CN101113424B

CN101113424B - Coleoptera pest efficient Bacillus thuringiensis cry8G gene, protein and uses thereof

Info

Publication number: CN101113424B
Application number: CN2007101182897A
Authority: CN
Inventors: 束长龙; 宋福平; 冯书亮; 张�杰; 王容燕; 黄大昉; 郎志宏
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences; Institute of Plant Protection Hebei Academy of Agricultural and Forestry Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences; Institute of Plant Protection Hebei Academy of Agricultural and Forestry Sciences
Priority date: 2007-07-04
Filing date: 2007-07-04
Publication date: 2010-12-29
Anticipated expiration: 2027-07-04
Also published as: WO2009003412A1; CN101113424A

Abstract

The invention relates to a highly effective bacillus thuringiensis cry8G gene and albumen for killing coleoptera pests and application thereof, pertaining to biological control technical field. The invention provides nucleotide sequences of the bacillus thuringiensis cry8G gene which is high poisonous to the coleoptera pests and amino acid sequences of the codes of the protein, and provides nucleotide sequences of the bacillus thuringiensis cry8G gene which is designed artificially and used for transgenic plants and amino acid sequences of the codes thereof. Therefore, toxicity to related pests is presented by the sequences through the genes or the artificial designed sequences of transgenic microbes and plants so as to overcome or delay the generation of drug-resistance of the pests for engineering bacteria and the transgenic plants.

Description

To coleopteran pest bacillus thuringiensis cry8G gene, albumen and application thereof efficiently

Technical field

The invention belongs to the biological control technical field, the present invention relates to nucleotide sequence to the cry8G gene of the high virulence of coleopteran pest, relate to proteinic aminoacid sequence to the high virulence of coleopteran pest, relate to, the nucleotide sequence of the cry8G gene that artificial design synthetic can be expressed in plant, the nucleotide sequence coded protein amino acid sequence that relates to the cr8G gene of this artificial design, relate to the recombinant bacterial strain that contains the cry8G gene, relate to and use this gene constructed expression carrier, also relate to and utilize the said gene sequence to carry out the method for Plant Transformation.

Background technology

Chafer belongs to Coleoptera Scarabaeoidea (Scarabaeidae), its larva (is commonly called as grub, also abbreviate " grub " below the present invention as) be the important worldwide distribution subterranean pest-insect of a class, can endanger various plants such as grain, cotton, oil crops, vegetables, sugar crop, tobacco, herbage, flowers, turfgrass, fruit tree.A large amount of investigation show that the harm of grub in subterranean pest-insect ranks first, and wherein mainly based on gill Scarabaeidae and Rutilidae larva, account for more than the 70-80% of total subterranean pest-insect amount.About 100,000,000 mu of area takes place in annual according to statistics national grub, and the serious time once reached 300,000,000 2 thousand ten thousand mu, and production loss is up to more than 20%, some plot even total crop failure.In recent years take place area maximum, generating capacity maximum be the Yellow River and Huai He River Haiti district, crops such as main harm grain, oil plant; Other geographic harm situation is also very serious, as the grub of harm sugarcane, in Guangdong, ground such as Guangxi, Yunnan, Sichuan, Fujian generally take place; In Tibet, west area such as Qinghai, Gansu, Xinjiang, the generation of grub also very serious (Wei Hongjun etc., " Chinese subterranean pest-insect ", Shanghai: Shanghai science tech publishing house, 1989,1-41; Wang Yongxiang etc., " region of no relief grub kind and integrated control technique in the Ji ", " Hebei Normal University's journal " (natural science edition), 1998,22 (2): 268-270).Being only second to rape with cultivated area in China oil crops, to occupy deputy peanut be example.The peanut yield of China accounts for about 35% of world's peanut ultimate production, occupies first place in the world, and a year outlet income reaches 20,700,000,000 dollars, calendar year 2001 whole nation peanut area (5,000,000 hectares) and gross output (1,450 ten thousand tons) all reach a record high.But grub is very serious to the harm of peanut.Be the harm of control grub, the general integrated control strategies such as agricultural, chemistry, physics that adopt though this has certain effect, are difficult to reach the effect of Sustainable Control.Therefore, seek the new method of effectively preventing and treating, become the task of top priority.

On the basis that obtains the high virulence Bt gene to grub, cultivating the transgenic plant that kill grub is new controlling ways that are worth exploration.

Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of distribution gram positive bacterium extremely widely.It is when forming gemma, can produce the parasporal crystal (parasporal crystal) of property of protein, to lepidopteran (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hymenoptera (Hymenoptera), Homoptera (Homoptera), Orthoptera (Orthoptera), Mallophaga various insects such as (Mallophaga), and nematode, mite class and protozoon have specific insecticidal activity (Schnepf, E.N.et al, Microbiol.And MolecularBiology Review, 1998,62:3775-806).(Insecticidal Crystal Proteins ICPs) claims delta-endotoxin (delta-endotoxin) again to this insecticidal crystal protein, and is free from environmental pollution to the person poultry harmless, thereby Bt has obtained using the most widely in the biological control of insect.

People have cloned the Bt killing gene of nearly 400 kinds of coded insect-killing crystallins at present, and they adhere to 157 kinds of pattern genes separately.In recent years the research trend of cry8 genoid is noticeable in the world.Studies show that this genoid has insecticidal action to multiple coleopteran pests such as Scarabaeidae, Culculionidae, Chrysomelidaes.1992, Ohba etc. filter out new bacterial strain (B.t.subsp.Japonensis the BuiBui) (Ohba that the chafer larva is had special insecticidal activity in the world first from the Bt bacterial strain, M.et al., A unique isolate of Bacillus thuringiensis serovar japonensis with a highlarvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology, 1992.14:54-57), Sato in 1994 etc. therefrom clone a kind of new killing gene cry8C (Sato, R.et al, Cloning, heterologous expression, and localization of a novel crystal protein genefrom Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeidinsects, Curr.Microbiol.1994.28:15-19.4).Found 11 kinds of cry8 genoids at present, encoded protein is made up of 1160-1210 amino acid, and molecular weight is between 128-137kDa.Detailed information sees Table 1 (Asano, S., Yamanaka, S.and Takeuchi, K., Protein having insecticidal activity, DNA encoding theprotein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186-A and JP 2002045186-A/2)).Wherein isolating Cry8Aa1 of U.S. Mycogen company and Cry8Ba1 have tangible insecticidal activity (Tracy E.Michaels to the various pests of Scarabaeidae, et al., Bacillusthuringiensis toxins active against scarab pests, 1994, USP5554534).The U.S. has separated two kinds of gene cry8Bb1 and cry8Bc1 gene from the Bt bacterial strain, discovery has significant insecticidal effect to west corn root leaf A (Western cornrootworm) and has been used for the exploitation (Abad of transgenic insect-resistant corn, Andre, R., DuckNicholas, B., Feng, Xiang, Flannagan Ronald, D., Kahn, Theodore, W., Sims, Lynne, E.Genes encoding novel proteins with pesticidal activity against coleopterans, 2002, WO 02/34774A2).In China; the screening successively in recent years of plant protection institute of Hebei province Academy of Agricultural Sciences and Agricultural University Of Hebei obtains the Bt bacterial strain that many strains have special insecticidal activity to yellowish-brown rutelian (Anomala exoleta) and anomala corpulenta (A.corpulenta) larva; the indoor biometrics mortality ratio all reaches 100% (Feng Shuliang etc.; " strain has the new strain isolated of Bacillus thuringiensis of insecticidal activity to cockchafer subclass larva "; " Chinese biological control "; 2000,16 (2): 74-78).

