WO2009003412A1

WO2009003412A1 - Bacillus thuringiensis stain, cry8g genes, proteins, which are all highly toxic to order coleoptera insect pests, and uses thereof

Info

Publication number: WO2009003412A1
Application number: PCT/CN2008/071514
Authority: WO
Inventors: 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: 2007-07-04
Filing date: 2008-07-01
Publication date: 2009-01-08
Also published as: CN101113424A; CN101113424B

Abstract

Bacillus thuringiensis strain, cry8G genes, proteins, which are all highly toxic to order coleoptera insect pests, and uses thereof are disclosed. The present invention provides a nucleotide sequence of Bacillus thuringiensis cry8G gene and an amino acid sequence of a protein encoded by the cry8G gene. Also provided are another nucleotide sequence of cry8G gene, which is artificially designed to transform plant, and its encoding amino acid sequence. By transformation of the above gene or the artificially designed sequence thereof, microorganism or plant shows toxic to the related insect pests, and thus overcomes or delays the generation of drug resistance of pests to engineering bacteria and transgenic plant.

Description

Bacillus thuringiensis strain, c y gene, protein and application thereof for coleopteran pests

The invention belongs to the technical field of biological control, and relates to a nucleotide sequence of a 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. Expressed

The nucleotide sequence of the Czy gene, which relates to the amino acid sequence of the protein encoded by the nucleotide sequence of the artificially designed gene, relates to a recombinant strain containing the gene, relates to the use of the gene to construct an expression vector, and to the use of the above gene sequence for plant transformation. method.

Background technique

The scarab belongs to the Scarabaei dae family, and its larvae (commonly referred to as "蛴螬" in the present invention) 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. A large number of investigations have shown that the hazards of cockroaches in underground pests are the highest, mainly larvae of the genus Stork and the genus Larva, accounting for 70-80% of the total number of underground pests. According to statistics, the annual occurrence of cockroaches is about 100 million mu, and the serious years have reached 320 million mu. The loss of production is as high as 20%. Some plots are even out of production. In recent years, 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. Occurred; in the western regions of Tibet, Qinghai, Gansu, Xinjiang, etc., the occurrence of cockroaches is also very serious (Wei Hongjun, etc., "China Underground Pests", Shanghai: Shanghai Science and Technology Press, 1989, 1-41; Wang Yongxiang, etc., "冀中"Types and Integrated Control Techniques in the Plain Area", Journal of Hebei Normal University (Natural Science Edition), 1998, 22 (2): 268-270). Take the flower planting area in China's oil crops second only to the rape flower in the second place. China's peanut production accounts for about 35% of the world's total peanut production, ranking first in the world, with an annual export income of 20.7 billion US dollars. In 2001, the national peanut area (5 million hectares) and total output (14.5 million tons) reached the highest level in history. However, the damage to peanuts is very serious. In order to control the hazards of cockroaches, comprehensive prevention and control strategies such as agriculture, chemistry and physics are generally adopted. Although this is effective, it is difficult to achieve the effect of continuous control. Therefore, it is imperative to find new and effective methods of prevention and control.

On the basis of obtaining the highly virulent Bt gene, the cultivation of acaricidal transgenic plants is a new and worthwhile approach to prevention.

Bacillus thuringiensis acillus thuringiensis (Bt) is an extremely widely distributed Gram-positive bacterium. It forms a protein-like parasporal crystal while forming spores, for Lepidoptera (Lepi doptera:), Diptera (Diptera:), Coleoptera: (Coleoptera:), Hymenoptera (Hymenoptera) :), Homoptera, Orthoptera, Mal lophaga and many other insects, as well as nematodes, mites and protozoa have specific insecticidal activity (Schnepf, EN et al , Microbiol. And Molecular Biology Revi ew, 1998, 62 : 3 775-806). Insecti ci dal 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. .

