by using the full-length crySEal pSS3612 plasmid DNA as a template, inserted into the Bt expression vector pSXY422b, transformed into Escherichia coli SCS11O, and the plasmid was extracted.
  • a full-length gene of 3.4 kb was obtained, inserted into the Bt expression vector pSXY422b, transformed into E. coli SCSU 0, and the plasmid was extracted and transformed into Bt crystalless mutant HD-73 by electroporation to obtain the engineering strain BioT8F.
  • the above two engineering bacteria 3CTC were cultured for 30 hours in beef extract medium (peptone 5 g, beef extract 3 g, glucose 10 g, water 1000 mL, 121 ° C, 20 minutes autoclaving), and 500 ⁇ bacteria solution was taken. To Eppendorf, ultrasonic disruption for 30 seconds (B.
  • Bt engineering strains were inoculated on common bacterial agar medium flask for 3 days.
  • the recipient strain HD-73_ was inoculated on a common bacterial agaric flask medium for 4 days.
  • the culture was washed, diluted 2 times in gradient concentration, 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% - 20%.
  • the dark scorpion golden tortoise was connected to 20 larvae of 15 days old.
  • the treatment with adding clear water was used as a blank control, and 28 ⁇ was infected.
  • the number of dead insects was checked in 14 days, and LC was calculated. The results are shown in Table 8, indicating that the engineered strain has a very high toxicity to the dark scorpion golden tortoise.
  • Both the Cry8E and CrySF proteins expressed have the activity of killing the larvae of the black scorpion.
  • Figure 1 Spores and crystal morphology of 185 strains.
  • Figure 2 Crystalline protein of 185 strain SDS-PAGE analysis.
  • Figure 3 Growth curve of 185 strain.
  • Figure 4 PCR-RFLP map of strain 185. among them:
  • Figure 5 PCR amplification products and restriction analysis of recombinant plasmids pSS 162 and pSS3162. among them:
  • Figure 7 Amino acid composition of the CrySEal protein.
  • Figure 8 Titration curve of Cry8Eal protein.
  • Figure 9 PCR amplification product and restriction analysis of recombinant plasmid pSS266. among them:
  • Figure 11 Amino acid composition of the Cry8Fal protein.
  • Figure 12 Titration curve of Cry8Fal protein.
  • the soil was collected from the apple orchard in Shunping County, Baoding City, Hebei province. 0.1-0.2 g of soil sample was placed in a test tube containing 10 ml of sterilized water and glass beads, shaken on a vortex shaker for 3 minutes, the soil particles were broken, and then placed on a 200 rpm shaker for 10 minutes, 75 ° C Water bath in a water bath for 17 minutes, fully kill the non-spore bacteria, wait for a little static _, then select 10 - 2 , 10_ 3 , 10- 4 three dilutions, respectively, take lOOul bacterial suspension on the BP plate, painted The cloth was evenly distributed and cultured at 37 ° C for three days, and a Bt colony smear microscopy was picked. A strain of Bt containing spherical crystals was found (see Figure 1).
  • the strain is a species of Bacillus, Bacillus thuringiensis.
  • the strain was submitted to the General Microbiology Center of the China Microbial Culture Collection and Management Committee for preservation. The date of deposit is November 5, 2004, and the deposit number is CGMCCN0.1242.
  • the information about the strain was identified as follows: It can form spores and form spherical parasporal crystals. SDS-PAGE electrophoresis showed that the insecticidal protein was about 130kDa (see Figure 2), and its crystal protein was in 14 hours.
  • the initial expression, and the growth curve indicates that it has entered the stagnation period for 14 hours (see Figure 3), indicating that the proteoprotein promoter may be spore-dependent; biological assays indicate that the strain is against the underground pest of the tortoise family.
  • the golden tortoise has obvious insecticidal effect, and the corrected mortality rate is 90% in 7 days, and it also has certain insecticidal activity against the coconut leaf beetle.
  • a pair of universal bows are designed according to the conserved regions of the gene-like genes.
  • S3un8 5-' CTGACTGATTTCCACCATCACG- 3.
  • results show a different map from the known TJ ⁇ gene, indicating that strain 185 may contain new insecticidal genes.
  • the total DNA of Bt strain was digested with Pstl and Kpnl, and two DNA fragment libraries were constructed with vector pBIuescript SK (+). Then, two DNA pools were detected by primer S5un8/S3un8 and PCR, and two positive clones pSS3612 and pSS162 were obtained. , insert a lpkb fragment of llkb and a Kpnl fragment of 2. Okb, respectively (see Figure 5).