Table 1 bacillus thuringiensis Cry8 class insecticidal crystal protein

Summary of the invention

The invention provides a kind of bacillus thuringiensis cry8G pattern gene order that Coleoptera important pests such as big black gill cockchafer is had high virulence, to be applied to transform microorganism and plant, make it to show toxicity, and overcome, delay the resistance generation of insect engineering bacteria and transgenic plant to relevant insect.

Bacillus thuringiensis bacterial strain HBF-18, its preserving number are CGMCC2070.

To coleopteran pest bacillus thuringiensis cry8Ga1 gene efficiently, its nucleotide sequence is shown in SEQ ID NO1.

A kind of engineering bacterial strain BioT8G is characterized in that containing the cry8Ga1 gene.

To coleopteran pest bacillus thuringiensis cry8Ga1 albumen efficiently, by above-mentioned cry8Ga1 coded by said gene, its aminoacid sequence is shown in SEQ ID NO2.

Cry8Ga1 albumen is murdered application in the coleopteran pest medicament in preparation.

A kind of albumen has above-mentioned albumen identical functions, and its aminoacid sequence is shown in SEQ ID NO4.

A kind of artificial reconstructed synthetic mcry8Ga1 gene, the albumen that its coding is above-mentioned, its nucleotide sequence is shown in SEQ ID NO3.

A kind of plant expression vector pBSmGN is characterized in that this plant expression vector is constructed by mcry8Ga1 gene order, composition type expression promoter or root-specific promoter, terminator and a kind of binary vector that can shuttle back and forth in intestinal bacteria and agrobacterium tumefaciens.

The application of mcry8Ga1 gene in the anti-coleopteran pest of plant.

The described plant expression vector pBSmGN that will contain the mcry8Ga1 gene that is applied as transforms plant or microorganism, makes it to produce the toxicity of anti-coleopteran pest.

Described plant is a tobacco.

The described protein Preparation that is applied as mcry8Ga1 genetic expression becomes medicament, is used to kill coleopteran pest.

The present invention obtains bacterial strain HBF-18 from the separation of Hebei soil, and its deposit number is CGMCC2070, and its biological characteristics is for can produce the brood cell in growth cycle, and generation simultaneously has the parasporal crystal of toxic action.

According to cry8 genoid conserved regions design a pair of universal primer:

SN5un85`-GTCCGAATAATCAGAATGAATATG-3`

SN3un85`-CGTTTCGCCTCTCTCACTGCAT-3`

Pcr amplification is identified the HBF-18 bacterial strain, amplification (seeing accompanying drawing 1), and it shows that band is all different with known cry8 genoid (seeing Table 3), shows and may contain new cry8 killing gene among the bacterial strain HBF-18.

Design a pair of full-length gene primer cry8G5/cry8G3 and be used for the full-length gene that increases.And introduce BamHI/SalI and be used for cloning and expression, primer is as follows to the sequence of cry8G5/cry8G3:

BamHI

cry8G5：5′-CGC GGATCCGAAATGAGTCCGAATAATCA-3′

SalI

cry8G3：5′-ACGC GTCGACCTCTTCTTCTAACACGAGT-3′

Total DNA with bacterial strain HBF-18 is a template, uses the pfuDNA polysaccharase, carries out pcr amplification, and result's (seeing accompanying drawing 2) shows the band that amplifies a 3.5Kb, is connected transformed into escherichia coli JM110 with carrier pET21b, obtains recombinant plasmid pSAS018 (accompanying drawing 2).Carry out sequencing analysis to inserting segment, obtain sequence SEQ ID NO 1 and be BamHI/SalI double digestion fragment among the pSAS018, sequence total length 3472bps, it contains open reading frame analysis revealed, the position of ORF1 is 1-3472, and GC content is 38.%, the albumen that 1157 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in the SEQ ID NO 2.This albumen of homology analysis revealed and Cry8 proteinoid have higher homology, and table 4 is its homology data.Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 58.2% (Cry8Bb1), by the called after Cry8Ga1 of Bt insecticidal crystal protein NK.

Primer cry8G5/cry8G3 introduces BamHI and SalI site respectively, with bacterial strain HBF-18 plasmid DNA is template, amplification obtains full-length gene, insert among the Bt expression vector pSTK and obtain recombinant plasmid pSK018 (seeing accompanying drawing 3), transformed into escherichia coli SCS110, extract plasmid, electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In (this mutant strain derives from Plant Protection institute, Chinese Academy of Agricultral Sciences Biotechnology Experiment chamber, can provide to the public, sees Li Haitao etc., Journal of Agricultural Biotechnology 2005Vol.13No.6P.787-791), obtain engineering bacteria BioT8G.

(glucose 10 restrains for peptone 5 grams, extractum carnis 3 grams in the extractum carnis substratum in 30 ℃ with above-mentioned engineering bacteria BioT8G, water 1000mL, 121 ℃, 20 minutes high pressure steam sterilizations) the middle cultivation, extraction albumen carries out the SDS-PAGE electrophoretic analysis, and (method is referring to Sambrook, J.et al, Molecular Cloning:A Laboratory Manual, 2nd ed.Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.1989), result's (seeing accompanying drawing 4).The result shows that the cry8Ga1 gene among the engineering bacteria Biot8G has obtained expression, and the molecular weight of representation is about 130kDa.