At present, nearly 400 Bt insecticidal genes encoding insecticidal crystal proteins have been cloned, which belong to 157 model genes. In recent years, the research trend of genes in the world has attracted people's attention. Studies have shown that these genes have insecticidal effects on a variety of coleopteran pests such as the chafer family, the genus Aphididae, and the leaf family. In 1992, Ohba et al. screened for the first time in the world a new strain (B. t. subsp. Japonensi s BuiBui ) with specific insecticidal activity against the larva of the chafer (Ohba, M. et al., A unique i solate) Of Baci l lus thuringiensis serovar japonensi s with a high larvi ci dal activity

Specific for scarabaeid beetles, Letters in Applied Microbiology, 1992.14:54-57), in 1994, Sato et al. cloned a new insecticidal gene ^^^ (Sato, R. et al, Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects, Curr. Microbiol. 1994. 28:15-19.4 At present, 11 kinds of crjW genes are found, the encoded protein is composed of 1160_1210 amino acids, and the molecular weight is 128- 137kDa. For detailed information, see Table 1 (Asano, S., Yamanaka, S. and Takeuchi, K., Protein having insecticidal activity, DNA encoding the protein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186 - A and JP 2002045186- A/2) ). Among them, Ciy8Aal and Cry8Bal isolated from Mycogen of the United States have obvious insecticidal activity against various pests of the family Tortoise (Tracy E. Michaels, et al., Bacillus thuringiensis toxins active against scarab pests, 1994, USP5554534) ₀ US from Bt strains Two genes isolated

The gene was found to have significant insecticidal effects on Western corn lOotworm and was 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, W002/34774 A2) ₀ in China, Institute of Plant Protection, Hebei Academy of Agricultural Sciences and Hebei Agricultural University In recent years, a number of Bt strains with specific insecticidal activity against Anomala exoleta and A. corpulenta larvae have been screened, and the indoor mortality rate has reached 100% (Feng Shuliang et al., "1 A new isolate of Bacillus thuringiensis that has insecticidal activity against the chafer larvae, "Chinese Biological Control," 2000, 16(2): 74-78).

Table 1 Bacillus thuringiensis Cry8 insecticidal crystal protein

1167 Asano, et al,

Cry8Da2 BD133574 130. 51 Bt scarabs

2002

1167 Asano, et al,

Cry8Da3 BD133575 130. 51 Bt scarabs

2002

Cry8Eal AY329081 131. 7 1165 BT185 scarabs Fu ing et al

Cry8Fal AY551093 133. 0 1174 BT185 scarabs Fu ing et al

The present invention provides a B. thuringiensis model gene sequence which is highly toxic to coleopteran important pests such as the Great Black Stork, and is applied to transform microorganisms and plants to exhibit toxicity to related pests, and to overcome and delay pests. Resistance to engineered bacteria and transgenic plants.

Bacillus thuringiensis strain HBF-18, the accession number is CGMCC2070.

The use of the strain in killing coleopteran pests.

The Bacillus thuringiensis gene having high efficiency against the coleopteran pest has a nucleotide sequence as shown in SEQ ID NO: 1. An engineered strain strain BioT8G, which is characterized by containing a cry8Gal gene.

The Bacillus thuringiensis protein which is highly efficient against Coleoptera pests is encoded by the above ^ gene, and its amino acid sequence is shown in SEQ ID NO: 2.

_{The application of C} r_r «% 3⁄4 ^ protein in the preparation of a medicament for killing coleopteran pests.

A protein having the same function as the above protein, the amino acid sequence of which is shown in SEQ ID N04.

A protein having the same function as the above-mentioned protein, and having an amino acid sequence having a homology to SEQ ID NO: 02 or SEQ ID N 04 of not less than 83.5%.

An artificially engineered synthetic gene encoding the above-described protein, the nucleotide sequence of which is shown in SEQ ID NO. A plant expression vector pBSmGN, characterized in that the plant expression vector consists of a 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 of mcry8Gal gene in plant anti-coleoptera pests.

The application is to transform a plant expression vector pBSmGN containing the mcrySGal gene into a plant or microorganism to produce toxicity against a sheathed pest.

The plant is tobacco.

The application is to prepare a protein expressed by the mcrySGal gene into a medicament for killing coleopteran pests.

A transgenic plant characterized in that a foreign gene is transformed in the plant, and the protein expressed by the foreign gene has the same function as any of the proteins described above.

A transgenic microorganism characterized in that a foreign gene is transformed in the microorganism, and the protein expressed by the foreign gene has the same function as the protein of any one of claims 5, 7, 8, and 9.

A biological preparation characterized by containing any of the above proteins.

The present invention isolates the strain HBF-18 from Hebei soil, and its storage number is CGMCC2070. Its biological characteristics are that it can produce spores in the growth cycle, and at the same time produce venomous companion crystals.

A pair of universal primers were designed based on the conserved regions of the gene:

SN5un8 5~ -GTCCGAATAATCAGAATGAATATG-3~

SN3un8 5 -CGTTTCGCCTCTCTCACTGCAT-3 ~

The HBF-18 strain was identified by PCR amplification, and the amplification results (see Figure 1) showed that the bands were different from the aphid genes (see Table 3), indicating that the strain HBF-18 may contain new insecticidal genes.

