CN1616486A - Pseudomonas pseudoalcaligenes disinsection protein gene - Google Patents

Abstract

The present invention provides a kind of new pesticidal protein gene and the gene coded protein. The pesticidal protein gene is originated from Pseudomonas pseudoalcalligenes, and its coded protein has wide and powerful intoxicating effect on grasshopper and Orthoptera insects, so that the pesticidal protein gene may be used in preventing and controlling pest of crop. The present invention also provides the Pseudomonas pseudoalcalligenes pesticidal protein separating and purifying method, and one method of producing pesticidal transgenic plant.

Description

The pseudomonas pseudoalcaligenes insecticidal protein gene

Technical field

The present invention relates to the insecticidal protein gene of pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes), described insecticidal protein gene is used in the host cell expresses.

Background technology

Over nearly 30 years, existing a lot of is that the microbial pesticide of main body composition is come out one after another with the entomopathogen.These microbial pesticides are owing to employed pathogenic micro-organism difference, thereby there is very big difference in insecticidal mechanism.The insecticidal mechanism that had confirmed germ insecticide already usually with bacteriotoxin albumen have very big relation (explain sub-ox, production and the application [M] of the golden spore bacillus of Su Yun. Beijing: agriculture press 1993).Since 1888 find that first toxin protein is diphtheria toxin, hundreds of bacteriotoxins in various bacteriums, have been found in succession.Wherein, existing a lot of bacteriotoxin albumen are studied in great detail.

So far bacteriotoxic research is mainly concentrated on the toxin protein of people and animals pathogenic bacterium, this anatoxic structure function and mechanism of toxication research have obtained the achievement that attracts people's attention.For example, confirmed that the diphtheria toxin (diphteria toxin) that diphtheria corynebacterium (Corynebacterium diphteriae) produces is the dimer that is made of two subunits of A, B, molecular weight is 62kD, and wherein the A molecular weight subunit is 24kD, and the B molecular weight subunit is 38kD.Diphtheria toxin has very strong toxicity, and a few microgram toxin just are enough to cause the people in desperately.This toxin is by the lysogenic phage genome encoding that parasitizes in the diphtheria corynebacterium, and its intoxicating mechanism is to suppress intracellular protein synthesis.Irreversible fixation can take place with peptide chain elongation factor EF2 in diphtheria toxin, thus the translocation of inhibition peptide chain (Shen is same, Wang Jingyan, biological chemistry [M]. Beijing: Higher Education Publishing House 1991).Pseudomonas aeruginosa exotoxin A (Pseudomonas aeruginosa exotoxin A) also is the extremely strong bacteriotoxin of a kind of virulence, a lps molecule enters tenuigenin just is enough to cell killing, this toxin protein is the single chain protein that is made of 613 amino acid, and molecular weight is 66kD.Three structure function districts of Pseudomonas aeruginosa exotoxin A all are positioned on the peptide chain, and the necessary cell combination of its cytotoxicity, transposition and three kinds of functions of ADP-ribosylation can be exercised by these three structure function districts.Its intoxicating mechanism is similar to diphtheria toxin, also is to produce cytotoxic effect by deactivation elongation factor EF2.The ADP-ribosylation takes place by the eukaryotic EF2 of catalysis in the Pseudomonas aeruginosa exotoxin A, thereby stop the peptide chain extension, stop the protein synthesis (Du Shihuo of cell, the structure function of Pseudomonas aeruginosa exotoxin A and recombinant toxin thereof [J]. biological chemistry and biophysics progress, 1995,22:112-117).

Bacteriotoxic structure functions such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A are studied in great detail, be based on these bacteriotoxins and having broad application prospects aspect the targeted therapy of tumor disease.By gene recombination technology, after the cell land of bacteriotoxin protein gene removed, merge with specific antibody gene again and form heterozygous genes, change cell such as intestinal bacteria over to produce immunotoxin, can be used for some disease such as tumor treatment.

So far the bacteriotoxin albumen of Fa Xianing has the intoxicating effect to people and animals mostly, and acting on the bacteriotoxin albumen of insect, specificity also knows little, and mainly concentrate on the bacterium of bacillus (Bacillus), as bacillus thuringiensis (Bacillusthuringiensis), Bacillus sphaericus (Bacillus sphaericus).Present most widely used germ insecticide is a sporeine preparation.The insecticidal action of bacillus thuringiensis mainly is by (Knowles B.H. due to a kind of toxin protein-insecticidal crystal protein that produces in the thalli growth process (insecticidal crystal protein), Mechanism of action of Bacillusthuringiensis insecticdal σ-endotoxins[J] .Adv insect Physiol, 1994,24:275-307).Found hundreds of bacillus thuringiensis insecticidal crystal proteins so far, every kind of insecticidal crystal protein all has specific insecticidal spectrum, can be at different target insect performance toxic actions.

Nearest studies show that, also there is a kind of toxin protein that insect is had the intoxicating effect in a kind of bacterium pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) of Rhodopseudomonas (Pseudomonas), in the thalline fermenting process, insecticidal proteins is secreted in fermented liquid, utilize this fermented liquid to carry out virulence experiment, discovery has stronger insecticidal activity (Yang Zhirong to locust, red legend, Ge Shaorong etc., the research [J] of pseudomonas pseudoalcaligenes control meadow locust. the Chinese biological control, 1996,12 (2): 55-57; Liu Shigui, red legend, Yang Zhirong etc., separation of a strain locust pathogenic bacteria and evaluation [J]. the microorganism journal, 1995,35:90-96).The discovery of bacterium insecticidal toxin protein and research are laid a good foundation for developing new and effective biotic pesticide.Increasingly mature along with development of molecular biology and genetic engineering technique; The encoding gene of existing a lot of bacterium insecticidal toxin proteins is cloned, for example from the various bacterial strains of Su Yun gold gemma, cloned more than 100 insecticidal crystalline gene (Feitelson J.S, Payne J., KimL., Bacillus thuringiensis:Insects and beyond[J] .Bio/Technology, 1992,10:271-275), from the various bacterial strains of Bacillus sphaericus, cloned dozens of toxin gene (Charles J.F, Niclson-LeRoux C, Delecluse A.Bacillus sphaericus toxins:molecular biology and mode of action[J] .Ann RevEntomol, 1996,41:389-410).These toxin genes have broad application prospects aspect pest control, utilized some bacillus thuringiensis insecticidal crystal proteins gene constructed the stronger efficient engineering strain of virulence, developed multiple novel thuringiensis cladosporioides bacillus insecticide.In addition, also utilize gene engineering to change the bacillus thuringiensis insecticidal crystal proteins gene over to various plants, cultivated transgenic pest-resistant crops such as transgene cotton, tobacco, paddy rice.

