CN101413007B - Bacillus thuringiensis cry8Ⅰ gene, protein and application thereof highly effective against coleopteran pests - Google Patents

Abstract

The invention relates to a thuringiensis cry8I gene, protein and application thereof, which belong to the field of biotechnology, wherein the thuringiensis cry8I gene and the protein have high efficiency on coleopteran pests. The thuringiensis cry8I gene which is efficient on the coleopteran pests is obtained through the separation from a wild BT-SU4 strain, a sequence of the thuringiensis cry8I gene is shown in SEQ ID NO1, and an amino acid sequence of a coding of the thuringiensis cry8I gene is shown in SEQ ID NO2. A gene expression product has higher virulence to the coleopteran pests, andcan overcome the disadvantages that Bt preparation products and gene species which are used for preventing the coleopteran pests in the world at present are single, make the pests easy to generate the resistance, have narrower insecticidal spectrum and lower virulence and so on; and the new gene can be applied to transforming microorganisms and plants, make the microorganisms and the plants show high virulence to related pests, and overcome and delay the occurrence of the drug resistance of the pests to engineering strains and transgenic plants.

Description

Bacillus thuringiensis cry8Ⅰ gene, protein and application thereof highly effective against coleopteran pests

technical field

The invention belongs to the technical field of biological control, and relates to the nucleotide sequence of the cry8I gene highly toxic to coleopteran pests, to the amino acid sequence of a protein highly toxic to coleopteran pests, to a recombinant strain containing the cry8I gene, and to the use of the cry8I gene Gene construction expression vector.

Background technique

Scarab belongs to Coleoptera Scarabaeidae (Scarabaeidae), and its larvae (commonly known as grubs, also referred to as "grubs" for short in the present invention hereinafter) are a class of important worldwide distribution underground pests, which can harm food, cotton, oil crops, vegetables, sugar Forage crops, tobacco, grass, flowers, lawn grass, fruit trees and other plants. A large number of surveys have shown that grubs are the most harmful underground pests, among which mainly the larvae of the family Beetleidae and Beetleidae, accounting for more than 70-80% of the total underground pests. According to statistics, the occurrence area of grubs in the whole country is about 100 million mu every year, and it once reached 320 million mu in severe years. The yield loss is as high as more than 20%, and some plots even go out of production. In recent years, the Huanghuaihai region has the largest occurrence area and the largest number of occurrences, mainly harming crops such as grain and oil crops; other regions are also very serious. For example, grubs that harm sugarcane are common in Guangdong, Guangxi, Yunnan, Sichuan, Fujian and other places. in Tibet, Qinghai, Gansu, Xinjiang and other western regions, the occurrence of grubs is also very serious (Wei Hongjun et al., "China's Subterranean Pests", Shanghai: Shanghai Science and Technology Press, 1989, 1-41; Wang Yongxiang et al., "Jizhong Species of grubs in plain areas and comprehensive control techniques", "Journal of Hebei Normal University" (Natural Science Edition), 1998, 22(2): 268-270). In order to control the harm of grubs in my country for many years, comprehensive control strategies such as agriculture, chemistry and physics have been generally adopted, which have played a positive role to a certain extent and have controlled the harm of this kind of pests to a low level. Since 2005, the prohibition of high-toxicity, high-residue and highly toxic chemical pesticides in China has brought new problems to the control of underground pests. Minor pests such as grubs have gradually developed into major pests, and they are rampant in some areas. , A direct threat to the safe production of my country's grain, oil and other crops.

At present, the planting area of peanuts ranks second in my country's oil crops, accounting for about 35% of the world's total peanut production, ranking first in the world, with an annual export revenue of 20.7 billion U.S. dollars. It was 14.34 million tons. With the soaring global grain and oil prices in the past two years, the high and stable yield of peanuts and other important oil crops is the top priority related to social stability, food and food security. At present, the biggest threat to peanut production is insect pests - grubs, which directly cause production reduction and affect the quality of peanut seeds, and the damage is very serious. Therefore, it is urgent to open up new effective prevention and treatment methods.

The key to solving this problem is to discover and utilize Bt insecticidal genes with high virulence to grubs.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium with a wide distribution. While forming spores, it can produce parasporal crystals of protein nature, which are suitable for Lepidoptera, Diptera, Coleoptera, Hymenoptera, and Homoptera. Various insects such as Homoptera, Orthoptera, and Mallophaga, as well as nematodes, mites and protozoa have specific insecticidal activity (Schnepf, E.N.et al, Microbiol.And Molecular Biology Review , 1998, 62:3775-806). This insecticidal crystal protein (Insecticidal Crystal Proteins, ICPs), also known as δ-endotoxin (delta-endotoxin), is harmless to humans and animals, and does not pollute the environment, so Bt has been the most widely used in the biological control of pests.