A pair of full-length gene primers, cJT<3⁄43⁄4 cJ <3⁄4a, were designed to amplify the full-length gene. And introduce 3⁄4MHI/Sail for cloning and expression, the sequence of primer pair CJT«% CJT5 is as follows:

BanRI

cry8G5 : 5 ' -CGCGGATCCGAAATGAGTCCGAATAATCA-3 '

Sail

cry8G3 : 5 ' -ACGCGTCGACCTCTTCTTCTAACACGAGT-3 '

The total DNA of the strain HBF-18 was used as a template, and PCR amplification was carried out using pfu DNA polymerase. The result (see Fig. 2) showed that a band of 3.5 Kb was amplified and ligated with the vector pET21b to transform Escherichia coli JM110. The recombinant plasmid pSAS018 was obtained (Fig. 2). Sequencing analysis of the inserted fragment revealed that the sequence SEQ ID NO 1 was a fragment of fe^HI/Sail in pSAS018, and 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 revealed the protein

The proteinoids have higher homology, 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 Cr_r«%3⁄4 by the Bt insecticidal crystal protein nomenclature committee.

The primers σ δ ΰ σ τδΰΰ were introduced into the 3⁄43⁄4sHI and S _a R sites respectively. The full-length gene was amplified by using the plasmid HBF-18 plasmid DNA as a template, and inserted into the Bt expression vector pSTK to obtain the recombinant plasmid pSK018 (see Figure 3). Escherichia coli SCS110, extracting plasmid, electroporation transformed 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 N0.6 P.787-791 ), obtained the engineering strain BioT8G.

The above-mentioned engineering strain BioT8G was cultured in 3CTC in beef extract medium (peptone 5 g, beef extract 3 g, glucose 10 g, water 1000 mL, 12 rC, autoclaved for 20 minutes), and the protein was extracted for SDS-PAGE electrophoresis analysis ( See Sambrook, J. et al, Molecular Cloning: A Labora tory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989), results (see Figure 4). The results showed that the CJ «3⁄43⁄4 gene in the engineered Biot8G was expressed, and the molecular weight of the expressed product was about 130 kDa.

The activity assay of Cir8Ga white showed that the expressed Cr_r«3⁄43⁄4^ had the activity of the larvae of the black-streaked and black-spotted golden tortoises.

The l-2040 bp sequence of the gene was optimized according to the difference in codon preferences between microorganisms and plants. The present invention performs whole-gene synthesis according to the artificially engineered sequence of the 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 CJ «%3⁄4 gene and the BCJ «%3⁄4 (modified cry8Cal) gene have a nucleotide sequence homology of only 86. 88%, and the G+C content is also increased from 37.6% of the original cry8Cal to 45.2%. The frequency of amino acid codon usage of the Cry8Gal protein was adjusted so that the frequency of amino acid codon usage of the mCrySGal protein was close to that of the plant, and the artificially engineered sequence of the CJT<%3⁄4 gene was introduced at both ends of the λ ΛΗΙ and feci sites (see SEQ ID NO 3), ligated into the PUC57 vector (this vector is a common vector, GenBank accession number is Y14837), and the recombinant plasmid is named pUC57-mcry8G.

In the artificially engineered Bt crj^ gene, the 2. Okb fragment was recovered by digesting the plasmid pUC57-mcry8G with 3⁄43⁄4sHI and >3⁄4cl, and the plasmid pBI121 was digested with the same endonuclease (this vector is a commonly used vector, GenBank accession number is AF485783). See Chen PY, et al, 2003, Mol. Breed, 11: 287-293), recovering the 12 kb fragment, ligating the two fragments, transforming 扉0 to obtain a positive transformant, and naming the new construct plasmid pBSmGN. This plasmid contains the constitutive expression promoter CaMV35S (ie one

The segment DNA sequence can drive the ligated gene fragment for transcription and then translate into protein. The constitutively expressed promoter can regulate the expression of the gene at any stage of growth and development and any tissue), mcry8Ga\ gene and NOS terminator ( A DNA sequence containing the 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 recombinant gene was transformed into Agrobacterium, and positive clones were prepared and transformed into tobacco. The biological activity of the transgenic tobacco showed that the transgenic plants showed good iHolotrichia paralleled performance.