Aspect the research of pseudomonas pseudoalcaligenes insecticidal proteins, confirmed already that this proteinic molecular weight was lower, be a kind of extracellular toxin albumen (Zhang Wen, Yang Zhirong, document received etc.The separation and purification of pseudomonas pseudoalcaligenes insect killing substance and evaluation [J]. the microorganism journal, 1998,38:57-62), and the research of related genera Pseudomonas alcaligenes insecticidal protein gene clone and transgenic plant there is no report both at home and abroad.

Summary of the invention

Insecticidal protein gene and application thereof

The present invention relates to a kind of insecticidal protein gene that derives from pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes), the protein of this genes encoding, described insecticidal protein gene is used in the host cell expresses.Pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) is a kind of Gram-negative bacteria.

Locust belongs to Orthoptera (Orthoptera) locust section (Locustidae), is a kind of global harmful insect.In China is national distribution and harm.Especially the most serious with the north, on northern grassland, become meadow first insect (Liu Shigui, Sichuan University's journal [J], 1992,29:2-8 with characteristics such as its " kind are many, quantity is big, distribution is wide, harm heavy ".) only northern grassland and Qinghai-Tibet meadow, nearly 1,000 ten thousand hectares of the annual area that takes place and endanger, the serious harm herbage growth makes meadow grass yield decline 30-70% (Li Kefu, Ma Yao, Du Wenliang, Chinese meadow [J], 1992, (1): 50-52.), cause the tremendous economic loss.The harm of locust still causes one of important factor of grassland degeneration, desertification, natural ecological environment worsening condition etc.

Found the worm that dies of illness naturally of many Ceracris kiangsuis (Ceracris kiangsu) in 1991 in Nikkei mountain forest field, Chongqing City, in the worm corpse, be separated to a kind of cause of disease mattress, through replying the infector host, can cause that former host is caused a disease and death, from the worm corpse, be separated to same pathogenic bacteria, prove the pathogenic bacterium that this cause of disease mattress is a locust self.Preliminary experiment results through infecting main meadow insect shows, this cause of disease mattress has higher infectivity to multiple meadow locust, 5 days deadly string is up to more than 90%, and grassland caterpillar (Gynephorap runergensis), the mythimna separata (Leucania separata) on meadow also had certain infectivity.And to multiple meadow locust have this bacterial strain of stronger infectivity through be accredited as pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) (Liu Shigui, red legend, Yang Zhirong, etc. microorganism journal [J], 1995,35 (2): 86-89).

In recent years to the deadly mechanism of its desinsection, insecticidal proteins (Zhang Wen, Yang Zhirong.Microorganism journal [J], 1998,38 (1): 57-62), security (Yang Zhirong, red legend, Ge Shaorong etc.Chinese biological control [J], 1996,12 (3): 114-116) etc. the aspect is furtherd investigate, and proves that this bacterium causes a disease to locust, is because due to a kind of insecticidal proteins that its metabolism produces.This molecular weight of albumen is 25100Da, belongs to reported first at home and abroad.Show that after deliberation this bacterium does not have gemma, itself have strong toxalbumin, be configured to the potentiality that genetically engineered bacteria is widely used in biological control so have.

An object of the present invention is to provide a kind of new insecticidal protein gene, it belongs to pseudomonas pseudoalcaligenes insecticidal protein gene (Pseudomonas pseudoalcaligenes insecticidal protein), derive from pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) (Liu Shigui, red legend, Yang Zhirong, etc. microorganism journal [J], 1995,35 (2): 86-89.), the albumen of this coded by said gene to orthopteran have widely, stronger toxic action.Can be used for the pest control of crop.Insecticidal protein gene complete sequence of the present invention, this gene be totally 276 amino acid, and the part of its coding toxicity peptide is a 247-1078 Nucleotide.Wherein 22 amino acid of C-terminal are the pseudomonas pseudoalcaligenes signal peptide, and the part of its coding toxicity peptide is a 247-313 Nucleotide.

Particularly, pseudomonas pseudoalcaligenes insecticidal protein gene of the present invention can obtain by the following method, but is not limited thereto.

Pseudomonas pseudoalcaligenes insecticidal protein gene of the present invention can be with separating such as the genome dna library sieve method.Be that available suitable carriers is directly cloned from the DNA library, or utilize known class Pseudomonas alcaligenes cDNA, DNA or its part to screen described pseudomonas pseudoalcaligenes insecticidal protein gene by hybridizing, or utilize the primer that designs according to pseudomonas pseudoalcaligenes cDNA or pseudomonas pseudoalcaligenes genomic dna and be that template is passed through polymerase chain reaction (PCR) and screened described insecticidal protein gene with the genome dna library of microorganism with the genome dna library of microorganism as probe.

Pseudomonas pseudoalcaligenes insecticidal protein gene of the present invention also can utilize the chemosynthesis of nucleic acid to produce by ordinary method, as phosphonic amide method [Mattencci, M.D.﹠amp; Caruthers, M.H., Journal of the American Chemical Society [(J.Am.Chem.Soc.) 103,3185 (1981)] and tris phosphite method [Nature 310,105 (1984) for Hunkapiller, M. etc.].

Another object of the present invention provides a kind of insect-killing protein and separation purification method.Proteinic sequence of the present invention under poor environment, pseudomonas pseudoalcaligenes can produce insecticidal proteins (insecticidal protein, IP).This protein is totally 253 amino acid, molecular weight 26KD, and the part that wherein has the toxicity peptide of insecticidal activity is a 22-278 amino acid.

Insecticidal protein gene of the present invention can be expressed in intestinal bacteria, and its expression product has toxic action to orthopteran.Utilize the wild type gene of total length or only use its 5 ' end 314-1078 Nucleotide toxicity region sequence of 765bp altogether, all can carry out plant nucleolus and transform.Gene order is carried out artificial reconstructed, make its codon be more suitable for to significantly improve expression amount or insecticidal effect in plant, expressing.

A further object of the present invention provides a kind of method of producing pest-resistant transfer-gen plant, comprises the step that insecticidal protein gene of the present invention is imported plant.

The method that foreign gene is imported plant is known, can use this method that gene construct of the present invention is inserted plant host, described method comprises biological and physics Plant Transformation method, and example is seen Niki etc., 1993, " foreign DNA is imported the method for plant ", molecular biology of plants and biotechnological means, Glick and Thompson compile, CRC publishing company, Boca Raton, p67-88.Selected method is different with the difference of host plant, comprises chemical infection protocol such as calcium phosphate, transgenosis such as Agrobacterium (Horsch etc., science, 227:1229-31,1985) that microorganism is instructed, electroporation, microinjection, particle gun and biolistic bombardment.