At present, more than 425 Bt insecticidal genes encoding insecticidal crystal proteins have been cloned (see http://www.biols.susx.ac.uk/home/Neil Crickmore/Bt/list.html ), and they belong to 180 model genes. In recent years, the international research trends of cry8 genes have attracted attention. Studies have shown that this type of gene has insecticidal effects on various Coleoptera pests such as scarabs, weevils, and phyllids. In 1992, Ohba et al. screened out a new strain (Btsubsp. Japonensis BuiBui) with specific insecticidal activity to scarab larvae from Bt strains for the first time in the world (Ohba, M.et al., A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeidbeetles, Letters in Applied Microbiology, 1992.14:54-57), in 1994 Sato etc. cloned a kind of new insecticidal gene cry8C from it (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, 20 kinds of cry8 genes have been found, and the encoded protein consists of 1160-1210 amino acids, and the molecular weight is between 128-137kDa. The detailed information of the model genes is shown in 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 JP2002045186-A/2)). Among them, Cry8Aa1 and Cry8Ba1 isolated from Mycogen Corporation of the United States have obvious insecticidal activity against various pests of the family Scarab (Tracy E. Michaels, et al., Bacillus thuringiensis toxins active against carab pests, 1994, USP5554534). Two genes, cry8Bb1 and cry8Bc1, were isolated from Bt strains in the United States, and they were found to have significant insecticidal effects on Western corn rootworm and have been used in the development of transgenic insect-resistant corn (Abad, Andre, R. , Duck Nicholas, B., Feng, Xiang, Flannagan Ronald, D., Kahn, Theodore, W., Sims, Lynne, E. Genes encoding novel proteins with pesticide activity against coleopterans, 2002, WO02/34774A2). In my country, we screened and obtained pairs of Anomala exoleta, A.corpulenta, Popillia quadriguttata, A.mongolica, and Proagopertha. lucidula) and other pest larvae have specific insecticidal activity of Bt strain HBF-1 (Feng Shuliang et al., "China Biological Control", 2000, 16(2): 74-78); "Plant Protection", 2006, 33(4): 417 -422). The Institute of Plant Protection, Chinese Academy of Agricultural Sciences cloned the cry8Ca2 gene from HBF-1 (Shu C, Liu R, Wang R, Zhang J, Feng S, Huang D, Song F. 2007. Current Microbiology. 55: 492-496). Subsequently, the Institute discovered a Bt185 strain containing two genes cry8Ea1 and cry8Fa1 with high activity against the dark gill beetle (Holotrichia parallela) (Yu H, Zhang J, Huang D, Song F. Current Microbiology.2006, 53: 13- 17). At present, cry8Ca2 gene (patent number: ZL200410009807.8), cry8Ea1, cry8Fa1 (patent number: ZL200410009808.2) genes have obtained national invention patent protection, cry8G (application number: 200710118289) and cry8H (application number: 200710120020) genes are under application Patent Protection.

Therefore, screening and cloning new, highly virulent Bt insecticidal protein genes will enrich insecticidal gene resources, provide new gene sources for the development of genetically engineered bacteria and insect-resistant plants, and then improve the insect-resistant effect of Bt transgenic products , and is expected to reduce the risk of pest resistance to Bt toxins, protect and prolong the service life of transgenic products, and have important economic, social and ecological benefits.

Table 1 Model genes of Cry8 insecticidal crystal proteins of Bacillus thuringiensis

Contents of the invention

Aiming at the shortcomings of the Bt preparation products used in the world to control coleopteran pests and the single gene variety, the pests are easy to develop resistance, the insecticidal spectrum is narrow and the toxicity is low, the present invention isolates and clones new Bt strains from domestic Bt strains. Gene cry8I, the gene expression product has high toxicity to coleopteran pests, and the amino acid sequence of the gene expression product provided by the present invention is significantly different from the known Bt Cry protein. This new gene can be applied to the transformation of microorganisms and plants, making them highly toxic to related pests, and overcoming and delaying the emergence of pest resistance to engineering bacteria and transgenic plants.

The nucleotide sequence of the Bacillus thuringiensis cry8Ia1 gene highly effective against coleopteran pests is shown in SEQ ID NO1.

An engineering bacteria strain BioT8I is characterized in that it contains a cry8Ia1 gene.

An expression vector pSTK-8I is characterized in that it is constructed by cry8Ia1 gene sequence and shuttle vector pSTK.

The Bacillus thuringiensis cry8Ia1 protein highly effective against coleopteran pests is encoded by the cry8Ia1 gene, and its amino acid sequence is shown in SEQ ID NO2.

Application of cry8Ia1 gene in plant resistance to coleopteran pests.

The application is characterized in that the cry8Ia1 gene is transformed into plants or microorganisms to produce toxicity against coleopteran pests.

The application is characterized in that the protein expressed by the cry8Ia1 gene is made into a medicament for killing coleopteran pests.

The present invention isolates the wild strain BT-SU4 from the soil in Hebei, and its preservation number is CGMCC2071 (see patent application 200710120020.2). Its biological characteristics are that it can produce spores during the growth cycle, and at the same time produce toxic parasporal crystals.

Two pairs of universal primers were designed according to the conserved region of the cry8 gene, and the 5-terminal primers of the two pairs of primers were the same:

S8Ca5:5`-GATGAGTCCGAATAATCAGAATGAAT-3`

S8Ca3:5`-TTACTCTTTCTTCTAACACGAGTTC-3`

S8Ea3: 5`-TTACTCTACGTCAACAATCAATTC-3`

The wild strain BT-SU4 was identified by PCR amplification, and the bands were amplified with S8Ca5 and S8Ea3 primer pairs, and analyzed by enzyme digestion with ScaI. The results of PCR-RFLP showed that the bands were different from the known cry8 genes, indicating that the wild Strain BT-SU4 contains a novel cry8 insecticidal gene.