Tobacco is a commonly used model plant for verifying gene function in plant genetic engineering. The performance of transgenic tobacco against Holotrichia parallela is due to the promoter of the artificially engineered gene in the plant, which expresses i _r«W Protein, the expression cassette in the binary vector - the constitutive promoter, the artificially modified ^ and the terminator can only be expressed in the genome of tobacco to express the foreign gene, so this binary vector can be used to transform any sputum to establish Agrobacterium transformation. In the method of the plant, the obtained transgenic plants all have the properties of the anti-dark black turtle iHolotrichia parallela). Biological preservation information:

Name of microorganism (scientific name of genus, species): Bacillus genus Bacillus thuringiensis iten'wgfew Depository: China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC)

Date of deposit: June 1, 2007

Deposit number: CGMCC 2070

DRAWINGS

Figure 1: PCR-RFLP map of strain HBF-18. among them:

M. DNA molecular weight standard

1. PCR product digestion

Figure 2: Recombinant plasmid pSAS018 restriction map. among them:

M. DNA molecular weight standard

1. PCR product BamHI/Sall digestion

2. Vector pET21bBamHI/SalI digestion

3. Recombinant plasmid pSAS018BamHI/Sal I restriction map

Figure 3: Recombinant plasmid pSK018 restriction map. among them:

M. DNA molecular weight standard

1. Vector pSTK BamHI/Sall

2. PCR product BamHI/Sall digestion

3. Recombinant plasmid pSK018 BamHI/Sal l digestion map

Figure 4: Expression of the cry8Gal gene in Bt amorphous mutants. among them:

M. Protein molecular weight standard

1. HBF-18

2. Biot8G

3. HD-73—

Figure 5: pBSmGN plasmid construction map

Figure 6: Molecular detection of transformed tobacco, where:

M. Protein molecular weight standard

1, 2. Test results for partially positive transgenic plants

detailed description

The present invention will be further described in detail below with reference to the embodiments.

Example 1

1.1 strain 185 gene identification

A pair of universal primers were designed based on the conserved region of the gene-like gene

SN5un8 5~ -GTCCGAATAATCAGAATGAATATG-3~

SN3un8 5 - CGTTTCGCCTCTCTCACTGCAT- 3 ~

Table 2 shows the homologous sequences of these genes and primers. Table 3 shows the size of the amplified fragments of the gene amplification products predicted by the pair of primers. These genes can be identified by this PCR-RFLP method.

Table 2 Pairing of the conserved regions of the primers and the cry8 genes and the position of the paired regions in the gene. Gene surface Jian Yanjian Primer

SN5un8 0GTTTO3⁄4CTCICrCACTGCA C

SN3un8 GTOOGAATM CAGAA GAATATG

Cry8Aa GTOCMATM CAAAA GAATATG 5-28 GATTOGG GAGAGAGGCAAAAOG 2164-2186

Cry8Ba GTOCMATM CAAAA GAATATG 5-28 GA GCAG GAGAGAGGCAAAAOG 2152-2174

Cry8Bb GTOCMATMTCAAAATGAATATG 5-28 GA GCAG GAGAGAGGCAAAAOG 2167-2189

Cry8Bc GTOCMATMTCAAAATGAATATG 5-28 GATGCAGTGAGAGAGGCAAAAOG 2179-2201

Cry8Ca GTOCMATM CAAAA GAGTATG 5-28 GA GCAG GAAAGAGG0GAAA0G 2155-2177

Cry8Da GTOCMATMTCAAAATGAATATG 5-28 GA GCAG GAAAGAGG0GAAA0G 2107-2129

Cry8Ea GTOCMATMTCAAAATGAATATG 3657-3680 GA GCAGTGAAAGAGGCAAAAOG 5786-5808

Cry8Fa GTOCMATMTCAAAATGAATATG 75-98 GA GCAGTGAAAGAGGCAAAACG 2234-2256 Table 3 PCR amplification products and restriction length polymorphisms of cry8

PCR (SN5un8/SN3un8)

Genotype ^

Kpnl and Dral digestion results

Gene product size

Digested with Kpnl and

Types Size (bp)

Dral (bp)

cry8Aa 2186 744, 772, 326, 310

cry8Ba 2170 1653,384, 133

cry8Bb 2185 1404,338,310, 133 cry8Bc 2197 2064, 135

cry8Ca 2173 1416,757

cry8Da 2125 2125

cry8Ea 2152 1817,335

cry8Fa 2182 1293,889

crySGa 2163 1267,570,326

The Bt strain 185 was identified using the following PCR reaction system (50 μυ:

lOxPCR buffer

MgCl ₂ (20mM)

dNTP (lOmM)

Primer pair (10 mM)

Template

Taq polymerase (5υ/μϋ 0. 5μΙ^

Add ultra-pure water to 5C^L, mix and centrifuge, add paraffin oil 3C^L.