The proteic determination of activity of Cry8Ga1 shows that the Cry8Ga1 of expression has the activity of killing holotrichia oblita and Holotrichia parallela larva.

According to different to codon preference of microorganism and plant, the sequence of the 1-2040bp of cry8Ga1 gene is optimized.The present invention has carried out full gene according to the artificial reconstructed sequence of cry8Ga1 gene and has synthesized, and new gene is seen the nucleotide sequence shown in the SEQ ID NO 3, and corresponding protein sequence is seen SEQ ID NO 4.The nucleotide sequence homology of cry8Ga1 gene and mcry8Ga1 (modified cry8Ca1) gene has only 86.88%, and G+C content also rises to 45.2% by 37.6% of original cry8Ca1.Adjusted the sub-frequency of utilization of the proteic amino acid code of Cry8Ga1, make the frequency of utilization in sub-frequency of utilization of the proteic amino acid code of mCry8Ga1 and the plant approaching, BamHI and KpnI, SacI site (seeing SEQ ID NO 3) are introduced in the artificial reconstructed sequence two ends of cry8Ca gene, (this carrier is a common carrier to be connected to the pUC57 carrier, the GenBank accession number is Y14837), recombinant plasmid called after pUC57-mcry8G.

When the Bt cry8G gene that synthetic is transformed, reclaim the 2.0kb fragment with BamHI and SacI digested plasmid pUC57-mcry8G, (this carrier is a common carrier, and the GenBank accession number is AF485783 with same endonuclease digestion plasmid pBI121.See Chen PY, et al, 2003, Mol.Breed 11:287-293), reclaims the 12kb fragment, and two fragments are connected, and transforms JM110, obtains positive transformant, this new structure plasmid called after pBSmGN.It (is the section of DNA sequence that this plasmid contains composition type expression promoter CaMV35S, can drive the gene fragment that is connected transcribes, and then translate into protein, the promotor of constitutive expression can regulatory gene all has expression in any stage of growing and any organizing), mcry8Ga1 gene and NOS terminator (the section of DNA sequence contains the termination signal of genetic expression).Plasmid construction figure sees accompanying drawing 5, and this plasmid can transform plant, obtains transgenic plant.

Gene mcry8Ga1 Agrobacterium-mediated Transformation with the synthetic transformation, prepare positive colony, transformation of tobacco again, the biological activity assay of transgene tobacco shows that transfer-gen plant has shown good anti-Holotrichia parallela (Holotrichiaparallela) performance.

Tobacco is a checking gene function model plant commonly used in the plant genetic engineering, the anti-Holotrichia parallela of transgene tobacco (Holotrichia parallela) performance is because there is promotor to start engineered mcry8Ga1 gene transcription in plant materials, expressed Cry8G albumen, expression cassette in the binary vector---constitutive promoter, engineered mcry8Ga1 and terminator only are incorporated in the genome of tobacco could expression alien gene, so can transform any plant of having set up conversion method for agrobacterium with this binary vector, the transgenic plant of acquisition all have anti-Holotrichia parallela (Holotrichiaparallela) performance.

Biological preservation information:

Microbial name (formal name used at school that belongs to, plants): bacillus thuringiensis Bacillus thuringiensis

Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center

Depositary institution address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City

Description of drawings

Preservation date: on June 1st, 2007

Deposit number: CGMCC No.2070

Fig. 1: the PCR-RFLP collection of illustrative plates of bacterial strain HBF-18.Wherein:

The M.DNA molecular weight standard

1.PCR the product enzyme is cut

Fig. 2: recombinant plasmid pSAS018 restriction enzyme mapping.Wherein:

The M.DNA molecular weight standard

1.PCR product B amHI/SalI enzyme is cut

2. carrier pET21bBamHI/SalI enzyme is cut

3. recombinant plasmid pSAS018BamHI/SalI restriction enzyme mapping

Fig. 3: recombinant plasmid pSK018 restriction enzyme mapping.Wherein:

The M.DNA molecular weight standard

1. carrier pSTK BamHI/SalI enzyme is cut

2.PCR product B amHI/SalI enzyme is cut

3. recombinant plasmid pSK018BamHI/SalI restriction enzyme mapping

Fig. 4: the cry8Ga1 gene does not have expression in the crystal mutant strain at Bt.Wherein:

M. protein molecular weight standard

1.HBF-18

2.Biot8G

3.HD-73 ^-

Fig. 5: pBSmGN plasmid construction figure

Fig. 6: the Molecular Detection of transformation of tobacco, wherein:

M. protein molecular weight standard

1,2. is the positive transfer-gen plant detected result of part

Embodiment

The present invention is described in further detail below in conjunction with embodiment.

Embodiment 1

1.1 cry gene identification in the bacterial strain 185

According to cry8 genoid conserved regions design a pair of universal primer

SN5un85`-GTCCGAATAATCAGAATGAATATG-3`

SN3un85`-CGTTTCGCCTCTCTCACTGCAT-3`

Table 2 is homologous sequences of these genes and primer, and table 3 is with this cry8 gene amplification product endonuclease bamhi size to the primer prediction, can identify respectively these genes by this PCR-RFLP method.

Table 2 primer and cry8 each gene conservative district pairing situation and position on gene, collochore

Pcr amplification product and the restriction enzyme digestion length polymorphism of table 3cry8

(50 μ L) identified Bt bacterial strain 185 with following PCR reaction system:

10×PCR?buffer 5μL

MgCl ₂(20mM) 6μL

dNTP(10mM) 1μL

Primer is to (10mM) 1 μ L/

Template 1uL

Taq polysaccharase (5U/ μ L) 0.5 μ L

Ultrapure water is mended to 50 μ L, and mixing is centrifugal, adds paraffin oil 30 μ L.

Amplification cycles: 94 ℃ of sex change 1 minute, 54 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.

To PCR product utilization KpnI and DraI restriction analysis, result's (accompanying drawing 1) shows that band is 1260bp, 570bp, and 320bp, different with the collection of illustrative plates (table 3) of known cry8 genoid, show and may contain new cry8 killing gene among the bacterial strain HBF-18.

1.2 the clone of cry8G gene among the bacterial strain HBF-18

BamHI

cry8G5：5′-CGC GGATCCGAAATGAGTCCGAATAATCA-3′

SalI

cry8G3：5′-ACGC GTCGACCTCTTCTTCTAACACGAGT-3′

Total DNA with bacterial strain HBF-18 is a template, uses the pfuDNA polysaccharase, carries out pcr amplification with following system.。

10×PCR?buffer	5μL?
		dNTP(10mM)?	1μL?
Primer is to (10mM)	1 μ L/
		Template	1uL?
PfuDNA polysaccharase (5U/ μ L)	0.5μL?