A pair of full-length gene primer cry8I5/cry8I3 was designed to amplify the full-length gene, and the cry8I5 primer was introduced into EcoRI for cloning and expression. The sequence of the primer pair cry8I5/cry8I3 is as follows:

cry8I5: 5-G ATG AGT CCG AAT AAT CAG AAT

cry8I3: CTC ATT TCT TCT ACA ATC AAT TCT ACA CTG TC

Using the total DNA of the wild strain BT-SU4 as a template, PCR amplification was carried out with high-fidelity DNA polymerase KOD-PLUS (product of Japan TOYOBO, purchased from Beijing Dingguo Biotechnology Co., Ltd.), and the results showed that the amplified The band of about 3.6kb was ligated with the blunt end of the cloning vector pBluescrip, and transformed into Escherichia coli JM110 to obtain the recombinant plasmid pB8I.

Sequencing analysis of the inserted fragment (as shown in SEQ ID NO 1) shows that it contains an open reading frame, the position of ORF1 is 1-3585 bp, the GC content is 38%, and it encodes a protein consisting of 1195 amino acids. After determination, its amino acid sequence is shown in SEQ ID NO 2. Homology analysis showed that the protein had high homology with Cry8 proteins. Since the amino acid homology with known Cry8 proteins is lower than 78%, the highest is only 55% (Cry8Ha1), it was named Cry8Ia1 by the Bt insecticidal crystal protein nomenclature committee.

A pair of primers were designed according to the 3585bp nucleotide sequence of the full-length cry8Ia1 gene. The primer cry8I5 was introduced into the EcoRI site, and the blunt end of cry8I3 was amplified by KOD-PLUS. The full-length gene was amplified using the wild strain BT-SU4 plasmid DNA as a template. , inserted into the Bt expression vector pSTK (8.5kb) cut by EcoRI, Eco1CR I (producing blunt ends), (this plasmid comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and can be provided to the public), and the recombinant The plasmid pSTK-8I was transformed into Escherichia coli SCS110, the plasmid was extracted, and transformed into the Bt amorphic mutant strain HD- ^73- (the The strains come from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, see Li Haitao et al., Journal of Agricultural Biotechnology 2005 Vol.13No.6P.787-791, available to the public). A variety of molecular tests were carried out on the positive transformants that could grow on the kanamycin-resistant plate, which proved that the transformation was successful, and the engineering bacteria BioT8I was obtained.

The above-mentioned engineering bacteria BioT8I was cultured in beef extract medium, and the protein was extracted for SDS-PAGE electrophoresis analysis. The results showed that the cry8Ia1 gene in the engineering bacteria BioT8I was expressed, and the molecular weight of the expressed product was about 135kDa, and it could form ellipsoidal crystals. According to the results of Western blot analysis, the cry8Ia1 gene can also normally express the target protein in the wild strain BT-SU4.

The Bt engineering strain BioT8I was inoculated in the liquid medium of beef extract protein building for 2 days, and the wild strain BT-SU4 was inoculated on the liquid medium of beef extract protein building for 3 days. Centrifuge and freeze-dry the bacterium, dilute with 2-fold gradient concentration, add 90ml of bacterial suspension to 500g of fine soil (sterilized by ultraviolet light) with uniform thickness of potato shreds, mix well, keep the soil water content at 18%, insert 45 scarab instar larvae were treated with adding water as a blank control, infected and reared at 28°C, and the number of dead insects was checked after 14 days to calculate the mortality rate. The activity determination of Cry8Ia1 protein showed that the expressed Cry8Ia1 had the activity of killing the larvae of the black beetle.

According to the routine methods of those skilled in the art, relevant microorganisms and plants can be transformed according to the cry8Ia1 gene, especially crops that are vulnerable to coleopteran pests, so that they can produce resistance, which can overcome or delay the resistance of insects to engineering bacteria and transgenic plants. The production of medicinal properties to increase the yield of crops. Through the combination of cry8Ia1 gene and cry1Ah, cry1Ba, cry3Aa7, cry2Ab and other gene expression products, the insecticidal spectrum against Lepidoptera, Coleoptera and Diptera pests can be expanded.

Description of drawings

Figure 1: PCR-RFLP profile of the wild strain BT-SU4. in:

M: Lambda DNA/Eco130I (bp): 19329, 7743, 6223, 4254, 3472, 2690, 1882, 1489, 925, 421;

1-5: PCR-RFLP of wild-type strains BT-SU4, cry8Ca2, cry8Ea1, cry8Fa1 and cry8Ga1

Figure 2: Restriction map of recombinant plasmid pB8I. in:

M: Lambda DNA/Eco130I (bp): 19329, 7743, 6223, 4254, 3472, 2690, 1882, 1489, 925, 421;

1:8I PCR product

2: Digestion of the recombinant plasmid pB8I EcoR I

3: Digestion of vector pBluscrip EcoR V

Figure 3: Restriction map of recombinant plasmid pSTK-8I. in:

M: Lambda DNA/Eco130I (bp): 19329, 7743, 6223, 4254, 3472, 2690, 1882, 1489, 925, 421;

1: PCR product of cry8Ia1 full-length gene

2: EcoR I single enzyme digestion of recombinant plasmid pSTK-8I

3: pSTK empty vector EcoR I single cutting

4: EcoR I and Sal I double digestion of recombinant plasmid pSTK-8I

5: pSTK-8I recombinant plasmid

Figure 4: Expression of cry8Ia1 gene in Bt amorphous mutant. in:

M: protein molecular weight standard (high range-212kDa, 116kDa, 97kDa, 66kDa, 45kDa)

1: HD- ^73-

2: BioT8H

3: BioT8I

4: BT-SU4

Figure 5: SEM results of spore crystals of wild strain BT-SU4 and engineered strain Bt BioT8I,

A is the wild strain BT-SU4, B is the engineered strain Bt BioT8I;

Figure 6: Detection of expression of cry8I gene in BT-SU4 strain by Western hybridization

A: SDS-PAGE;

B: Western blot;

H: protein molecular weight standard (high range-212 _kDa , 116 _kDa , 97 _kDa , 66 _kDa , 45 _kDa );

1: Wild strain BT-SU4;

2: Cry8H;

3: HD- ^73- ;

4: Purified Cry8I digested by protease;

Detailed ways

Examples of the present invention are described below. It should be noted that the embodiments of the present invention are only illustrative but not limiting to the present invention.

Example 1, identification of cry gene in wild strain BT-SU4

Two pairs of universal primers were designed according to the conserved regions of cry8 genes. According to the current database analysis, all cry8 genes can be amplified with two pairs of primers, S8Ca5/S8Ca3 and S8Ca5/S8Ea3. The primer sequences are as follows:

S8Ca5:5`-GATGAGTCCGAATAATCAGAATGAAT-3`

S8Ca3:5`-TTACTCTTTCTTCTAACACGAGTTC-3`

S8Ea3: 5`-TTACTCTACGTCAACAATCAATTC-3`

Table 2 shows the homologous sequences of these genes and primers, and Table 3 shows the size of the digested fragments of cry8 gene amplified products predicted by this pair of primers. These genes can be identified respectively by this PCR-RFLP method.

Table 2 The pairing of the primers with the conserved regions of cry8 genes and the position of the paired regions on the genes

Table 3 PCR amplification products and restriction enzyme length polymorphisms of cry8

Make up to 50 μL with ultrapure water, mix well and centrifuge, add 30 μL of paraffin oil.

Amplification cycle: denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 4 minutes, 30 cycles, and finally extension at 72°C for 10 minutes.

Utilize ScaI digestion analysis to PCR product, result (accompanying drawing 1) shows that band is 3585bp, is different from the map (table 3) of known cry8 class gene, shows that bacterial strain wild may contain new cry8 insecticidal gene. Embodiment 2. Cloning of cry8I gene in wild strain BT-SU4

A pair of full-length gene primers cry8I5/cry8I3 was designed to amplify the full-length gene. And the introduction of an EcoRI restriction site at the 5-end for cloning and expression, the sequence of the primer pair cry8I5/cry8I3 is as follows:

cry8I5: gg aat tcg atg agt ccg aat aat cag aat

cry8I3: cgc gtc gac tta cat ttc ttc tac aat caa ttc

Using the total DNA of the wild strain BT-SU4 as a template, use KOD-PLUS DNA polymerase (product of TOYOBO, Japan, purchased from Beijing Dingguo Biotechnology Co., Ltd.) to perform PCR amplification with the following system:

10×PCR buffer 5μL dNTP (10mM) 1μL Primer pair (10mM) 1μL/piece template 1uL KOD-PLUS DNA Polymerase (5U/μL) 0.5μL

Make up to 50 μL with ultrapure water, mix well and centrifuge, add 30 μL of paraffin oil.

Amplification cycle: Denaturation at 95°C for 5 minutes, annealing at 54°C for 1 minute, extension at 68°C for 3 minutes and 50 seconds, 30 cycles, and finally extension at 68°C for 10 minutes. The result (see attached figure 2) shows that a band of about 3.6 kb was amplified , and transformed into E. coli JM110 by connecting with the blunt end of the vector pBluscrip to obtain the positive transformant pB8I. The transformant pB8I was digested with EcoRI, and two bands of about 3kb and 3.6kb were obtained, indicating that cry8I had been successfully inserted into the pBluscrip vector, and it was inserted in the forward direction. Sequence analysis was performed on the inserted fragment, and the sequence SEQ ID NO 1 was obtained. The full length of the sequence was 3585bps. The analysis showed that it contained an open reading frame, the position of ORF1 was 1-3585, the GC content was 51%, and it encoded a protein consisting of 1195 amino acids. After determination, its amino acid sequence is shown in SEQ ID NO 2. The results of Blast show that Cry8Ia1 has three conserved domains and loop of Cry-like proteins, and the corresponding sequence is as follows:

Domain I (70-291):

FAGLSITAKI MDAFDVPGGDIFNGLLLEIIG ILWDLQDDTW EAFMEQVEVL IDQKIAEYAR

NLALTNLKGL ENSYKLYLEA LADWKQNPTS SSQERVRTR FRDTDDDSLTV FMPSFAVKGY

EVPLLAVYAQ AANLHLLLLR DSVAYGLGWG LSQLNVNDNY NRQVRLTGEY TNHCVSWYTT

GLEKLRGSNA QSWIKFNRYR REMTVMVLDI VALFPNYDAR R

Loop I: YPQ

Domain II (296-516):