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 PCR product was analyzed by Ι and Dral digestion. The results (Fig. 1) showed that the bands were 1260 bp, 570 bp, and 320 bp, which are different from the known T<? gene map (Table 3), indicating that the strain HBF-18 May contain a new "<? Insecticidal gene. 1. 2 clone of crj^C ¹ gene in HBF-18 strain

Design of a pair of full-length gene primers

And introduce SMHI/Sail for cloning and expression, primer pairs

The sequence is as follows:

BanRI

cry8G5 : 5 ' -CGCGGATCCGAAATGAGTCCGAATAATCA-3 '

Sail

cry8G3 : 5 ' - ACGCGTCGACCTCTTCTTCTAACACGAGT-3 '

Using the total DNA of strain HBF-18 as a template, pfuDNA polymerase was used for PCR amplification using the following system.

Add ultra-pure water to 5C^L, mix and centrifuge, add paraffin oil 3C^L.

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 (see Fig. 2) 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 pSAS018. Sequencing the inserted fragment to obtain the sequence SEQ ID NO 1 as pSAS018

Sail double-cleavage fragment, the full length of 3472bps, analysis showed that it contains an open reading frame, 0RF1 position is 1-3472, GC content is 38.%, encoding a protein 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 proteinoid is less than 78%, the highest is only 58.2% (Cr_r&3⁄4W), named Cry8Gal by the Bt insecticidal crystal protein nomenclature committee.

Table 4 Cry8Gal and Cr _y 8 protein homologous comparison data

Cry8Aal Cry8Bal Cry8Bbl Cry8Bcl Cry8Cal Cry8Dal Cry8Eal Cry8Fal

Cry8Gal 54 53 49 49 62 60 53 54

The present invention further analyzed the amino acid composition of the Cry8Gal protein (see Table 6), and found that its molecular weight was 56 kDa, and the isoelectric point was pH 4.735 (see Table 5), and the biochemical indicators of the protein were analyzed (see Table 5).

Table 5 <3⁄43⁄4 protein biochemical characteristics

Analysis content data

Full length of protein 1157 aa

Molecular weight 131663. 17 m. w.

1 milligram of protein moles 7. 595 pMoles

1 mole protein racemization coefficient 215030

Protein concentration of lod A ^ 0. 61 mg/ml

1 mg/ml A280 absorbance 1. 63 AU

Isoelectric point 5. 15

Charge value at pH 7 -25. 28 Table 6 Amino acid composition of Crj^3⁄4 protein

Amino acid ΪΪ mass percent frequency percent

counting

Charged (RKHYCDE) 335 34. 24 28. 95

Acidic (DE) 139 12. 82 12. 01

Basic (KR) 113 12. 05 9. 77

Polar (NCQSTY) 395 34. 49 34. 14

Hydrophobic 358 30.94

(AILFWV)

A Ala 69 4. 03 5. 96

C Cys 8 0. 64 0. 69

D Asp 64 5. 59 5. 53

E Glu 75 7. 24 6. 8

F Phe 41 4. 44 3. 54

G Gly 71 3. 50 6. 14

H His 10 1. 02 0. 86

I He 63 5. 42 5. 45

K Lys 47 4. 51 4. 06

L Leu 97 8. 34 8. 38

M Met 20 1. 96 1. 73

N Asn . 86 7. 45 7. 43

P Pro 50 3. 77 4. 32

Q Gin 61 5. 85 5. 27

R Arg 66 7,. 54 5. 70

S Ser 90 6. 20 7. 78

Replacement page (Article 26) T Thr 85 6. 64 7. 35

V Val 65 4. 99 5. 62

W Trp 23 3. 08 1. 99

Y Tyr 65 7. 72 5. 62

B Asx 150 13. 04 12. 96

Z Glx 136 13. 08 11. 75

1. 3 crj^ gene expression

Primer

The full-length gene was amplified by inserting the full-length cry8Gal strain HBF-18 plasmid DNA into the 3⁄43⁄4sHI and 53⁄4 1 locus, inserting the Bt expression vector pSTK (see Figure 3), transforming Escherichia coli SCS110, and extracting the plasmid. , electroporation transformed Bt crystalless mutant HD-73-, obtained engineering bacteria BioT8G