Metaplasia of the expression vector of vegetable cell and extracorporeal culturing method or plant and regeneration are known, can obtain, example is seen Gruber etc., 1993, " plant conversion carrier ", molecular biology of plants and biotechnological means, Glick and Thompson compile, CRC publishing company, Boca Raton, p89-119.

Insecticidal protein gene of the present invention can be directly used in Plant Transformation without transforming.When carrying out Plant Transformation, this gene (under the driving of plant gene promoter) fixed point can be inserted the general plasmid pCAMBIA2301G (ChenL of single dicotyledons, Zhang S, Beachy RN, Fauquet CM (1998) A protocol for consistent, large-scale productionof fertile transgenic rice plants.Plant Cell Reports 18:25-31V) between genomic Tnos gene of plant chloroplast and the P35s gene.

When the insecticidal protein gene among the present invention was used for the plant nucleolus conversion, all ordinary methods all can be used.

The figure explanation

Fig. 1 is that pseudomonas pseudoalcaligenes insecticidal protein gene recombinant expression vector pT7-6-IP makes up.

Fig. 2 is that the pCAMBIA2301G-IP conversion carrier makes up.

Embodiment

Hereinafter will the present invention will be further described by embodiment, but embodiment does not limit the present invention in any way.

Embodiment 1

The separation and purification of pseudomonas pseudoalcaligenes insecticidal proteins

1.1 medium preparation

With class produce the false unit cell mattress of alkali be inoculated in broth culture (Zhou Deqing. microbiology laboratory manual [M], Science and Technology of Shanghai press, 1985), nutrient media components (g/L): extractum carnis 5g, peptone 10g, NaCl 5g, pH7.2.Behind 35 ℃ of cultivation 12h, change being inoculated in the same substratum again, 35 ℃, 120r/min, shaking culture 36h is standby.

1.2 the insecticidal activity of pseudomonas pseudoalcaligenes culture detects

The pseudomonas pseudoalcaligenes culture obtains bacterial cultures supernatant liquor and bacterial precipitation in 4 ℃, the centrifugal 10min of 6000g.Bacterial precipitation carries out homogenate behind deionized water wash 3 times, institute's homogenate that obtains is collected the homogenate supernatant liquor after 10000g is centrifugal, and its protein concn is 43mg/ml after measured.After with anti-dialysis method the bacterial cultures supernatant liquor being carried out suitably concentrating, recording its protein concentration is 8.3mg/ml.Two kinds of solution all are diluted to 5mg/ml, and adding sucrose to concentration is 20%, is used for insecticidal activity assay.Measurement result shows, the bacterial cultures supernatant liquor to 3 age locust insecticidal action is arranged, bacterium homogenate supernatant liquor and then do not have an insecticidal activity through the bacterial cultures supernatant liquor that Proteinase K is handled, the prompting insecticidal proteins is present in the bacterial cultures supernatant liquor, be a kind of extracellular toxin albumen of thalline excretory, this result with bibliographical information is consistent.

1.3. the separation and purification of insecticidal proteins

1.3.1 the preparation of thick insecticidal proteins extract

The pseudomonas pseudoalcaligenes that picking is frozen is inoculated on the LB agar plate, in 35 ℃ of cultivation 16h, makes actication of culture.Separate good bacterium colony for picking 3-4 then, be inoculated in extractum carnis-protein culture medium and cultivate 12h, and then be transferred to the same cultivation of substratum relaying persistent oscillation (rotating speed, 140r/min in 35 ℃ of vibrations (rotating speed is 140r/min); Temperature, 35 ℃) 36h.

Bacterial cultures is sub-packed in the centrifuge tube, and 4 ℃ of centrifugal (6000g) 20min discard bacterial precipitation, collect supernatant liquor.The liquid that obtains directly carries out ultrafiltration with the Millipore ultrafiltration system.Ultrafiltration system through 0.5mol/L HCL washing once, is washed with distilled water to neutrality in advance then, with 0.5mol/L NaOH washing once, is washed with distilled water to neutrality again.When ultrafiltration, the first employing above proteinic filter membrane of 40kD that can dam carries out ultrafiltration, and contained protein all below 40kD, is collected filtrate in the filtrate, measures insecticidal activity.Use the following proteinic filter membrane of the 10kD that to dam again instead, the filtrate that the ultrafiltration of last step is obtained is carried out ultrafiltration, collect unfiltered part and measure insecticidal activity.The dialysis tubing of again this part protein solution being packed into places the PEG2000 powder, is concentrated into certain volume, obtains thick insecticidal proteins extract.

1.3.2DEAE-Mierocrystalline cellulose-32 column chromatography

After the abundant swelling of DEAE-Mierocrystalline cellulose-32 usefulness distilled water, remove floating matter, handle 20min, be washed with distilled water to neutrality with 0.5mol/L NaOH.Soak 20min with 0.5mol/L HCl then, washing is to neutral.Handle once and wash with 0.5mol/L NaOH again, drain moisture, place 50mmol/L Tris-HCl (pH7.8) damping fluid with suction method to neutral.

With DEAE-Mierocrystalline cellulose-32 pack into chromatography column (2.5 * 30cm), with level pad (50mmol/LTris-HCl, pH7.8) abundant balance.Thick insecticidal proteins extract is splined on DEAE-Mierocrystalline cellulose-32 post after balance, and with level pad wash-out foreign protein, flow velocity is 30ml/h, treats A ₂₈₀After being lower than 0.02, using the level pad that contains 0-0.6mol/L NaCl instead and carry out linear gradient elution, wash speed and be 30ml/h, the fraction collection elutriant is measured corresponding insecticidal activity.Merging the elutriant contain insecticidal activity then, is that 7.8 50mmol/L Tris-HCl damping fluid is fully dialysed at 4 ℃ to pH, to remove the NaCl in the protein soln.Be concentrated into certain volume with PEG20000 again.

1.3.3 Sephadex G-100 gel-filtration

After Sephadex G-100 powder adds 50mmol/L Tris-HCl (pH7.8), in boiling water bath, heat 5h, make its abundant swelling, remove the fine particle that swims in damping fluid with decantation, wait to be chilled to pack into after the service temperature chromatography column (1.6 * 80cm), with this chromatography column of 50mmol/L Tris-HCl (pH7.8) balance of 3 times of column volumes, to be splined on this chromatography column through the insecticidal proteins sample behind DEAE-Mierocrystalline cellulose-32 purifying again, use the level pad wash-out, the fraction collection elutriant is measured A ₂₈₀And insecticidal activity, merge the elutriant that contains insecticidal activity, at 4 ℃ distilled water is fully dialysed, concentrate with PEG20000 again.