ATTTEL TRLIYTDPHG YTIYSPTSQT TIPWYEYGQS FSEIENVAIQ APRLFRWAQE MQIYTKFVRH

APQESHYWSA HTFSFHHTRD NTKTTLTYGD TSDPISVGTA DLSDLDIYKV SSLVASSWGS

GVRLLVTKAK FEVIYTFNQL WEFNYEPPGI SNFFNQWKNT DTELPIQIVD PPTFGDPNQY

SHRVAYISHA PIQPYSGAFR NYGLVPVFGW SHVSVD

Loop II: RNNT LYADRI

DomainIII (526-665):

TQIP AVKAVQLSGE PYPVVRGPGF TGGDIARLPN NPNVGLLFNS KVESPSENKR YRVRIRYACA

SGARAIFGGL

Homology analysis of 601 YLPITVQFNQ TMSTTTPTRY EDFQYVDVSG SFILGNTNVS FSLVPQSANQ TNNLFIDKIE FIPEN showed that this protein had high homology with Cry8 proteins, and Table 4 was its homology data. Since the amino acid homology with known Cry8 proteins is lower than 78%, the highest is only 55% (Cry8Ha1), it was named Cry8Ia1 by the Bt insecticidal crystal protein nomenclature committee.

Table 4 Homologous comparison data of Cry8Ia1 and Cry8 proteins

Cry8Aa1 Cry8Ba1 Cry8Ca1 Cry8Da1 Cry8Ea1 Cry8Fa1 Cry8Ga1 Cry8Ha1 Cry81a1 48% 48% 48% 47% 49% 49% 50% 55%

The present invention further analyzes the amino acid composition of the Cry8Ia1 protein (see Table 6), and learns that its molecular weight is 135.51kDa, and the isoelectric point is pH4.93 (see Table 5), and the biochemical index of the protein is analyzed (see Table 5)

Table 5 Biochemical properties of Cry8Ia1 protein

analysis content data full length protein 1195aa molecular weight 135514.11mw 1 mg protein moles 7.379 pMoles 1 molar protein racemization coefficient 189470 1od A₂₈₀ protein concentration 0.72mg/ml 1mg/ml A₂₈₀ absorption value 1.40AU Isoelectric point 4.93 Charged value at pH7 -41.55

Table 6 Amino acid composition of Cry8Ia1 protein

amino acid number mass percentage frequency percentage Charged (RKHYCDE) 332 32.90 27.78

Acidic (DE) 144 12.87 12.05 Basic (KR) 101 10.53 845 Polar (NCQSTY) 409 34.57 34.23 Hydrophobic (AILFWV) 384 31.42 32.13 A Ala 68 3.86 5.69 C Cys 6 0.46 0.50 D Asp 70 5.93 5.86 E Glu 74 6.93 6.19 F Phe 49 5.15 4.10 G Gly 68 3.25 5.69 H His 18 1.78 1.51 I Ile 68 5.68 5.69 K Lys 38 3.54 3.18 L Leu 93 7.77 7.78 M Met 17 1.62 1.42 N Asn 94 7.91 7.87 P Pro 53 3.89 4.44 Q 62 5.77 5.19 R Arg 63 6.99 5.27 S Ser 89 5.96 7.45 T Thr 95 7.21 7.95 V Val 87 6.49 7.28 W Trp 19 2.47 1.59 Y Tyr 63 7.27 5.27 B Asx 164 13.84 13.72 wxya 136 12.70 11.38

Example 3, construction of cry8Ia1 full-length gene shuttle vector

Design primers cry8I5/cry8I3, in which the 5-primer is added with an EcoR I restriction site, and the 3-end is amplified by high-fidelity KOD-PLUS polymerase. The PCR amplification conditions are 94°C for 1 minute, 54°C 1 minute, 3 minutes at 68°C, 30 cycles, extension at 68°C for 10 minutes, amplified with high-fidelity KOD-PLUS polymerase. A 3.6kbcry8Ia1 full-length fragment was obtained, as shown in lane 1 in Fig. 3 . After the complete reaction of EcoR I enzyme digestion, electrophoresis was carried out, and the fragment was recovered from the gel, and the Bt-E.Coli shuttle vector pSTK vector (this plasmid came from China Agricultural Science and Technology Co., Ltd. Biotechnology Laboratory, Institute of Plant Protection, Academy of Sciences, available to the public) Ligation reaction, 4°C, 12 hours; take 5 μL of the ligation product to transform into E.coli recipient strain JM110, and use a kanamycin resistance plate to screen for positive recombinant plasmids. The obtained positive recombinant plasmids were respectively inoculated into liquid LB test tubes, cultured at 37°C and 230rpm for 12 hours, and the plasmids were extracted by the commonly used alkaline hydrolysis method. Fig. 3), the enzyme digestion result is consistent with the sequence prediction result of the software, indicating that it is a positive transformant, and the clone is sequenced, and the result shows that the constructed expression vector is correct, named pSTK-8I.

Embodiment 4, the construction of Bt engineering bacteria BioT8I and the detection of expressed protein

The recombinant expression clone pSTK-8I from JM110 was transformed into the E.coli methylated mutant strain SCS110 by electric shock method. The electric shock conditions were: 2500V, 400Ω, 25μF, 100μL competent cells, and 1μL plasmid DNA. Incubate at 37°C for 1 hour, 100 rpm; extract the demethylated pSTK-8I plasmid from SCS110.