The above two engineering strains 3CTC were cultured in beef extract medium for 30 hours, and 50 C ^ L of bacterial liquid was taken into an Eppendorf tube, and ultrasonically disrupted for 30 seconds (B. Braun U Labsoni c, 230 V, T = 0.5 seconds). Add 25μΙ^ to 25μΙ^ new with 0. 5Ν NaOH at 25 °C for 5 minutes; add 65 L 3 X sample buffer (925μΙ^ loading buffer + 75μΙ^β-mercaptoethanol), boil for 10 minutes at 10CTC. The precipitate was removed by centrifugation. Load lOuL for SDS-PAGE analysis. The results (Fig. 4) showed that the CJT<3⁄43⁄4^ gene in the engineered organism Biot8G was expressed, and the molecular weight of the expressed product was about 130 kDa.

1. 4 Crj^6 protein activity assay

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. The infection was carried out at 28 °C, and the number of dead insects was checked in 14 days, and LC _{5 was} calculated. . The results (see Table 8) indicate that the engineered strains of the North Black Stork

oWiis) and the dark cockroach {Holotrichia parallela) have extremely high toxic activity. The Cry8G protein expressed by it has the activity of killing the black cockroach and the black cockroach.

Table 7 Bt engineering bacteria and HBF-18 strains

Obli ta) , dark cockroach (Holotrichia parallela) larvae insecticidal activity assay

Note: In the concentration unit of the result, X 107g soil and X 107ml are converted, and 36ml of the bacterial suspension is added per 200g of soil. Example 2

2. 1 Artificially designed synthetic nucleotide sequence of a gene that can be expressed in plants

The l_2040bp sequence of the ^ gene was optimized according to the difference in codon preference between microorganisms and plants. The present invention performs whole gene synthesis according to the artificially engineered sequence of the gene, and the new gene is shown in the nucleotide sequence shown in SEQ ID NO 3. The nucleotide sequence homology of the CJT «%3⁄4 gene with the BCJ «%3⁄4 (modifi ed cry8Cal) gene was only 86. 88% 6+ (the content was also increased from the original £;/^ ₍ 3⁄43⁄4 of 37.6%) 45. 2% (Table 8). Adjusted the amino acid codon of Cry8Gal protein

The frequency of use makes the amino acid codon usage frequency of the mCry8Gal protein close to that used in plants (Table 9). The artificially engineered sequence of the cry8Ca gene was introduced into the Kpnl site and ligated into the pUC57 vector. The recombinant plasmid was named p!JC57-mcry8G.

Table 8 Comparison of cry8Gal gene and mcr _y 8Ga + C content with polyadenylation signal sequence

crySGal mcry8Gal optimization results

Number of bases Percentage Base Number of bases Percentage GC content Remarks

A 682 33. 4 A 603 29. 6

Removal of 10 polygons C 350 17. 2 C 482 23. 6 increase 37. 6%

Glycosylated signal G 417 20. 4 G 440 21. 6 reached 45. 2%

Sequence

T 591 29 T 515 25. 2 !

Frequency of amino acid codon usage in 9 plants, Cry8al and mCry8Gal

Codon usage

Password

Amino Acid Plant Cry8Gal Cry8Gal mCry8Gal mCry8Gal

Percentage percentage number of percentages

Ala gcc 32 12 6 16 32

Ala gcg 3 16 8 2 3

Ala get 41 29 14 20 41

Ala gca 23 43 21 11 23

Arg egg 5 0 0 2 5

Arg agg 23 9 3 7 23

Arg cga 7 9 3 2 7

Arg cgc 11 9 3 4 11

Arg aga 29 34 11 9 29

Arg cgt 25 38 12 8 25

Asn aac 72 33 15 33 72

Asn aat 28 67 31 13 28

Asp gac 52 17 6 19 52

Asp gat 48 83 30 17 48

Cys tgc 78 0 0 1 78

Cys tgt 22 100 1 0 22

Gin cag 36 32 10 13 18

Gin caa 64 68 21 18 26

Glu gag 48 21 8 27 39

Glu gaa 52 79 30 11 16

Gly ggc 20 10 4 8 20

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L f Οΐ 6ΐ L 3OB 丄

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2.2 Construction of plant expression vector

The 2.0 kb fragment was recovered by BanRl and the digested plasmid pUC57-mcry8G (preserved by the Biotechnology Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences), and the plasmid pBI121 was digested with the same endonuclease (this vector is a commonly used vector, GenBank accession number is AF485783. See Chen PY, et al, 2003, Mol. Breed, 11: 287-293), recovering a 12 kb fragment, ligating the two fragments, transforming JM110 to obtain a positive transformant, and naming the newly constructed plasmid pBSmGN. The plasmid contains a constitutively expressed promoter, CaMV35S (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 is expression), the mcrySGa 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 express a foreign gene in the plant.