Amplification cycles: 94 ℃ of sex change 1 minute, 54 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.Result's (seeing accompanying drawing 2) shows the band that amplifies 3.5Kb, is connected transformed into escherichia coli JM110 with carrier pET21b, obtains positive transformant pSAS018.Carry out sequencing analysis to inserting segment, obtain sequence SEQ ID NO 1 and be BamHI/SalI double digestion fragment among the pSAS018, sequence total length 3472bps, it contains open reading frame analysis revealed, the position of ORF1 is 1-3472, and GC content is 38.%, the albumen that 1157 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in the SEQID NO 2.This albumen of homology analysis revealed and Cry8 proteinoid have higher homology, and table 4 is its homology data.Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 58.2% (Cry8Bb1), by the called after Cry8Ga1 of Bt insecticidal crystal protein NK.

Table 4Cry8Ga1 and Cry8 albumen homology comparative data

The present invention has further analyzed the proteic amino acid of Cry8Ga1 and has formed (seeing Table 6), learns that its molecular weight is 131.56kDa, and iso-electric point is pH4.735 (seeing Table 5), has analyzed proteic biochemical indicator (seeing Table 5)

The proteic biochemical characteristic of table 5Cry8Ga1

The proteic amino acid of table 6Cry8Ga1 is formed

1.3cry8G expression of gene

Primer cry8G5/cry8G3 introduces BamHI and SalI site respectively, with the bacterial strain HBF-18 plasmid DNA that contains total length cry8Ga1 is template, amplification obtains full-length gene, insert among the Bt expression vector pSTK (seeing accompanying drawing 3), transformed into escherichia coli SCS110, extract plasmid, electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In, obtain engineering bacteria BioT8G.

Respectively above-mentioned two strain engineering bacterias were cultivated 30 hours in the extractum carnis substratum for 30 ℃, got 500 μ L bacterium liquid to the Eppendorf pipe, ultrasonic disruption 30 seconds (B.Braun U Labsonic, 230V, T _{At interval}=0.5 second); Get 100 μ L and add 25 μ L and newly join 0.5NNaOH, 25 ℃ of effects 5 minutes; Add 65 μ L3 * sample buffers (925 μ L sample-loading buffers+75 μ L beta-mercaptoethanols), 100 ℃ were boiled 5 minutes.The centrifugal precipitation of removing.Last sample 10uL carries out SDS-PAGE electrophoretic analysis result.Result's (accompanying drawing 4) shows that the cry8Ga1 gene among the engineering bacteria Biot8G has all obtained expression, and the molecular weight of representation is about 130kDa.

1.4Cry8G proteic determination of activity

The Bt engineering strain is seeded on the common bacteria agar Kolle flask substratum cultivated 3 days.With F-strain HD-73 ^-Be seeded on the common bacteria agar Kolle flask substratum and cultivated 4 days.Culture is washed, 2 times of gradient concentration dilutions, the 40ml bacteria suspension is joined 200g to be had in the fine earth (ultraviolet disinfection) of even thickness potato silk, and mixing makes soil moisture content remain on 18%-20%.Insert 20 of 15 days instar larvaes of Holotrichia parallela, adding the blank that is treated to of clear water, 28 ℃ are infected and raise, and check in 14 days to calculate LC by dead borer population ₅₀Result's (seeing Table 8) shows that engineering strain has high cytotoxicity to holotrichia oblita (Holotrichiaoblita) and Holotrichia parallela (Holotrichia parallela).The Cry8G albumen of its expression has the activity of holotrichia oblita, Holotrichia parallela worm extremely.

Table 7Bt engineering bacteria and HBF-18 bacterial strain are to holotrichia oblita (Holotrichia oblita), Holotrichia parallela (Holotrichia parallela) larva insecticidal activity assay

Annotate: in result's concentration unit, * 10 ⁸/ g soil and * 10 ⁸The conversion of/ml adds the 36ml bacteria suspension in every 200g soil.

Embodiment 2

2.1 manually design the nucleotide sequence of the cry8G gene that synthetic can express in plant

According to different to codon preference of microorganism and plant, the sequence of the 1-2040bp of cry8Ga1 gene is optimized.The present invention has carried out full gene according to the artificial reconstructed sequence of cry8Ga1 gene and has synthesized, and new gene is seen the nucleotide sequence shown in the SEQ ID NO 3.The nucleotide sequence homology of cry8Ga1 gene and mcry8Ga1 (modified cry8Ca1) gene has only 86.88%, and G+C content also rises to 45.2% (table 8) by 37.6% of original cry8Ca1.Adjusted the sub-frequency of utilization of the proteic amino acid code of Cry8Ga1, made frequency of utilization in sub-frequency of utilization of the proteic amino acid code of mCry8Ga1 and the plant near (table 9).BamHI and KpnI site are introduced in the artificial reconstructed sequence two ends of cry8Ca gene, be connected to the pUC57 carrier, recombinant plasmid called after pUC57-mcry8G.

Table 8cry8Ga1 gene and mcry8Ga1G+C content compare the signal sequence situation with polyadenylation

The sub-frequency of utilization of proteic amino acid code among table 9 plant, Cry8a1 and the mCry8Ga1

2.2 the structure of plant expression vector

Reclaim the 2.0kb fragment with BamHI and SacI digested plasmid pUC57-mcry8G (Plant Protection institute, Chinese Academy of Agricultral Sciences's biotechnology group is preserved); (this carrier is a common carrier, and the GenBank accession number is AF485783 with same endonuclease digestion plasmid pBI121.See Chen PY, et al, 2003, Mol.Breed 11:287-293), reclaims the 12kb fragment, and two fragments are connected, and transforms JM110, obtains positive transformant, this new structure plasmid called after pBSmGN.The promotor CaMV35S that this plasmid contains constitutive expression (is the section of DNA sequence, can drive the gene fragment that is connected transcribes, and then translate into protein, the promotor of constitutive expression can regulatory gene all has expression in any stage of growing and any organizing), mcry8Ga gene and NOS terminator (the section of DNA sequence contains the termination signal of genetic expression).Plasmid construction figure sees accompanying drawing 5, this plasmid can be in plant expression alien gene.