Preparation of Bt crystal-free mutant strain HD-73 ^- competent (this bacterial strain comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, see Li Haitao et al., Journal of Agricultural Biotechnology 2005Vol.13No.6P.787-791, available to the public Provided), the demethylated pSTK-8I plasmid was introduced into it by electric shock transformation method. Electric shock conditions: 2200V, 1000Ω, 25μF, 100μL competent cells, 1μL plasmid DNA. Incubate at 30° C. for 2 hours, 100 rpm; select positive transformants with 50 μg/mL kanamycin plate. After extracting the plasmid, restriction analysis, PCR amplification and other molecular tests, a Bt-positive transformant containing the pSTK-8I expression plasmid was obtained, and the transformant was named as the engineering bacterium BioT8I.

The Bt engineering strain BioT8I was inoculated in the beef extract protein building liquid medium and cultured for 48 hours, and the wild strain BT-SU4 was inoculated on the beef extract protein building liquid medium and cultured. When the release of spores and the formation of crystals were observed, 1000 μL of bacteria Collect the bacteria by centrifugation, suspend after washing with ultrapure water once, take 10 μL of cell crystal mixture for observation and recording by scanning electron microscope, the results (accompanying drawing 5) show that the cry8I gene can be expressed normally in the Bt crystal-free mutant strain HD- ^73- , forming ellipsoidal crystals. The other suspensions were centrifuged again, suspended by adding 50 μL of ultrapure water, adding 25 μL of 3× sample buffer (925 μL of loading buffer + 75 μL of β-mercaptoethanol), and boiling at 100°C for 10 minutes. The precipitate was removed by centrifugation. Load 10uL of the sample for SDS-PAGE electrophoresis analysis. The results (accompanying drawing 4) showed that all the cry8Ia1 genes in the engineering bacteria BioT8I were expressed, and the molecular weight of the expressed product was about 135 kDa.

In order to further prove that the cry8Ia1 gene was expressed in the wild strain BT-SU4, Western hybridization analysis was carried out. The wild strain BT-SU4, the engineered strain BioT8I and the control strain were boiled at 100°C for 10 min, followed by SDS-PAGE, and Western hybridization with the prepared and purified Cry8I protein antibody. The results of Western hybridization showed that there were hybridization bands in the BT8 strain, but there were no hybridization bands in Cry8H and HD- ⁷³ as negative controls. The results of this experiment show that the cry8Ia1 gene can express a protein of 135kDa in the wild strain BT-SU4 (Fig. 6).

The insecticidal activity assay of embodiment 5, Cry8I protein

The Bt engineering strain BioT8I was inoculated in the liquid medium of beef extract protein building for 2 days, and the wild strain BT-SU4 was inoculated on the liquid medium of beef extract protein building for 3 days. Centrifuge and freeze-dry the bacterium, dilute with 2-fold gradient concentration, add 90ml of bacterial suspension to 500g of fine soil (sterilized by ultraviolet light) with uniform thickness of potato shreds, mix well, keep the soil water content at 18%, insert 45 scarab instar larvae were treated with adding water as a blank control, infected and reared at 28°C, and the number of dead insects was checked after 14 days to calculate the mortality rate. The results (see Table 7) show that the engineering strain Holotrichia parallela has very high poisonous activity.

Table 7 Determination of the insecticidal activity of Bt engineering bacteria and wild strain BT-SU4 strains on the larvae of the black gill beetle (Holotrichia parallela)

Note: In the concentration units of the results, the concentration is X mg of bacterial powder per gram of soil.

Attachment: DNA sequence and protein sequence involved in the present invention

SEQ ID NO 1 (nucleotide sequence of cry8Ia1 gene)