1.3.4 the determination of activity of insecticidal proteins

The droplet feeding method of (1995) such as employing de Leon is measured, and laboratory animal is a Ceracris kiangsui.After the Ceracris kiangsui pieces of an egg that pick up from Jiang'an, Sichuan Zhu Hai are cleaned with 0.1% potassium permanganate solution, use twice of aqua sterilisa rinsing again, dry and be placed on thermostat container, in 28 ℃ of hatchings, larval growth to 3 age promptly is used for the determination of activity of insecticidal proteins, gets 30 μ l insecticidal proteins solution (containing 20% sucrose) with microsyringe, the 3 hungry instar larvaes of feeding, control group 20% sucrose solution of then feeding.Locust through feeding transfers to 5d on the fresh corn seedling, each 25 locust of control group and experimental group, and the death condition of locust in the statistics 5d, this experiment repeats 3 times, calculates mortality ratio to identify the insecticidal activity of testing sample according to experimental result.

1.3.5 SDS-polyacrylamide gel electrophoresis

The method of employing Ausubel etc. (Ausubel F.M, Brent R, Kingston R.E, et al.Current Protocolsin Molecular Biology[M] .USA:John Wiley ﹠amp; Sons Inc.1997), carries out discontinuous vertical tabular electrophoresis.Resolving gel concentration is 12%, and concentrated gum concentration is 40%.The glue section that cutting concentrates insecticidal proteins reclaims with buffer solution elution.

1.4 the-terminal amino acid sequential analysis of insecticidal proteins

Purifying insecticidal proteins dry powder is put on pvdf membrane after being dissolved in special agent, Beckman LF3000 automatic protein sequencer is measured after fixing the processing, measurement result shows, terminal 10 the amino acid whose sequences of the desinsection egg N-of purifying are Gly ValTrp Gln His Gln Ser His Ala ALa, can be used for the design of oligonucleotide probe in view of the above.

Embodiment 2

Pseudomonas pseudoalcaligenes insecticidal protein gene clone and prokaryotic expression and insecticidal test.

2.1 the structure of pseudomonas pseudoalcaligenes genomic library

Extract the pseudomonas pseudoalcaligenes genomic dna by the CTAB method, the genomic dna that takes a morsel then carries out the Sau3AI enzyme and cuts preliminary experiment, under this condition genomic dna to be carried out Sau3AI partially digested for enzyme slitting band when the random dna fragment that determine to produce concentrated on 2-10kb, enzyme is cut product and is separated by 0.5% agarose gel electrophoresis, reclaims the fragment of 2-10kb from sepharose.Adopting pUC19 plasmid (Norrander, J.et al., Gene (1983) 26:101-106) is cloning vector, is cloning site with the BamHl site of this plasmid vector.When pUC19 through the BamHl complete digestion, change linear molecule into, remove the 5 ' phosphate group at linear molecule two ends with calf intestinal alkaline phosphatase, obtain the dephosphorylation linear plasmid.

The dephosphorylation linear plasmid is mixed with the partially digested fragment of genomic dna, adding the T4 dna ligase connects, make the BamHI site of the genomic DNA fragment insertion pUCl9 of pseudomonas pseudoalcaligenes, form recombinant plasmid, be used for transformed into escherichia coli DH5a then, converted product is coated the LB flat board that contains 100 μ g/ml penbritins, obtains about 15000 bacterium colonies altogether behind 37 ℃ of cultivation 16h.

In order to determine the recombination frequency of bacterium colony, 160 bacterium colony dibblings of picking are in containing IPTG and X-gal penbritin-LB flat board at random, behind 37 ℃ of cultivation 16h, being positioned over 4 ℃ develops the color a few hours, 109 white colonies appear, these white colonies are the reorganization bacterium colony that carries recombinant plasmid, account for the ratio of total bacterium colony according to white colony, and calculating recombination frequency is 68%.

20 white colonies of picking are used to extract plasmid to detect the insertion fragment length of plasmid at random.Through limit restriction enzyme digestion and electrophoretic analysis, find that inserting segmental mean length is about 4.5kb.N=ln (1-p)/ln (1-f) by formula, calculating any given dna sequence dna probability of occurrence (P) is 0.99 o'clock required reorganization bacterium colony number, assesses the library quality with this.Bacterial genomes DNA size below 5000kb, should be 0.09% because the mean length of reorganization bacterium colony is inserted the ratio (f) of fragment in genome usually at least, and required reorganization bacterium colony number (N) is at most 5115.

Surplus the reorganization bacterium colony that this experiment obtains has reached 10,000, thereby comprised all dna sequences of pseudomonas pseudoalcaligenes, reached requirement fully as genomic library.All transformants of gained wash from penbritin-LB flat board during with transformed into escherichia coli with aqua sterilisa, and abandoning supernatant after centrifugal is collected thalline, adds the LB substratum that 10ml contains 50% glycerine, preserves these genomic libraries in-20 ℃.

2.2 the screening of insecticidal protein gene positive colony and evaluation

Take out the genomic library of-20 ℃ of preservations, draw a small amount of bacterium liquid, and be diluted to every milliliter of about 10000 bacteriums with aqua sterilisa, coat the penbritin-LB flat board that contains IPTG and X-gal then, 37 ℃ of cultivations directly are put into media surface with nylon membrane when bacterium colony reaches 0.2mm, bacterium colony is duplicated on the nylon membrane.Nylon membrane is after cracking, sex change, neutralization and vacuum are done roasting fixing, and the DNA original position that bacterium colony discharges is incorporated on the nylon membrane, and the nylon membrane of this moment is bacterium colony original position cracking film, and available oligonucleotide probe is hybridized.

Employed oligonucleotide probe is according to the design of the sequence of amino acid of insecticidal proteins N-terminal in the experiment; Probe length is 17 Nucleotide, and its sequence is GGNGTNTGGCARCAYCA, and wherein N represents any base, and Y represents pyrimidine, and R represents purine.This oligonucleotide probe sequence puts in order corresponding to 6 amino-acid residues of the N-terminal of insecticidal proteins.Hybridize with this probe and the former firmly cracking film of bacterium colony, occur 16 positive hybridization signals altogether.