2.3 Agrobacterium-mediated Transformation

Get the competent cell of a pipe 200 μ l Agrobacterium LBA4404, add 1 μ g pBSmGN plasmid DNA, quick-frozen is 1 minute in the liquid nitrogen, and 37 ℃ are recovered to cultivate 5 minutes, add 1ml YEB liquid nutrient medium, 28 ℃ at a slow speed the vibration (＜100rpm) 4 hours, 1, centrifugal 30 seconds kinds of 000rpm, abandon supernatant, add 100 μ l YEB liquid nutrient medium re-suspended cells, coat on the flat board of the YEB substratum that contains kantlex 100 μ g/ml and Streptomycin sulphate 125 μ g/ml, cultivated 48 hours for 28 ℃.Clone with on the YEB resistance culture medium flat plate shakes bacterium, extracts plasmid, uses PCR method and detects positive colony.

2.4 tobacco transforms

The Agrobacterium clone that will contain the pBSmGN plasmid is inoculated in the YEB liquid nutrient medium that contains kantlex 100 μ g/ml and Streptomycin sulphate 125 μ g/ml, and 28 ℃ of shaking culture are to OD ₆₀₀Be 0.6-0.8,1/50 is inoculated in the MS salt (pH7.0); The tobacco aseptic seedling is cut into 0.4 * 0.6cm ²Fritter, tobacco leaf is immersed in the MS salt, Agrobacterium was infected 10 minutes; Take out tobacco leaf, blot bacterium liquid with sterilization filter paper, be placed in the MS substratum that is covered with one deck filter paper, 28 ℃ of dark cultivations 3 days, after 3 days tobacco leaf is transferred to MS and screens division culture medium (MS substratum+100 μ g/ml kantlex+500 μ g/ml carboxylic benzyl mycin+3mg/ml 6-BA+0.2mg/ml NAA), 28 ℃, light/dark=16 hours/8 hours, after 2 weeks, there is green callus point to occur at blade edge, after 1 week, the callus point is divided into plantlet, the plantlet cutting-out is moved to root media (MS substratum+100 μ g/ml kantlex+500 μ g/ml carboxylic benzyl mycins) take root, after the root development stalwartness, move to flowerpot continued growth in soil (common soil: nutrition soil: vermiculite=2: 1: 1).

2.5 the Molecular Detection of transformation of tobacco

Extract the genomic dna of transformation of tobacco, get 1 μ g genomic dna and do template, primer sequence is as follows:

8GF1：5′-TTCAGTTGTCCACTCCGCCTA

8GR1：5′-GCCATTCACAGCCTTCTTTGC

The reaction conditions of pcr amplification is: 94 ℃, and 5 minutes, 1 circulation; 94 ℃, 1 minute, 53 ℃, 1 minute, 72 ℃, 2 minutes, 30 circulations.The product electrophoresis.As shown in Figure 6, positive transformant amplifies the big or small fragment of 640bp that is.

2.6 the biological activity assay of transgene tobacco

Gather 2 kinds of holotrichia oblitas (Holotrichia oblita), Holotrichia parallela (Holotrichiaparallela) from the field in the more serious chafer adult colony of northern China hazard ratio, take back indoor, put into the raising box of 40 * 40 * 50cm respectively, put the thick humidity of the 5-8cm fine earth that sieves at the bottom of the box, the fresh elm blade of feeding, 26-28 ℃ of raising.After treating that it lays eggs, ovum is chosen, be placed in the moist soil and make hatching.The larva that has just hatched is chosen, and (Φ 8cm H5cm) is put 5 of larvas to each little raising box, and feed tender of potato block and fresh corn are treated that larva is long to use for giving birth to survey after certain length of time.

Adopt colony's plantation and colony to connect the method for worm, carry out biological assay.Adopt the kind method for planting of plantation respectively and mixed planting transfer-gen plant and unconverted plant in the soil of land for growing field crops, each handles 12 strains.

According to the hazard rating grade scale of setting up, holotrichia oblita (Holotrichia oblita) larva is as follows to hazard rating, the hazard index statistics for the preliminary operation gene plant, sees Table 10.

The harm statistics of table 10. transfer-gen plant and non-transgenic strain.

From table 10 statistics, the hazard ratio that changes the pBSmGN plant is 62.5%, and hazard index is 22.9; Non-transgenic plant hazard ratio 100%, hazard index are 72.9.Commentaries on classics pBSmGN plant has shown the characteristic of good anti-holotrichia oblita (Holo trichia oblita).

Holotrichia parallela (Holotrichiaparallela) larva is as follows to the hazard rating statistics for the preliminary operation gene plant.Changeing pBSmGN plant hazard ratio is 50%, and hazard index is 12.5, and being lower than non-transgenic plant hazard ratio is 100%, and unconverted plant hazard index 75.0 is suitable.As seen change the pBSmGN plant and shown good anti-Holotrichia parallela (Holotrichiaparallela).

According to above information, utilization can make this gene obtain expressing at plant root in the promotor of the special open gene of root, thereby only in the resistance of plant root acquisition to target pest Holotrichia parallela (Holotrichia parallela).

Appendix: dna sequence dna involved in the present invention and protein sequence

SEQ ID NO 1 (nucleotide sequence of cry8Ga1 gene)