 AGTCCGA ATAATCAGAA TGAGTTTGAT ATTATAGATG TACCATCTAA TATTTCTGTA1

AGTCCGA ATAATCAGAA TGAGTTTGAT ATTATAGATG TACCATCTAA TATTTCTGTA

61 TCCAATAGTT TTGTTAGATA TCCTTTCGCA AATGATCCCA ATCGTACATT ACAAAATACA

121 AATTATAAAG ATTTTTTAAC AATGTCTGAA AAATCTAATT CTGGATATTT AACAGATCCT

181 GAAGCTTTTG ATGATGTCGG TTCAGCGATT TTTGCTGGAC TTAGTATTAC TGCTAAAATC

241 ATGGATGCTT TCGATGTTCC TGGGGGAGAC ATATTTAATG GGTTACTTGA AATCATTGGT

301 ATCCTCTGGG ATCTTCAAGA TGACACATGG GAAGCTTTTA TGGAACAAGT AGAAGTACTG

361 ATTGATCAAA AAATAGCAGA GTATGCAAGA AATCTTGCAC TTACAAATTT AAAAGGATTA

421 GAAAATAGTT ATAAATTATA TTTAGAAGCA CTAGCAGATT GGAAACAAAA TCCTACTAGT

481 CCAAGTTCTC AAGAACGTGT AAGAACAAGG TTCCGCGATA CTGATGATTC TTTAACAGTA

541 TTTATGCCAT CTTTTGCAGT TAAGGGATAT GAAGTTCCCT TATTAGCAGT ATATGCGCAA

601 GCTGCGAATC TGCATTTATT GTTATTGAGA GATTCCGTTG CTTATGGATT AGGATGGGGG

661 CTTTCCCAAC TTAATGTGAA TGATAATTAT AATCGACAAG TACGACTCAC AGGGGAATAT

721 ACAAATCATT GTGTATCATG GTATACAACG GGTTTAGAAA AATTAAGAGG TTCGAATGCT

781 CAGAGTTGGA TTAAATTTAA TCGTTATCGT AGGGAAATGA CAGTGATGGT ACTAGATATA

841 GTTGCTTTAT TTCCTAATTA TGATGCGCGT AGATACCCTC AAGCAACGAC TACAGAACTA

901 ACCAGATTAA TTTATACGGA TCCACATGGT TATACAATTT ATTCACCAAC TTCTCAAACA

961 ACCATACCGT GGTATGAGTA TGGCCAATCT TTCTCAGAAA TAGAAAATGT TGCAATTCAG

1021 GCGCCTAGAC TTTTTAGGTG GGCGCAAGAA ATGCAGATTT ATACAAAATT TGTAAGACAC

1081 GCCCCACAAG AATCTCATTA TTGGTCAGCC CATACCTTTT CATTTCATCA TACTAGAGAT

1141 AATACCAAAA CCACACTAAC ATATGGAGAT ACTTCAGATC CTATATCTGT GGGTACAGCT

1201 GATCTAAGCG ATTTAGATAT ATATAAAGTT TCATCATTGG TTGCATCGAG TTGGGGGTCA

1261 GGAGTTAGAT TACTTGTTAC TAAAGCTAAA TTTGAGGTTA TTTATACATT TAACCAATTA

1321 TGGGAATTTA ATTATGAACC TCCAGGCATA TCCAATTTTT TTAATCAATG GAAAAATACT

1381 GACACAGAAT TACCTATCCA GATAGTCGAC CCACCTACTT TTGGAGACCC TAATCAGTAT

1441 AGTCATAGGG TAGCTTATAT TTCCCACGCT CCAATACAAC CATATTCAGG GGCTTTTAGA

1501 AATTATGGGT TGGTTCCTGT ATTTGGATGG TCGCATGTGA GTGTGGATAG AAATAATACA

1561 CTTTATGCAG ACAGAATTAC TCAAATTCCT GCGGTGAAAG CTGTACAGTT AAGTGGTGAA

1621 CCATATCCAG TTGTACGTGG ACCTGGATTT ACTGGAGGAG ATATAGCCCG ATTACCTAAT

1681 AATCCAAACG TAGGATTATT GTTTAATAGT AAAGTAGAAT CACCTTCAGA AAATAAGAGA

1741 TATCGTGTAC GAATAAGGTA CGCTTGTGCT TCTGGAGCTC GAGCAATTTT TGGTGGATTA

1801 TACTTACCTA TAACAGTCCA ATTTAACCAA ACCATGTCGA CTACTACTCC TACAAGATAT

1861 GAAGATTTTC AATATGTAGA TGTAAGTGGC TCTTTTATAT TAGGAAATAC TAATGTAAGT

1921 TTTTCTTTAG TACCCCAAAG TGCTAATCAA ACTAATAATT TATTTATTGA TAAAATTGAA

1981 TTTATTCCAG AAACCCAGC ACTTGAAGCA GAAAGCGATT TAGAGGCTGC AAAGAAAGCA

2041 GTAAATGCCT TGTTTACGAA TAGTAAAGAT ACTTTACAGA TAGGTGTGAC AGACTATCAA

2101 ATCAATCAAG CTGCAAATTT AATTGAATGC GTATCCGATG AGGTATATCC AAATGAAAAG

2161 CGATTGTTAT TTGATGCAGT AAAAGAGGCA AAACGACTTA GTACGGCACG TAACTTGCTT

2221 GAAGATACGG ATTTTCATAC AATAAATGGG GGAAATGGAT GGACGGGAAG TACGGGTATT

2281 GAAATTGTGG AAGGAGATAT TCTCTTTAAA GATCGCTCGC TTCGACTTCC AAGTGCGAGA

2341 GAAATAGAGA GAGAAACTTA TCCAACATAT ATCTATCAAA AAATAGAGGA ATCACGATTA

2401 AAACCAAATA CAAGATATAG TTTGAGAGGG TTTATCGGAA GTAGCCAAGA TTTAGAGATA

2461 TATGTCCTTC GTCATCAGGC ATATCGTGTT ATTAAAAATG TTTCAGATAA TCTGTTACCA

2521 AATATGCGCC CTATCAACGC TTGTGGAGGA GTTGACCGAT GCAGTCAACA AAAATATGTG

2581 AATAATTCAT TAGAAGTGAA TAGCGGTTTA TCAAATGGAA TTGGAGCGGC TGACTCTCAT

2641 GAGTTTTTCAA TTCATGTGGA TACAGGAGAA CTGAATTATA ATGAGGATAT GGGTATTGGG

2701 GTTGTATTTA AAATCACAAC TACAGATGGG TACGCAACAG TAGGGAATAT TGAATTGGTG