2.3 Agrobacterium transformation

Take a tube of 200μ 1 Agrobacterium LBA4404 competent cells, add 1 μg of pBSmGN plasmid DNA, freeze in liquid nitrogen for 1 minute, restore culture at 37 °C for 5 minutes, add 1ml YEB liquid medium, slow oscillation at 28 °C ( <100 rpm) After 4 hours, centrifuge at 30 rpm for 30 seconds, discard the supernatant, resuspend the cells in ΙΟΟμ Ι YEB liquid medium, and apply to 100 μg/ml containing kanamycin and 125 μg/ml of streptomycin. The plate of YEB medium was cultured at 28 ° C for 48 hours. Clones on YEB-resistant medium plates were shaken, and plasmids were extracted, and positive clones were detected by PCR.

2.4 Tobacco conversion

The Agrobacterium clone containing the PBSmGN plasmid was inoculated into YEB liquid medium containing kanamycin 100 μg/ml and streptomycin 125 μg/ml, 28°. Shake the culture to 03⁄4. . 0.6_0.8, 1/50 was inoculated into MS salt (pH7.0); the tobacco sterile seedling was cut into 0.4X0.6cm ² pieces, the tobacco leaves were immersed in MS salt, and Agrobacterium infected for 10 minutes. Remove the tobacco leaves, blot the bacterial solution with sterile filter paper, place in MS medium with a layer of filter paper, and incubate for 3 days at 28 °C. Transfer the tobacco leaves to MS Screening Differentiation Medium (MS medium) after 3 days. +100pg/ml kanamycin +500 μg/ml carboxymycin +3mg/ml 6-BA+O.2mg/ml NAA), 28 °C, light/dark = 16 hours / 8 hours, 2 weeks After that, green callus appeared at the edge of the leaf. After 1 week, the callus differentiated into plantlets, and the plantlets were excised and transferred to rooting medium (MS medium + 100 μg/ml kanamycin + 500 μ). g/ml carboxymycin) rooting, root development is strong, moved to the pot to continue to grow in the soil (common soil: nutrient soil: vermiculite = 2: 1: 1).

2.5 Molecular detection of transformed tobacco

The genomic DNA of the transformed tobacco was extracted, and 1 μg of genomic DNA was used as a template. The primer sequences were as follows:

8GF1: 5' -TTCAGTTGTCCACTCCGCCTA

8GR1: 5' - GCCATTCACAGCCTTCTTTGC

The reaction conditions for PCR amplification were: 94 ° C, 5 minutes, 1 cycle; 94 ° C, 1 minute, 53 ° C, 1 minute, 72 ° C, 2 minutes, 30 cycles. The product was electrophoresed. As shown in Fig. 6, the positive transformant amplified a fragment of 640 bp in size.

2.6 Detection of biological activity of transgenic tobacco

From the field, AWoiWcA ij oblita and Motrichia parallels are two species of chafers that are more harmful in northern China. They are brought back indoors and placed in a 40X40X50TM servo box. Bottom the 5-8cm thick wet sifted fine soil, serve the fresh eucalyptus leaves, 26-28 Ό support. After the eggs are laid, the eggs are picked and placed in moist soil to incubate. Pick out the newly hatched larvae, put 5 larvae in each small squash box (®8cm, H5cm), feed the potatoes

Blocks and fresh corn tender roots, until the larvae grow to a certain age to feed the test.

Bioassays were performed using population planting and colony methods. In the field soil, 12 plants were treated for each planting and mixed planting of transgenic plants and untransformed plants.

According to the established classification criteria of hazard degree, the damage degree and hazard index of Woir _C A _S oblita larvae on the tested transgenic plants are as follows, see Table 10.

Table 10. Hazard statistics for transgenic and non-transgenic plants.