Behind the pairing bacterium colony of definite positive hybridization signal, preserve flat board with these the suspicious bacterium colony dibblings of sterilization toothpick picking accordingly in dull and stereotyped the reaching of penbritin-LB of covering nylon membrane, be cultured in 37 ℃ and peel off nylon membrane when bacterium colony reaches 0.2mm and carry out the original position cracking, prepared original position cracking film is used further to second and takes turns screening by hybridization.When second took turns screening by hybridization, hybridization temperature was brought up to 50 ℃ from 48 ℃ of first round screening, and the rinsing temperature also improves 2 ℃ carries out hybridization and rinsing under stricter condition.Take turns second and to obtain 5 positive colonies in the screening altogether.Obtain cloning the segmental plasmid pSC1-1 of 1.4kb through subclone and Southern hybridization checking, this fragment is the encoding insecticidal proteins gene fragment.Obtain full length sequence by determined dna sequence, this fragment coding total length is the aminoacid sequence of 26kD.

2.3 the pseudomonas pseudoalcaligenes insecticidal protein gene is at expression in escherichia coli:

Cut out insecticidal protein gene with EcoRI and BamHI from cloning vector pSC1-1, be connected to the prokaryotic expression carrier pT7-6[Tabor that cuts with same enzyme, S., (1990), Current protocols in molecular biology (F.A.Ausubcl et al., eds) pp.16.2.1-16.2.11.Greene publishing and Wiley-Interscienee, New York] the last pT7-6-IP (see figure 1) that makes up, transformed into escherichia coli also screens recon on the AMP flat board, extract plasmid then and carry out enzyme and cut evaluation.

Cultivate inducible gene expression through the recombinant bacteria of identifying that contains insecticidal protein gene at 37 ℃, and the concrete grammar reference literature (Ceng Qing etc., viral journal, 1992,8:205-209).The expression product of insecticidal proteins detects by the SDS-polyacrylamide gel electrophoresis and the Western engram analysis (is explained sub-ox, Sun Ming, Liu Ziduo etc., classification of bacillus thuringiensis and biological activity protein gene [J]. the Chinese biological control, 1996,12:85-89) prove that this expression strain produces the insecticidal proteins molecule of 26kD.

2.4 insecticidal test

By SDS-polyacrylamide gel electrophoresis and Western engram analysis, the intestinal bacteria of the verified pT7-6-IP of carrying recombinant plasmid have higher insecticidal proteins content after inducing 3h.Therefore, adopt the thalline of abduction delivering to carry out the insecticidal activity detection.The thalline of centrifugal collection 100ml saturated culture adds the 4ml20% sucrose solution and carries out homogenate, by the centrifugal bacterial debris of removing, collects supernatant liquor.Adopt the droplet feeding method measure the homogenate supernatant liquor to 3 age locust insecticidal activity, with the intestinal bacteria homogenate supernatant liquor that contains pT7-6 is contrast, sample sets and control group are 100 locusts in the experiment, the death condition of locust in the 5d behind the statistics feeding, the result shows, dead 36 of sample sets, dead 11 of control group, the foreign protein that escherichia coli expression is described has certain insecticidal activity, and this confirms that further the foreign gene that the reorganization bacterium is carried is an insecticidal protein gene.

Embodiment 3

Insecticidal protein gene is used for clover and transforms and cultivate pest-resistant clover

3.1 vector construction:

3.1.1 the structure of carrier pCAMBIA2301G

The building process of carrier pCAMBIA2301G as shown in Figure 2.At first, plant expression vector pBI121 and pCAMBIA2301 are carried out EcoRI, HindIII double digestion.Then, reclaim the fragment about pBI121 3Kb, the fragment about pCAMBIA2301 11Kb, and use T ₄Dna ligase connects, and obtains carrier pCAMBIA2301G.

3.1.2 the structure of carrier pCAMBIA2301G-IP

With carrier pSC1-1 is template pcr amplification pseudomonas pseudoalcaligenes insecticidal protein gene fragment, with XbaI, SacI double digestion, reclaims the 1.4kb fragment.Simultaneously, carrier pCAMBIA2301G is also with XbaI, SacI double digestion and reclaim fragment about 12Kb, is connected with pseudomonas pseudoalcaligenes insecticidal protein gene fragment after enzyme is cut then, obtains expression vector pCAMBIA2301G-IP (see figure 2).

3.2 Agrobacterium-mediated Transformation

3.2.1 intestinal bacteria CaCl ₂The preparation of method competent cell and conversion

The preparation of intestinal bacteria (Esherichia coli) DH5 α competent cell.The single bacterium colony of picking DH5 α on the LB flat board, 37 ℃ of overnight incubation in 2ml LB substratum.Get the 0.5ml culture, 28 ℃ are continued to be cultured to OD in the adding 50ml LB substratum ₆₀₀≈ 0.5.Add in the 1.5ml centrifuge tube 4 ℃ of centrifugal collection thalline.With 500 μ l 0.1mol/L ice CaCl ₂Resuspended, ice bath is centrifugal again after 30 minutes, with 200 μ l0.1mol/L ice CaCl ₂Resuspended, be used for after 30 minutes to 24 hours transforming in 0 ℃ of preservation.

Colibacillary conversion.Add 5-10 μ l plasmid in 200 μ l competent cells, ice bath was put into 42 ℃ of water-bath heat shocks 2 minutes rapidly after 30 minutes, took out back 0 ℃ rapidly and placed 2 minutes.Add 800 μ l LB then, cultivated 1 hour for 37 ℃.Last centrifugal 2 minutes, thalline is coated on the antibiotic flat board of LB+ 37 ℃ of overnight incubation.

3.2.2 the preparation of Agrobacterium competent cell and conversion (Wang Guanlin etc., plant genetic engineering philosophy and technique, Science Press, 1999).

Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105, the preparation of competent cell.Agrobacterium tumefaciens bacterium liquid is cultured to OD in 28 ℃ ₆₀₀During ≈ 0.5,4 ℃ of centrifugal collection thalline are iced CaCl with 0.1mol/L ₂Resuspended, ice bath is centrifugal after 30 minutes, ices CaCl with 0.1mol/L ₂After resuspended, in 0 ℃ of preservation.

Freeze-thaw method transforms agrobacterium tumefaciens.Add 5-10 μ l plasmid in 200 μ l competent cells, ice bath is after 30 minutes, and in liquid nitrogen quick-frozen 1-2 minute, take out rapidly, put into 37 ℃ of water-baths and dissolve.Dissolving back fully adds 800 μ l LB, cultivates 3～5 hours for 28 ℃.Last centrifugal 2 minutes, thalline is coated on LB+ Rifampin (40mg/L)+Streptomycin sulphate (25mg/L)+kantlex (75mg/L) flat board, cultivated 2 days for 28 ℃.