2761 GAAGTGGAAA CGTTATCCGG AGAAGCATTA GAACGTGTGC AAAGACAAGA AAAGCAGTGG

2821 CAGGGCCAAT TGGCGACAAG ACGTAAAGAA ACAGAAACGA GATACGGGCC AGCAAAAACAA

2881 GCTATTGATC GTTTATTCGT AGATTTTCAA GATCAACAAC TATCTCGTAG TACAGAAATC

2941 TCAGATTTGA CGGAAGCGCA AAATCTAGTA CAGTCTATTC CTTACGTATA CAATGATATG

3001 TTACCAGAAA TCCCGGGAAT GAACTATACC AGTGTTACAG AGTTAACAAA CAGACTTCAA

3061 CAAGCATGGA ATTTGTATGA CCAGAGAAAT AGCATACAAA ATGGTGATTT CCGAAATGAT

3121 GTAAGTAATT GGAATGTTAC AACTGAAGTC AATATTCAAC AAATGAATGA TACGTCTGTC

3181 CTTGTGATTC CAAATTGGGA TTCGCAAGTG TCACAACAAA TTACCGTTCA ACCAAATCGA

3241 AGATATGTGT TACGTGTTAC CGCAAGAAAA GAAGGAAATG GAGATGGCTA TGTAACCATC

3301 CGTGATGGAG CAAACCATAC AGAAACGCTG ACATTTAATA CATGTGATTA TGATGGAAAT

3361 AGTGTATATA ATAATCAACC ATTAAATGCA AATAATAATG TATATACTAC AAAATCATCA

3421 AATACCAATA GCTATCATAC AAATGGTGTG TATCATGACC AAACTAGCTA TGTTACAAAA

3481 ACAATGGAAT TTACTCCGTA TACGGAGCAA GTCTGGGTTG AGATGAGTGA AACAGAAGGA

3541 GTATTTTATA TAGACAGTGT AGAATTGATT GTAGAAGAAA TGTAA

SEQ ID NO 2 (amino acid sequence of Cry8Ia1 protein):

1 MSPNNQNEFD IIDVPSNISV SNSFVRYPFA NDPNRTLQNT NYKDFLTMSE KSNSGYLTDP

61 EAFDDVGSAI FAGLSITAKI MDAFDVPGGDIFNGLLLEIIG ILWDLQDDTW EAFMEQVEVL

121 IDQKIAEYAR NLALTNLKGL ENSYKLYLEA LADWKQNPTS SSQERVRTR FRDTDDSLTV

181 FMPSFAVKGY EVPLLAVYAQ AANLHLLLLR DSVAYGLGWG LSQLNVNDNY NRQVRLTGEY

241 TNHCVSWYTT GLEKLRGSNA QSWIKFNRYR REMTVMVLDI VALFPNYDAR RYPQATTTEL

301 TRLIYTDPHG YTIYSPTSQT TIPWYEYGQS FSEIENVAIQ APRLFRWAQE MQIYTKFVRH

361 APQESHYWSA HTFSFHHTRD NTKTTLTYGD TSDPISVGTA DLSDLDIYKV SSLVASSWGS

421 GVRLLVTKAK FEVIYTFNQL WEFNYEPPGI SNFFNQWKNT DTELPIQIVD PPTFGDPNQY

481 SHRVAYISHA PIQPYSGAFR NYGLVPVFGW SHVSVDRNNT LYADRITQIP AVKAVQLSGE

541 PYPVVRGPGF TGGDIARLPN NPNVGLLFNS KVESPSENKR YRVRIRYACA SGARAIFGGL

601 YLPITVQFNQ TMSTTTPTRY EDFQYVDVSG SFILGNTNVS FSLVPQSANQ TNNLFIDKIE

661 FIPENPALEA ESDLEAAKKA VNALFTNSKD TLQIGVTDYQ INQAANLIEC VSDEVYPNEK

721 RLLFDAVKEA KRLSTARNLL EDTDFHTING GNGWTGSTGI EIVEGDILFK DRSLRLPSAR

781 EIERETYPTY IYQKIEESRL KPNTRYSLRG FIGSSQDLEI YVLRHQAYRVIKNVSDNLLP

841 NMRPINACGG VDRCSQQKYV NNSLEVNSGL SNGIGAADSH EFSIHVDTGE LNYNEDMGIG

901 VVFKITTTDG YATVGNIELV EVETLSGEAL ERVQRQEKQW QGQLATRRKE TETRYGPAKQ

961 AIDRLFVDFQ DQQLSRSTEI SDLTEAQNLV QSIPYVYNDM LPEIPGMNYT SVTELTNRLQ

1021 QAWNLYDQRN SIQNGDFRND VSNWNVTTEV NIQQMNDTSV LVIPNWDSQV SQQITVQPNR

1081 RYVLRVTARK EGNGDGYVTI RDGANHTETL TFNTCDYDGN SVYNNQPLNA NNNVYTTKSS

1141 NTNSYHTNGV YHDQTSYVTK TMEFTPYTEQ VWVEMSETEG

1181 VFYIDSVELI VEEM ^*

Claims (5)

1. to coleopteran pest bacillus thuringiensis cry8Ia1 gene efficiently, its nucleotide sequence is shown in SEQ ID NO.1.

2. an engineering bacterial strain BioT8I is characterized in that containing the described cry8Ia1 gene of claim 1.

3. a shuttle expression carrier pSTK-8I is characterized in that by described cry8Ia1 gene order of claim 1 and shuttle vectors pSTK constructed.

4. to coleopteran pest bacillus thuringiensis Cry8Ia1 albumen efficiently, by the described cry8Ia1 coded by said gene of claim 1, its aminoacid sequence is shown in SEQ ID NO.2.

5. the application of the described cry8Ia1 gene of claim 1 in the anti-coleopteran pest of plant, it is characterized in that the albumen of described cry8Ia1 genetic expression is made medicament, be used to murder coleopteran pest, described coleopteran pest refers to Holotrichia parallela (Holotrichia parallela).

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