  • the pSS3612 was digested with restriction enzymes, and the 7 kb Pstl and Kpnl double-cleavage fragments contained the full-length gene of cry8Eal.
  • the fragment was subcloned with pBIuescript S (+) to obtain PSS3612-7, and the 4 kb Kpnl fragment was subcloned to obtain pSS3612- 4 (such as Figure 6).
  • the inserted sequences of pSS3612-4 and pSS3612-7 were sequenced to obtain the sequence SEQ ID NO 1.
  • the sequence was a double-cut fragment of Pst. and Kpnl in pSS3612.
  • the full length of the sequence was 7276 bp S.
  • the position of 0RF1 was 3658-7152, and the position of 0RF2 was 2799-3377.
  • the position of 0RF1 is 3658-7152, the GC content is 38.03%, and the protein of 1164 amino acids is encoded, and the amino acid sequence thereof is determined as SEQ ID NO 2. Homology analysis indicated that the protein has high homology with Cry8 proteins (see Table 5). 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 Cry8Eal by the Bt insecticidal crystal protein nomenclature committee.
  • the present invention further analyzed the amino acid composition of the Cry8Eal protein (see Table 6 and Figure 7) and found that its molecular weight was 131.56 kDa and the isoelectric point was pH 4.735 (see Figure 8).
  • the inserted sequence of PSS162 plasmid was analyzed.
  • the fragment was 2.3 kb in length and had a complete 3' end sequence. It was completely homologous to the C ⁇ 3 ⁇ 43 ⁇ 47 sequence, but the 5' end was quite different, and the complete reading frame was lacking.
  • a pair of primers (5-185-KpnI: TTGGTATGGCGTTTCGTTG; and 3-185-Kpnl: TATTGCAGGTCCAGGATTCAC) were designed to clone the full-length gene, and 185 plasmid DNA was digested with Xbal, and the vector pBluescript SK was designed.
  • (+) ligation screening for a positive clone pSS266 inserted into an exogenous fragment of about 9Kb (see Figure 9).
  • a further restriction analysis revealed that the 3.0 kb fragment generated by Clal digestion contained a 5' reading frame (see Figure 10).
  • the fragment was subcloned with pBluescript SK (+), and the positive clone was named pSS266-3.
  • the fragment was sequenced, and the resulting sequence was spliced with the inserted sequence in PSS162 to obtain a 3.9 kb fragment.
  • the nucleic acid fragment was sequenced to obtain a nucleotide sequence as shown in SEQ ID NO 3.
  • the present invention further analyzed the amino acid composition of the Cry8Fal protein using bioanalysis software Bioedit (see Table 7 and Figure 11). The results showed that the protein had a molecular weight of 133.08 kDa and an isoelectric point of pH 4.565 (see Figure 12).
  • ⁇ Example 5 £ ⁇ and expression of the CTW gene' ⁇ Based on the full-length sequences of the cloned c'y8Ea and c' ⁇ F a genes, primers for expression of the two genes were designed. The sequences are as follows:
  • Primers 8E1 and 8E2 were introduced into the ZtonHI and Sal ⁇ sites, respectively, to the full-length ay8Eal pSS3612 plasmid.
  • DNA was used as a template to amplify the full-length gene, inserted into the Bt expression vector P SXY422b, transformed into Escherichia coli SCS110, and the plasmid was extracted and electroporated into Bt crystalless mutant HD-73 to obtain the engineering strain BioT8E.
  • primers 8F1 and 8F4 were introduced in overlapping primers 8F2 and 8F3. Introduced BatnHl and Xhol, respectively.
  • the pSS266 plasmid DNA containing the full-length 'ySFal gene as a template the 0.3 kb and 3.1 kb products were amplified with 8F1 and 8F2, 8F3 and 8F4, respectively, and they were used as templates, respectively, and amplified by primers 8F1 and 8F4, respectively.
  • the full-length gene of kb was inserted into the Bt expression vector pSXY422b, transformed into Escherichia coli SCSI 10, and the plasmid was extracted and electroporated into Bt-free mutant strain HD-73 to obtain the engineering strain BioT8F.
  • the Bt engineering strain was inoculated on a common bacterial agar medium flask for 3 days.
  • the recipient strain HD-73- was inoculated on a common bacterial agaric flask medium for 4 days.
  • the culture was washed, diluted 2 times in gradient concentration, 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%.
  • Twenty-five-day-old larvae of the dark-spotted golden tortoise were connected to the larvae, and the treatment with adding clear water was used as a blank control, and 28 ⁇ was infected, and the number of dead insects was checked 4 days, and LC 5 was calculated.
  • the results indicate that the engineered strain has extremely high toxic activity against the dark scorpionfish. Both of the expressed Cry8E and CrySF proteins have the activity of killing the larvae of the black cockroach.