1

TCCGA?ATAATCAGAA?CGAATATGAA?ATTATAGATG?CGTCATCACC?TACTTCTGTA

61 TCTAATAACT?CAGTGAAATA?CCCTTTAGCA?AGTGATCAAA?CGACCACATT?ACAAAATATG

121 AACTATAAAG?ATTATCTGAG?AATGTCTGAG?GGAGAGAATC?CTGAATTATT?TGGAAATCCA

181 GAGACGTTTA?TTAGTGCGCA?GGATGCGGTT?GGAACTGGGA?TTGATATTGT?GAGTAAACTA

241 CTAGGTAGTT?TAGGGGTTCC?ACTTGTTGGG?CAAGCCGCAA?CGGCACTTAA?ATGGATTATA

301 GGTAAATTGT?GGCCTTCTTC?AGGAAACCCG?TGGGATGATT?TGATGACGGC?AGTAGAAGAA

361 CTCATAAATC?AAAAAATAGA?AGCATATGCA?AGAAGTAAGG?CACTTGCTGA?ATTGGGTGTT

421 TCGGGAAGAG?CTGTAAAATC?CTATCAAACC?GCACTTGAAG?AGTGGCAAAA?AAACCCGAAT

481 AACGCGCGAA?GCGCAGCACT?TGTAAGGGAA?AGATTTTCAG?ATGCAGAACA?TACATTGCGT

541 ACTCAAATGA?GTTTATTTAC?CGTTCGTGGT?TATGAAATTC?CGCTTTTAGC?AACATATGCA

601 CAAGCTGCCA?ATTTGCATTT?GTTTGTAATG?AAGGATATTC?AAATTTACGG?GAGAGAATGG

661 GGATATACTC?AGGGAGATAT?TAACCTTTTC?TATCGAGAAC?AAGTAGAATT?TACAGGGGAA

721 TACTCTGATT?ATTGTGTTAA?GTGGTACAAT?GCTGGCTTAG?ATAAATTAAG?AGGCTCGACT

781 GCTCTACAAT?GGATTAACTA?TAATCGTTTC?CGCAGAGAAA?TGACAGTGAT?GGCACTGGAT

841 ATAGTTGCAT?TATTCCCAAA?TTATGACATA?CGCATGTATC?CAATGAAAAC?AACCGCAGAA

901 TTAACGCGAA?GAATTTATAC?AGATCCGCTT?GGTTATACGG?GAAGTGGGTC?TAACACGCCA

961 CCATGGTATA?ATTATGGATA?TTCTTTCTCA?TGGATAGAAA?ATAATGCCGT?GCCAGCACCT

1021 GGATTGTTCC?AGTGGTTACA?AGGAATTGGG?ATTTATACTA?AATTTGCTCG?TATAACTCCA

1081 TTTTATGCGA?ACTATTGGTC?AGGACATACT?GTATTTTATA?AATTTACTAA?CGATTCTACT

1141 GAGAGACGTG?TTCAGTATGG?AGATACAGAT?ACTCCAGAAT?TAGATAGTTC?TTCCTTTGAA

1201 AATGTTGACA?TTTATAAGGT?TTCAGCATCA?GTTGGTTCGT?ACAAAAGTAA?TACCGTACTA

1261 TTACCAACTT?TTAAAGCTAC?TTTTGAGGGG?GTAAATCAAA?ATAATCAGTT?AAAGACCTTT

1321 AGGTATCAAA?AAGAATCTAA?TGTCCCAAGT?CAAACGAAAA?ACTCAACCAC?AGAGCTGCCT

1381 GTTCAGTTAT?CAACTCCGCC?TACTTACGGA?GATTCTGAAC?AGTACAGTCA?TAGACTAGCC

1441 TATGTTTTTG?ATGCCCCAAT?CGATTCATAT?ACAGGCATAT?ATCGCATGTA?TGGATTTGCC

1501 CCTATTCTTG?GTTGGACACA?TATTAGTGTA?AGTCGTGACA?ATAGGATTGA?TCCAGATAAA

1561 ATTACTCAAA?TTCCAGCTGT?AAAGGCATAT?GCTGAGGGTC?TTGCTAATTA?TATCAAAGAT

1621 CCGGGGTTTA?CAGGAGGAGA?TTTATTAGCT?TTAGGTAGAA?ACTCAAATAC?TTCATTGATT

1681 GTCAATTTTT?CGAAGCCTCA?AACATACCGT?ATTCGTATTC?GTTATGCTGC?TAGTAAAACT

1741 TCGTATTTTC?AACTACGTGG?GCTGCATAAT?ATAGCTCAGT?CTCAGCGTTT?CGAAGCGACG

1801 TATTCTAATA?AAAATGAAAA?CGATTTGACA?TTTAACGATT?TTAAATATGT?AGAAATTCAA

1861 AAAACTGTTT?CAATAGACAA?TCCATCAGAA?AGTCGTAGTA?TAAGTATATA?CACTCAATCA

1921 GATACAGAAT?ACTTATTATG?GACAAATCGA?ATTCATCCCA?GTAGATGCAA?CATTTGGAGC

1981 GGAACAAGAC?CTAGATGTGG?CAAAGAAAGC?GGTGAATGGC?TTGTGTACCA?ATACAAAAGA

2041 TGCCTTACAG?ACAAGTGTAA?CGGATTATCA?AGTCAATCAA?GCGGCAAACT?TAGTAGAATG

2101 CCTATCGATG?AGTTATACCC?AAATGAAAAA?CGCATGTTAT?GGGATGCAGT?GAAAGAGGCG

2161 AAACGACTTG?TTCAGGCACG?TAACTTACTC?CAAGATACAG?GCTTTAATGT?AATAAATGGA

2221 GAAAACGGAT?GGACGGGAAG?TACGGGAATT?GAGGTTGTGG?AAGGGGATGT?TCTGTTTAAA

2281 GATCGTTCGC?TTCGTTTGCC?AAGTGCGAGA?GAGATTGATA?CAGAAACATA?TCCAACGTAT