3.3 leaf dish method transforms clover

3.3.1 agrobacterium tumefaciens leaf dish method transforms clover (Lv Deyang, Deng. the high sulfur-containing amino acid protein transgene of clover plant regeneration [J]. Acta Genetica Sinica, 2000,27 (4): 331-337.Lu D.Y, et al.Acomparison of the cultural bahaviour ofprotoplasts from leaves, cotyledons and roots of Medicago sativa[J] .Plant Sci.lett, 1983,31:87-99).

Be taken at MS ₀The cotyledon of the alfalfa of seedling age 5-7d on the substratum (Medicago Sative L.) (this clover does not have gus gene genetic background) aseptic seedling cuts 1/3 altogether from two ends, immerses 2/3 cotyledon of remainder centrifugal and uses liquid MS ₀In the resuspended agrobacterium tumefaciens bacterium liquid, shaking table is cultivated 30min, and the medication bale-out removes surperficial bacterium liquid for inhaling on the aseptic filter paper, is inoculated on the UM substratum that adds lid layer filter paper, behind the dark cultivation 60h, use sterilized water, sterilized water+Pyocianil (500mg/L) and MS successively ₀+ Pyocianil (500mg/L) is washed cotyledon surface Agrobacterium off, forwards on the screening culture medium (UM+50mg/L kantlex+100mg/L cephamycin), about 25 ℃, carries out after the illumination cultivation .30d of 16h/d the indefinite bud that induces being gone to MSD ₄Root induction on the+100mg/L cephamycin forwards MS to behind about 30d ₀On, induce whole plant regeneration.

3.3.2 the screening of transformed plant and transplanting cut the blade of regeneration plant, more in two, are inoculated on the UM+100mg/L kantlex.After the plant of kalamycin resistance cultivated well developed root system, move into 1/10MS ₀Cultivate a week in the liquid nutrient medium and practice seedling, move to again in the flowerpot that nutrition soil is housed.

3.4 the evaluation of transgenic alfalfa

3.4.1 Southern hybridization

Southern is hybridized with improved CTAB method (Yuan Qinghua; cassia twig etc.; sativa genomic dna extracts and preferred [J] meadow of RAPD reaction conditions journal, and 2001,9 (2): 99-105) the total DNA of extracting alfalfa plants; take by weighing the fresh and tender clover blade of 100mg; it is shredded be placed in the 1.5mL centrifuge tube, add liquid nitrogen, grind to powder with pestle; 2 * CTAB the damping fluid that adds 900 μ l65 ℃ preheatings, 65 ℃ of following water-bath 20min (shaking up once every 2min) take out the back and cool.Add 500 μ l chloroform isoamyl alcohol mixed solutions (24: 1).Shake up 4 ℃ of centrifugal 10min of following 7500r/min.Get supernatant liquor and place the 1.5ml centrifuge tube, add 500 μ l chloroform isoamyl alcohols.4 ℃ of centrifugal 10min of following 7500r/min behind the mixing.Supernatant liquor is moved in the new centrifuge tube, add 1/10 volume 3mol/L NaAc and isopyknic Virahol, jog is to flocks occurring.After the abandoning supernatant.Clean twice with 100 μ l, 75% ethanol, dry 1h under the room temperature is dissolved in the TE damping fluid (pH8.0).Select the restriction enzyme EcoRI of no recognition site in the insecticidal protein gene sequence to carry out complete degestion, the concentration of DNA sample.With pCAMBIA2301G-IP is that template is made pcr amplification, and product is after low melting-point agarose reclaims purifying.Carry out probe mark by digoxigenin labeled and detection kit operational manual.The commentaries on classics film of DNA and fixation procedure by the literary composition (Yan Ziying. Wang Hailin is translated, fine works molecular biology experiment guide [M] Beijing. Science Press, 1998, method 35-36) is carried out.Prehybridization, hybridization and process color are undertaken by digoxigenin labeled and detection kit operational manual.Southern is hybridized positive signals person, is confirmed as pseudomonas pseudoalcaligenes insecticidal protein gene transformant.

3.4.2 the pest-resistant experiment of insecticidal protein gene transformant

The blade of getting insecticidal protein gene transformant (T1 generation) the bamboo locust of feeding was added up larval mortality after four days.The result shows that clover insecticidal protein gene transformant has very high insect-resistance.This result has proved that the pseudomonas pseudoalcaligenes insecticidal protein gene among the present invention can efficiently express in clover, and insecticidal effect is remarkable.

Biological material specimens

Preservation date: on September 10th, 2003

Deposit number: 1001

Classification name: Pseudomonas Pseudoalcaligenes

Depositary institution's title: 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, Institute of Microorganism, Academia Sinica's nucleotide sequence table

＜110〉open outstanding Zhao and build Yang Zhirong

＜120〉pseudomonas pseudoalcaligenes insecticidal protein gene

<160>1

<170>PatentIn?Version?2.1

<210>1

<211>1377

<212>DNA

＜213〉pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes)

<220>

<221>gene

<222>(1)…(1377)

<220>

<221>Promoter

<222>(1)…(246)

<220>

<221>sig_peptide

<222>(247)…(313)

<220>

<221>mat_peptide

<222>(314)…(1075)

<220>

<221>CDS

<222>(247)…(1078)

<220>

<221>gene

<222>(1)…(1367)