Strain: pBSmGN Strain: Non-transgenic plants Hazard class Number of plants Hazard index Hazard level Number of plants Hazard index

0 5 0 0

1 4 1 0

2 3 22. 9 2 4 72. 9

3 0 3 5

4 0 4 3

0。 The hazard rate of the non-transgenic plants was 100%, and the hazard index was 72.9. The transgenic pBSmGN plants showed good resistance to the North China Blackbird

The characteristics of oblita).

Dark cockroach (yo oir cA s

. The hazard rate of transgenic plants with pBSmGN was 50%, the hazard index was 12.5, the hazard rate was less than 100% for non-transgenic plants, and the untransformed plant hazard index was 75. 0. It can be seen that the transgenic pBSmGN plants showed good resistance to Motrichia parallels.

According to the above information, a promoter which can specifically transcribe a gene at the root can be used to express the gene at the root of the plant, thereby obtaining resistance to the target pest olotrichia parallels only at the root of the plant.

1 /1

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-1 PCT/R0/134 Form (SAFE)

Microorganisms or other biological materials related to preservation

Description of the material (Article 133 bis of the PCT Regulations)

-1 -1 Software version PCT-SAFE

Version 3. 51. 021. 196 MT/F0P 20070401/0. 20. 5. 9

-2 International Application No. PCT/CN2008/071514

-3 File number of the applicant or agent PC08277/ZWB The following instructions are in this manual.

Deposited microorganisms or other raw

Material related:

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-3 Preservation

-3-1 Name of the depository unit CGMCC China Microbial Culture Collection Management Committee General Microbiology Center-3-2 Depository Address China Microbial Culture Collection Committee, Beijing 2714, China Postcode: 100080, Beij ing (CN).

-3-3 Date of Deposit June 01, 2007 (01. 06. 2007)

-3-4 Deposit No. CGMCC 2070

-5 This note is for the designated countries listed below. All designated countries are filled out by the receiving Office -4 This form is received with the international application:

(Yes or no)

-4-1 Authorized officials to be completed by the International Bureau -5 The date of receipt of this form by the International Bureau:

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Claims

Rights request

1. Bacillus thuringiensis strain HBF-18, the accession number is: CGMCC2070.

2. Use of the strain of claim 1 for killing coleopteran pests.

3. The Bacillus thuringiensis gene highly efficient against Coleoptera pests, the nucleotide sequence of which is shown in SEQ ID NO.

An engineered strain strain BioT8G, which comprises the cry8Gal gene of claim 3.

5. A Bacillus thuringiensis cry8Gal protein highly efficient against a coleopteran pest, encoded by the gene of claim 3, having an amino acid sequence as shown in SEQ ID NO: 2.

6. Use of the crySGal protein of claim 5 for the preparation of a medicament for killing a coleopteran pest.

A protein, as claimed in claim characterized by having the _{C r_r} 5 «¾¾ ^ same protein function, and SEQ ID N02 sequence homology with the amino acid sequence is not less than 83.5%.

8. A protein having the same function as the protein of claim 4, the amino acid sequence of which is shown in SEQ ID N04.

A 5%. The protein having the same function as the protein of claim 8 and having an amino acid sequence of not less than 83.5%.

10. An artificially engineered synthetic gene encoding the protein of claim 8 having a nucleotide sequence such as SEQ ID

N03 is shown.

A plant expression vector pBSmGN, characterized in that the plant expression vector consists of the niceSGal gene of claim 10, a constitutive expression promoter or a root-specific promoter, a terminator and a cell capable of producing Escherichia coli and root cancer A binary vector shuttled in Agrobacterium was constructed.

Application of coleopteran pests in _C ry8Gal 12. The gene of claim 10 in a plant anti.

The use according to claim 12, characterized in that the plant expression vector pBSmGN containing the nicrySGal gene is transformed into a plant or a microorganism to produce toxicity against a coleopteran pest.

14. The use according to claim 12, wherein the plant is tobacco.

15. Use according to claim 12, characterized in that the gene expressed protein is prepared into a medicament for killing the pteridophyte pest.

A transgenic plant characterized in that a foreign gene is transformed in the plant, and the protein expressed by the foreign gene has the same function as the protein according to any one of claims 5, 7, 8, and 9.

A transgenic microorganism characterized in that a foreign gene is transformed in the microorganism, and the protein expressed by the foreign gene has the same function as the protein according to any one of claims 5, 7, 8, and 9.

18. A biological preparation comprising the protein of any one of claims 5, 7, 8, and 9.