2341 CTCTATCAAC?AAATAGATGA?ATCGCTTTTA?AAACCATATA?CAAGATATAG?ACTAAGAGGT

2401 TTTATAGGAA?GTAGTCAAGA?TTTAGAGATT?AAATTAATAC?GTCATCGGGC?AAATCAAATT

2461 GTCAAAAATG?TACCGGATAA?CCTCTTGCCA?GATGTACGCC?CTGTCAATTC?TTGTGGTGGA

2521 GTCGATCGCT?GCAGTGAACA?ACAGTATGTA?GACGCGAATT?TAGCACTCGA?AAACAATGGA

2581 GAAAATAGAA?ATATGTCTTC?TGATTCCCAT?GCATTTTCTT?TCCATATGGA?TACAGGTGAA

2641 ATAGATTTAA?ATGAAAATAC?AGGTATTTGG?GTCGTATTTA?AAATTCCGAC?AACAAATGGA

2701 TACGCAACAT?TAGGAAACCT?TGAATTGGTA?GAAGAGGGGC?CATTATCAGG?AGACGCACTA

2761 GAACGCTTGC?AAAGAGAAGA?ACAGCAGTGG?AAGCTTCAAA?GAACCAAAAG?ACGTGAAGAG

2821 ACGGATAGAA?AATATATGGC?AGCAAAACAA?GCCATTGATC?GTTTATTCGC?AGATTATCAA

2881 GACCAACAAC?TCAATTCTGG?TGTAGAAATG?TCAGATTTGC?TTGCAGCTCA?AAACCTTGTA

2941 CAGTCCATTC?CTTATGTGTA?TAACGAAATG?TTCCCAGAAA?TCCCTGGAAT?GAACTATACA

3001 AATTTCACAG?AGTTAACAAA?CAGACTCCAA?CAAGCATGGA?ATTTGTATGA?TCTTCGAAAT

3061 GCTATACCAA?ATGGAGATTT?TCGAAATGGA?TTAAGTGATT?GGAATGCAAC?ATCAGATATA

3121 AATGTGCAAC?AACTAAACGA?TACATCTGTC?CTTGTCATTC?CAAACTGGAA?TTCTCAAGTG

3181 TCACAACAAT?TTACAGTTCA?ACCGAATTAT?AGATATGTAT?TACGTGTCAC?AGCGAGAAAA

3241 GAGGGAGCAG?GAGACGGATA?TGTGATCATC?CGTGATGGTA?CAAATCAGAC?AGAAACACTC

3301 GCATTTAATA?CATGTGATAA?TGATGCAGGT?GTTTTATCTA?CTAATCAAGC?TAGCTATATC

3361 ACAAAAACAG?TGGAATTCAC?GCCATCTACA?GAGCAAGTTT?GGATTGACAT?GAGTGAGACC

3421 GAAGGTGTAT?TCAACATAGA?AAGTGTAGAA?CTCGTGTTAG?AAGAAGAGTA?AG

SEQ ID N02 (the proteic aminoacid sequence of Cry8Ga1):

1 MSPNNQNEYE?IIDASSPTSV?SNNSVKYPLA?SDQTTTLQNM?NYKDYLRMSE?GENPELFGNP

61 ETFISAQDAV?GTGIDIVSKL?LGSLGVPLVG?QAATALKWI?I?GKLWPSSGNP?WDDLMTAVEE

121 LINQKIEAYA?RSKALAELGV?SGRAVKSYQT?ALEEWQKNPN?NARSAALVRE?RFSDAEHTLR

181 TQMSLFTVRG?YEIPLLATYA?QAANLHLFVM?KDIQIYGREW?GYTQGDINLF?YREQVEFTGE

241 YSDYCVKWYN?AGLDKLRGST?ALQWINYNRF?RREMTVMALD?IVALFPNYDI?RMYPMKTTAE

301 LTRRIYTDPL?GYTGSGSNTP?PWYNYGYSFS?WIENNAVPAP?GLFQWLQGIG?IYTKFARITP

361 FYANYWSGHT?VFYKFTNDST?ERRVQYGDTD?TPELDSSSFE?NVDIYKVSAS?VGSYKSNTVL

421 LPTFKATFEG?VNQNNQLKTF?RYQKESNVPS?QTKNSTTELP?VQLSTPPTYG?DSEQYSHRLA

481 YVFDAPIDSY?TGIYRMYGFA?PILGWTHISV?SRDNRIDPDK?ITQIPAVKAY?AEGLANYIKD

541 PGFTGGDLLA?LGRNSNTSLI?VNFSKPQTYR?IRIRYAASKT?SYFQLRGLHN?IAQSQRFEAT

601 YSNKNENDLT?FNDFKYVEIQ?KTVSIDNPSE?SRSISIYTQS?DTEYLLWTNR?IHPSRCNIWS

661 GTRPRCGKES?GEWLVYQYKR?CLTDKCNGLS?SQSSGKLSRM?PIDELYPNEK?RMLWDAVKEA

721 KRLVQARNLL?QDTGFNVING?ENGWTGSTGI?EVVEGDVLFK?DRSLRLPSAR?EIDTETYPTY

781 LYQQIDESLL?KPYTRYRLRG?FIGSSQDLEI?KLIRHRANQI?VKNVPDNLLP?DVRPVNSCGG

841 VDRCSEQQYV?DANLALENNG?ENRNMSSDSH?AFSFHMDTGE?IDLNENTGIW?VVFKIPTTNG

901 YATLGNLELV?EEGPLSGDAL?ERLQREEQQW?KLQRTKRREE?TDRKYMAAKQ?AIDRLFADYQ

961 DQQLNSGVEM?SDLLAAQNLV?QSIPYVYNEM?FPEIPGMNYT?NFTELTNRLQ?QAWNLYDLRN

1021?AIPNGDFRNG?LSDWNATSDI?NVQQLNDTSV?LVIPNWNSQV?SQQFTVQPNY?RYVLRVTARK

1081?EGAGDGYVII?RDGTNQTETL?AFNTCDNDAG?VLSTNQASYI?TKTVEFTPST?EQVWIDMSET

1141?EGVFNIESVE?LVLEEE*

SEQ ID N03 (manually designing the nucleotide sequence of gene)