<400>1

1?GGATCCCGGC?TTGCGGCCCC?CCTTTGTTTC?GCAAGACGGC?GTGGTCTTTG

51?AACTGGAAAC?CTTGGACCCG?CGACCCTGGC?GCGCTTTTTG?GGAAGCAGTG

101?GGCGTACCGG?CCGATCTGGC?TGGTCAGGCC?TGGAAAGCGT?TTTTGCTGCG

151?CTATGCCAAG?GCGGTGTGCC?CTGTGCCAGC?GGCATGTTTA?AGCAGTCTGC

201?AAGCCCTGAA?CTTTGCCCGT?TTGCAGGAAC?TGGCCCTGCA?GACGGGA

248?ATG?GCT?ATC?TTG?CCG?GTA?CGT?ACC?CCG?GCT?CAA?CGC?CAG?AGC?GTT

1?MET?Ala?Ile?Leu?Pro?Val?Arg?Thr?Pro?Ala?Gln?Arg?Gln?Ser?Val

293?AGT?GAT?TAC?GCG?GCC?TTG?GCC?GGT?GTG?TGG?CAG?CAT?CAG?AGT?CAT

16?Ser?Asp?Tyr?Ala?Ala?Leu?Ala?Gly?Val?Trp?Gln?His?Gln?Ser?His

338?GCC?GCG?CCC?GGC?TCA?TTG?AGC?ACC?TTG?CCG?TCC?AAG?GCC?GAT?TTG

31?Ala?Ala?Pro?Gly?Ser?Leu?Ser?Thr?Leu?Pro?Ser?Lys?Ala?Asp?Leu

383?CCC?CTG?CGT?GGT?TTG?CGG?GTG?GTG?GAG?TCC?TGC?CGT?CGG?ATT?CAA

46?Pro?Leu?Arg?Gly?Leu?Arg?Val?Val?Glu?Ser?Cys?Arg?Arg?Ile?Gln

428?GGG?CCT?ATT?GCC?GGT?CAC?TTG?CTG?GCG?CTG?CTG?GGG?GCG?GAA?GTG

61?Gly?Pro?Ile?Ala?Gly?His?Leu?Leu?Ala?Leu?Leu?Gly?Ala?Glu?Val

473?ATT?CGT?CTG?GAA?CCG?CCC?GGT?GGT?GAT?CCC?TTG?CGT?GCC?ATG?CCG

76?Ile?Arg?Leu?Glu?Pro?Pro?Gly?Gly?Asp?Pro?Leu?Arg?Ala?MET?Pro

518?CCG?TGC?GTG?GAT?GGC?TGC?TCG?GTG?CGC?TTT?GAT?GCG?CTC?AAT?CAA

91?Pro?Cys?Val?Asp?Gly?Cys?Ser?Val?Arg?Phe?Asp?Ala?Leu?Asn?Gln

563?TTC?AAG?ACG?GTG?CAG?GAA?GTG?GAC?ATC?AAA?TCG?GCT?CAA?GGC?CGC

106?Phe?Lys?Thr?Val?Gln?Glu?Val?Asp?Ile?Lys?Ser?Ala?Gln?Gly?Arg

608?CAG?GCC?ATT?TAC?GAA?TTG?GTG?AGC?CAG?TCT?GAT?GTA?TTT?CTG?CAT

121?Gln?Ala?Ile?Tyr?Glu?Leu?Val?Ser?Gln?Ser?Asp?Val?Phe?Leu?His

653?AAC?TGG?GCA?CCC?GGC?AAG?GCG?GCC?GAG?CTG?CAA?CTG?GAT?GCC?CAG

136?Asn?Trp?Ala?Pro?Gly?Lys?Ala?Ala?Glu?Leu?Gln?Leu?Asp?Ala?Gln

698?GAC?TTG?CAC?GCG?GTG?CGC?CCG?GAT?CTG?GTC?TAC?GCC?TAT?GCG?GGC

151?Asp?Leu?His?Ala?Val?Arg?Pro?Asp?Leu?Val?Tyr?Ala?Tyr?Ala?Gly

743?GGT?TGG?GGT?CAG?GAG?CAA?GTG?GAC?GCA?CCG?GGC?ACG?GAC?TTC?ACG

166?Gly?Trp?Gly?Gln?Glu?Gln?Val?Asp?Ala?Pro?Gly?Thr?Asp?Phe?Thr

788?GTG?CAA?GCC?TGG?TCG?GGT?ATT?GCT?CAC?ACC?ATT?TCT?CAA?ACC?TCG

181?Val?Gln?Ala?Trp?Ser?Gly?Ile?Ala?His?Thr?Ile?Ser?Gln?Thr?Ser

833?GAT?GCA?CGG?GGC?GGG?TCG?TTG?TTT?ACG?GTG?CTG?GAT?GTG?CTG?GGC

196?Asp?Ala?Arg?Gly?Gly?Ser?Leu?Phe?Thr?Val?Leu?Asp?Val?Leu?Gly

878?GGG?GTG?ATG?GCC?GCG?CTG?GGT?ATC?AGT?GCC?GCC?TTG?CTG?CGC?CGG

211?Gly?Val?MET?Ala?Ala?Leu?Gly?Ile?Ser?Ala?Ala?Leu?Leu?Arg?Arg

923?GGC?CTG?AGC?GGG?TCG?GGC?TTG?CGG?GTA?GAC?AGC?TCC?TTG?TTG?GCC

226?Gly?Leu?Ser?Gly?Ser?Gly?Leu?Arg?Val?Asp?Ser?Ser?Leu?Leu?Ala

968?ACG?GCC?GAT?CAT?CTG?GCC?CAG?GCC?GTT?TCT?CCC?ATC?AGT?AAA?ACT

241?Thr?Ala?Asp?His?Leu?Ala?Gln?Ala?Val?Ser?Pro?Ile?Ser?Lys?Thr

1013?GGC?GTG?TCG?GCG?GTG?TTC?CAG?ACG?GGC?GAG?GGC?TTC?ATC?GTC?ATC

256?Gly?Val?Ser?Ala?Val?Phe?Gln?Thr?Gly?Glu?Gly?Phe?Ile?Val?Ile

1058?GAC?TGC?CAG?GAT?CAA?ACG?TGA

271?Asp?Cys?Gln?Asp?Gln?Thr? ^***

1079?CT?GCATGCCCTG?GCCGGGTGGC

1101?TGAATGTGTC?GCCCGATGCT?GTCTGGACGG?TTTTGCCGGA?TCGTCTTCTG

1151?TCCCAGTCTG?CCCGTAGCCT?GGAAACGCAA?CTGGATGTGC?TGGGCATACC

1201?GGCCCGCCGC?GTCCATAGCA?ATCTGGCGCA?ACTGCGTGCT?GATCCACGTC

1251?TGACGTCTCA?TTTCCATGAC?AAGGGCTATT?CGTCTGTTAA?TTCTCCCTGG

1301?AGGTTTTTAT?GAATCACGCT?GGCATCATCG?ATCTGGTCCC?TGCGTCATTG

1351?CGCCAACGCT?GGATAGAGGA?CGGTACC

Claims (8)

1. insect-killing protein, it has aminoacid sequence as follows:

1?MET?Ala?Ile?Leu?Pro?Val?Arg?Thr?Pro?Ala?Gln?Arg?Gln?Ser?Val

16?Ser?Asp?Tyr?Ala?Ala?Leu?Ala?Gly?Val?Trp?Gln?His?Gln?Ser?His

31?Ala?Ala?Pro?Gly?Ser?Leu?Ser?Thr?Leu?Pro?Ser?Lys?Ala?Asp?Leu

46?Pro?Leu?Arg?Gly?Leu?Arg?Val?Val?Glu?Ser?Cys?Arg?Arg?Ile?Gln

61?Gly?Pro?Ile?Ala?Gly?His?Leu?Leu?Ala?Leu?Leu?Gly?Ala?Glu?Val

76?Ile?Arg?Leu?Glu?Pro?Pro?Gly?Gly?Asp?Pro?Leu?Arg?Ala?MET?Pro

91?Pro?Cys?Val?Asp?Gly?Cys?Ser?Val?Arg?Phe?Asp?Ala?Leu?Asn?Gln

106?Phe?Lys?Thr?Val?Gln?Glu?Val?Asp?Ile?Lys?Ser?Ala?Gln?Gly?Arg

121?Gln?Ala?Ile?Tyr?Glu?Leu?Val?Ser?Gln?Ser?Asp?Val?Phe?Leu?His

136?Asn?Trp?Ala?Pro?Gly?Lys?Ala?Ala?Glu?Leu?Gln?Leu?Asp?Ala?Gln

151?Asp?Leu?His?Ala?Val?Arg?Pro?Asp?Leu?Val?Tyr?Ala?Tyr?Ala?Gly

166?Gly?Trp?Gly?Gln?Glu?Gln?Val?Asp?Ala?Pro?Gly?Thr?Asp?Phe?Thr

181?Val?Gln?Ala?Trp?Ser?Gly?Ile?Ala?His?Thr?Ile?Ser?Gln?Thr?Ser

196?Asp?Ala?Arg?Gly?Gly?Ser?Leu?Phe?Thr?Val?Leu?Asp?Val?Leu?Gly

211?Gly?Val?MET?Ala?Ala?Leu?Gly?Ile?Ser?Ala?Ala?Leu?Leu?Arg?Arg

226?Gly?Leu?Ser?Gly?Ser?Gly?Leu?Arg?Val?Asp?Ser?Ser?Leu?Leu?Ala

241?Thr?Ala?Asp?His?Leu?Ala?Gln?Ala?Val?Ser?Pro?Ile?Ser?Lys?Thr

256?Gly?Val?Ser?Ala?Val?Phe?Gln?Thr?Gly?Glu?Gly?Phe?Ile?Val?Ile

271?Asp?Cys?Gln?Asp?Gln?Thr

2. coding claim 1 described proteinic nucleotide sequence.

3. according to the described nucleotide sequence of claim 2, it has following sequence:

1?GGATCCCGGC?TTGCGGCCCC?CCTTTGTTTC?GCAAGACGGC?GTGGTCTTTG

51?AACTGGAAAC?CTTGGACCCG?CGACCCTGGC?GCGCTTTTTG?GGAAGCAGTG

101?GGCGTACCGG?CCGATCTGGC?TGGTCAGGCC?TGGAAAGCGT?TTTTGCTGCG

151?CTATGCCAAG?GCGGTGTGCC?CTGTGCCAGC?GGCATGTTTA?AGCAGTCTGC

201?AAGCCCTGAA?CTTTGCCCGT?TTGCAGGAAC?TGGCCCTGCA?GACGGGAATG

251?GCTATCTTGC?CGGTACGTAC?CCCGGCTCAA?CGCCAGAGCG?TTAGTGATTA

301?CGCGGCCTTG?GCCGGTGTGT?GGCAGCATCA?GAGTCATGCC?GCGCCCGGCT

351?CATTGAGCAC?CTTGCCGTCC?AAGGCCGATT?TGCCCCTGCG?TGGTTTGCGG

401?GTGGTGGAGT?CCTGCCGTCG?GATTCAAGGG?CCTATTGCCG?GTCACTTGCT

451?GGCGCTGCTG?GGGGCGGAAG?TGATTCGTCT?GGAACCGCCC?GGTGGTGATC

501?CCTTGCGTGC?CATGCCGCCG?TGCGTGGATG?GCTGCTCGGT?GCGCTTTGAT

551?GCGCTCAATC?AATTCAAGAC?GGTGCAGGAA?GTGGACATCA?AATCGGCTCA

601?AGGCCGCCAG?GCCATTTACG?AATTGGTGAG?CCAGTCTGAT?GTATTTCTGC

651?ATAACTGGGC?ACCCGGCAAG?GCGGCCGAGC?TGCAACTGGA?TGCCCAGGAC

701?TTGCACGCGG?TGCGCCCGGA?TCTGGTCTAC?GCCTATGCGG?GCGGTTGGGG

751?TCAGGAGCAA?GTGGACGCAC?CGGGCACGGA?CTTCACGGTG?CAAGCCTGGT

801?CGGGTATTGC?TCACACCATT?TCTCAAACCT?CGGATGCACG?GGGCGGGTCG

851?TTGTTTACGG?TGCTGGATGT?GCTGGGCGGG?GTGATGGCCG?CGCTGGGTAT

901?CAGTGCCGCC?TTGCTGCGCC?GGGGCCTGAG?CGGGTCGGGC?TTGCGGGTAG

951?ACAGCTCCTT?GTTGGCCACG?GCCGATCATC?TGGCCCAGGC?CGTTTCTCCC

1001?ATCAGTAAAA?CTGGCGTGTC?GGCGGTGTTC?CAGACGGGCG?AGGGCTTCAT

1051?CGTCATCGAC?TGCCAGGATC?AAACGTGACT?GCATGCCCTG?GCCGGGTGGC

1101?TGAATGTGTC?GCCCGATGCT?GTCTGGACGG?TTTTGCCGGA?TCGTCTTCTG

1151?TCCCAGTCTG?CCCGTAGCCT?GGAAACGCAA?CTGGATGTGC?TGGGCATACC

1201?GGCCCGCCGC?GTCCATAGCA?ATCTGGCGCA?ACTGCGTGCT?GATCCACGTC

1251?TGACGTCTCA?TTTCCATGAC?AAGGGCTATT?CGTCTGTTAA?TTCTCCCTGG

1301?AGGTTTTTAT?GAATCACGCT?GGCATCATCG?ATCTGGTCCC?TGCGTCATTG

1351?CGCCAACGCT?GGATAGAGGA?CGGTACC

4. an insecticidal proteins separation purification method comprises the step of the described insecticidal proteins of claim 1 being used DEAE-Mierocrystalline cellulose-32 column chromatography, Sephadex G-100 gel-filtration, the separation and purification of SDS-polyacrylamide gel electrophoresis.

5. a method of producing pest-resistant transfer-gen plant comprises the step that the described insecticidal protein gene of claim 3 is imported plant.

6. in accordance with the method for claim 5, it is characterized in that described insecticidal protein gene is fixed a point to insert intermediate carrier pCAMBIA2301G, by the During Agrobacterium plant.

7. an insect-resistant transgenic plant is characterized in that, it closes the requirement 3 described insecticidal protein genes of having the right.

8. according to the described plant of claim 7, it is characterized in that it is an alfalfa plants.

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