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Abstract

La bactérie Bacillus thuringiensis et les gènes à haute activité larvicide sur les coléoptères font partie des agents de lutte biologique. L’invention a pour objet une souche de Bacillus thuringiensis à haute activité larvicide sur les coléoptères, la séquence nucléotide de cry 8E et cry 8F, ainsi que la séquence amino-acide de la protéine pour laquelle cry 8E et cry 8F codent. L’invention a également pour objet la production expresse des gènes cry8E et cry8F de la bactérie Bacillus thuringiensis et de tous les deux. L’utilisation ou l’utilisation coopérative des gènes ci-dessus ou de leur association leur confère une toxicité plus importante pour les coléoptères que les souches d’origine ou que les productions expresses de gènes simples, étend leur activité larvicide aux lépidoptères, coléoptères et diptères, permet de déployer leur toxicité sur les nuisibles apparentés et empêche ou diffère la résistance générée par les souches obtenues par génie génétique et les végétaux transgéniques en utilisant ces derniers pour transformer le microbe et les végétaux. Parallèlement, les agents larvicides biologiques obtenus à partir des protéines pour lesquelles les gènes cry 8E et cry 8F codent étendent le champ d’application de la protéine.
PCT/CN2005/001133 2004-11-16 2005-07-27 Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres Ceased WO2006053473A1 (fr)

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US9567381B2 (en) 2012-03-09 2017-02-14 Vestaron Corporation Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
US11447531B2 (en) 2016-10-21 2022-09-20 Vestaron Corporation Cleavable peptides and insecticidal and nematicidal proteins comprising same
US11692016B2 (en) 2012-03-09 2023-07-04 Vestaron Corporation High gene expression yeast strain

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US7329736B2 (en) * 2006-04-14 2008-02-12 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
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
CN101531980B (zh) * 2009-04-13 2010-05-12 四川农业大学 苏云金芽孢杆菌hs18-1及其应用
CN102659933B (zh) * 2012-04-21 2013-12-25 中国农业科学院植物保护研究所 苏云金芽胞杆菌基因cry8like、与cry8G组合及其应用
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

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US9567381B2 (en) 2012-03-09 2017-02-14 Vestaron Corporation Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
US10669319B2 (en) 2012-03-09 2020-06-02 Vestaron Corporation Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
US11472854B2 (en) 2012-03-09 2022-10-18 Vestaron Corporation Insecticidal peptide production, peptide expression in plants and combinations of cysteine rich peptides
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