BamHI

1 CGC GGATCCGCGATGAGTCCGAATAACCAGAACGAATATGAGATCATCGATGCTTCCTCC

61 CCCACCTCTGTTTCTAATAACTCAGTGAAGTACCCTCTTGCAAGCGACCAGACGACCACA

121 TTGCAAAACATGAACTACAAAGATTACCTGCGGATGTCTGAGGGAGAGAATCCTGAACTT

181 TTTGGAAATCCCGAGACGTTCATTAGTGCTCAGGACGCTGTTGGCACTGGTATTGATATT

241 GTGAGCAAGCTGTTGGGTAGTTTGGGGGTTCCACTTGTTGGTCAGGCCGCCACGGCACTT

301 AAATGGATCATAGGTAAGTTGTGGCCTTCTTCCGGAAACCCCTGGGACGATTTGATGACG

361 GCCGTAGAAGAACTCATCAACCAGAAGATAGAAGCGTATGCCAGAAGCAAGGCACTTGCT

421 GAATTGGGTGTTAGCGGACGGGCTGTGAAGTCCTACCAAACCGCCCTTGAAGAGTGGCAG

481 AAGAACCCCAATAACGCTCGAAGCGCAGCCCTTGTCAGGGAAAGATTTTCAGACGCAGAA

541 CACACATTGCGTACCCAAATGAGTTTGTTCACCGTTCGTGGTTACGAAATTCCGCTTTTA

601 GCCACATATGCACAAGCTGCCAATTTGCACTTGTTTGTGATGAAGGATATTCAAATTTAC

661 GGTAGAGAATGGGGATACACTCAGGGCGACATCAACCTTTTCTACAGGGAACAAGTAGAA

721 TTCACAGGGGAATACTCTGATTATTGCGTTAAGTGGTACAACGCTGGCTTGGACAAACTT

781 AGAGGCAGCACCGCTCTACAATGGATTAACTACAATAGGTTCCGCAGAGAAATGACAGTG

841 ATGGCCCTGGATATCGTTGCACTTTTCCCCAATTACGACATCCGCATGTATCCAATGAAG

901 ACAACCGCAGAACTTACGCGAAGAATTTACACAGACCCCCTTGGTTACACGGGAAGCGGT

961 TCTAACACGCCACCATGGTATAACTACGGATACTCTTTCTCCTGGATAGAAAACAACGCC

1021 GTGCCAGCCCCTGGATTGTTCCAGTGGTTGCAAGGCATTGGGATCTATACCAAGTTTGCT

1081 CGTATCACTCCATTTTACGCGAACTACTGGTCAGGACATACTGTCTTCTACAAATTTACT

1141 AACGATTCTACTGAGAGACGTGTTCAGTATGGAGACACAGATACTCCAGAATTAGACAGT

1201 TCTTCCTTCGAAAACGTTGACATTTATAAGGTTTCCGCCTCAGTTGGTTCGTACAAGAGC

1261 AATACCGTGTTGTTACCAACTTTTAAAGCTACTTTCGAGGGTGTCAATCAGAACAATCAG

1321 CTTAAGACCTTTAGGTATCAAAAGGAATCTAATGTCCCAAGCCAAACGAAGAACTCAACC

1381 ACAGAGCTGCCTGTTCAGTTGTCCACTCCGCCTACTTACGGAGACTCTGAACAGTACAGT

1441 CACAGACTAGCCTACGTTTTCGATGCCCCAATCGACTCATATACAGGCATATACCGCATG

1501 TACGGCTTTGCCCCTATTCTTGGTTGGACACACATTAGCGTGAGTAGGGACAACAGGATC

1561 GATCCAGACAAGATTACTCAAATTCCAGCTGTAAAGGCATATGCTGAGGGTCTTGCTAAT

1621 TACATCAAAGATCCCGGGTTCACAGGAGGCGACTTACTTGCTTTAGGTAGAAACTCCAAT

1681 ACTTCATTGATTGTCAACTTTTCGAAGCCTCAAACATACCGTATCCGTATTAGGTACGCT

1741 GCTAGCAAAACTTCGTATTTCCAACTACGTGGTCTGCACAATATCGCTCAGTCTCAGCGT

1801 TTCGAAGCTACGTACTCTAATAAGAACGAAAACGATTTGACATTTAACGACTTCAAGTAC

1861 GTCGAAATTCAAAAAACTGTTTCCATCGACAATCCATCAGAAAGCAGGAGTATATCCATC

1921 TACACTCAATCAGATACAGAACTTCTTATTATAGACAAAATCGAATTCATCCCAGTAGAC

1981 GCCACATTTGAAGCGGAACAAGACCTAGATGTGGCAAAGAAGGCTGTGAATGGCTTGTTC

KpnI SacI

2041 ACGAACACAAAGTAACGG GGTACCCCGA GCTCG

SEQ ID N04 (manually designing the proteic aminoacid sequence of gene mCry8Ga1)

1 MSPNNQNEYE?IIDASSPTSV?SNNSVKYPLA?SDQTTTLQNM?NYKDYLRMSE?GENPELFGNP

61 ETFISAQDAV?GTGIDIVSKL?LGSLGVPLVG?QAATALKWII?GKLWPSSGNP?WDDLMTAVEE

121 LINQKIEAYA?RSKALAELGV?SGRAVKSYQT?ALEEWQKNPN?NARSAALVRE?RFSDAEHTLR

181 TQMSLFTVRG?YEIPLLATYA?QAANLHLFVM?KDIQIYGREW?GYTQGDINLF?YREQVEFTGE

241 YSDYCVKWYN?AGLDKLRGST?ALQWINYNRF?RREMTVMALD?IVALFPNYDI?RMYPMKTTAE

301 LTRRIYTDPL?GYTGSGSNTP?PWYNYGYSFS?WIENNAVPAP?GLFQWLQGIG?IYTKFARITP

361 FYANYWSGHT?VFYKFTNDST?ERRVQYGDTD?TPELDSSSFE?NVDIYKVSAS?VGSYKSNTVL

421 LPTFKATFEG?VNQNNQLKTF?RYQKESNVPS?QTKNSTTELP?VQLSTPPTYG?DSEQYSHRLA

481 YVFDAPIDSY?TGIYRMYGFA?PILGWTHISV?SRDNRIDPDK?ITQIPAVKAY?AEGLANYIKD

541 PGFTGGDLLA?LGRNSNTSLI?VNFSKPQTYR?IRIRYAASKT?SYFQLRGLHN?IAQSQRFEAT

601 YSNKNENDLT?FNDFKYVEIQ?KTVSIDNPSE?SRSISIYTQS?DTEYLLWTNR?IHPSRCNIWS

661 GTRPRCGKES?GEWLVYQYKR

Claims

1. bacillus thuringiensis bacterial strain HBF-18, its preserving number is: CGMCC2070.

2. to coleopteran pest bacillus thuringiensis cry8Gal gene efficiently, its nucleotide sequence is shown in SEQ ID NO.1.

3. an engineering bacterial strain BioT8G is characterized in that containing the described cry8Gal gene of claim 2.

4. to coleopteran pest bacillus thuringiensis cry8Gal albumen efficiently, by the described cry8Gal coded by said gene of claim 2, its aminoacid sequence is shown in SEQ ID NO.2.

5. the described cry8Gal albumen of claim 4 is murdered application in the coleopteran pest medicament in preparation.

6. an albumen has the described albumen identical functions of claim 4, and its aminoacid sequence is shown in SEQ ID NO4.

7. artificial reconstructed synthetic mcry8Gal gene, the described albumen of its coding claim 6, its nucleotide sequence is shown in SEQ ID NO.3.

8. a plant expression vector pBSmGN is characterized in that this plant expression vector is constructed by the described mcry8Gal gene order of claim 7, composition type expression promoter CaMV35S, NOS terminator and a kind of binary vector pBI121 that can shuttle back and forth in intestinal bacteria and agrobacterium tumefaciens.

9. the application of the described mcry8Gal gene of claim 7 in the anti-coleopteran pest of plant.

10. application according to claim 9 is characterized in that the described plant expression vector pBSmGN of claim 8 is transformed plant, makes plant produce the toxicity of anti-coleopteran pest.

11. application according to claim 10, described plant is a tobacco.

12. according to the described application of claim 9, it is characterized in that the protein Preparation of mcry8Gal genetic expression is become medicament, be used to kill coleopteran pest.