CN1398970A - Glacial nuclear active gene and its expression vector and engineering bacterium - Google Patents

Abstract

The present invention is glacial nuclear active gene and its expression vector and engineering bacterium, belonging to the field of biological prevention and treatment technology. The present invention relates to a kind of glacial nuclear gene sequence and its coded glacial nuclear protein amino acid sequence as well as pronucleus expression vector mob-Tn5-iceA capable of being transferred inside host body. Using the vector fo the present invention can introduce glacial nuclear gene into other bacterial strain, and integrate it to chromosome DNA of host bacterium to realize high-efficiency expression of the glacial nuclear gene. The present invention has high application value to promoting freeze killing of pest.

Description

Ice nucleation activity gene, expression vector and engineering bacteria

Technical field of the present invention:

The invention belongs to the biological control technical field.Further, the present invention relates to ice nucleation activity gene, its cloning process, expression vector and engineering bacteria.Further, the present invention relates to the aminoacid sequence of ina gene sequence, ice-nucleating activity protein, the expression vector mob-Tn5-iceA of many hosts' transfers of ina gene and chromosomal integration.

Research background of the present invention:

Some bacteriums can be created in the ice-nucleus of tool catalytic activity more than-5 ℃, and these bacteriums are called as ice nucleating bacteria.It is reported that many kinds of 20 in the bacterium or mutation can produce the strongest heterogeneous ice-nucleus of nature activity; The ice nucleation activity of ice nucleating bacteria is gene-determined by ice nucleation activity.Clone's bacterial ice nuclei protein gene, can in environmental safety (environmentally safe) or hereditary safety (genetically regarded as safe) bacterial strain, efficiently express, can be used for bacterial cell surface display, high quick detection and the gene of making reports, show the applications well prospect.The ice nucleation protein that ice nucleating bacteria and ina gene are expressed can be applied in the short aspects such as freeze concentration, lyophilize and quality processing of freezing desinsection, artificial snow, ice making, food at catalysis water freezing under the comparatively high temps (1～-5 ℃).

From Orser (Orser, C. etc., J.Bacteriol., 1985,164:359-366) from Erwinia herbicola, be cloned into since first ina gene iceE, successively have 7 bacterial ice nuclei genes successively to obtain clone and order-checking.Two kinds of schemes are taked in the bacterial ice nuclei gene clone usually, early stage people such as Orser (Orser, C. etc., J.Bacteriol., 1985,164:359-366) made up the gene library of P.syringae and E.herbicola, and directly screened the ice nucleation activity positive colony with duplicating freezing technology (replica freezing technique).The another kind clone approach of bacterial ice nuclei gene is to utilize homology ina gene produced in fragments probe, from genomic library, angle and get target gene, as (Michigami such as Michigami, Y. etc., Biosci.Biotech.Biochem., 1994,58:763-764) from E.uredovora KUIN-3, be separated to a new ina gene inaU.At present, also nobody utilizes the method for PCR to be cloned into the ice nucleation activity gene, also not the transgenic engineered bacteria of ice nucleation activity gene integration to the chromosomal DNA.

Therefore, obtain ina gene, and ina gene is incorporated into obtains on the chromosomal DNA that transgenosis is short freezes the killing gene engineering bacteria, just have very strong practicality with the method for PCR.

Content of the present invention:

Purpose of the present invention:

The regional important agricultural fruit tree pest insect of China " three Norths " includes twenties kinds of Pyrausta nubilalis (Hubern)., bollworm, loxostege sticticalis, eating-core bean worm, pine moth, Anoplophora glabripenis etc., also has storage pest in addition, and they often cause enormous economic loss.After people recognized all drawbacks that simple chemical dispensary brings, bio-control method was just paid attention to.These insects cold winter overwintering mortality height, season warm winter mortality ratio low, and overwintering mortality just is the important factor of decision hazard rating in next year, forcing down the worm source radix of surviving the winter is the effective measure that Pest Control takes place.The short desinsection theory of freezing of ice nucleating bacteria is authentic, but being difficult to use existing these wild-type ice nucleating bacterias freezes extremely field and storage insect survives the winter, major cause has two: one, be used for the short ice nucleating bacteria that freezes desinsection research at present and in insect gut, can not stablize surely and grow, easily excreted after use in the field and lose and freeze effect extremely by polypide; Its two, these ice nucleating bacterias colonization ability of growing nonparasitically upon another plant on plant materials is strong, easily brings out frost after use in the field.This two aspects reason has become the short global problem that freezes in the pesticidal applications of obstruction.

The present invention utilizes PCR method to obtain the bacterial ice nuclei gene, utilizes this gene constructed a kind of mob-Tn5-iceA expression vector then; Further, utilize this carrier and the host bacterium that is fit to has made up to be suitable for freezing and kills the field wintering insect and the short of storage pest frozen the killing gene engineering bacteria, and finish Pyrausta nubilalis (Hubern). and bollworm are frozen the test job of effect extremely.

Technical scheme of the present invention:

1. the clone of iceA gene in pineapple Erwinia 110 bacterial strains

Extract the chromosomal DNA of pineapple Erwinia (Erwiniaananas) 110, electrophoresis detection (Fig. 1) with reference to " fine works molecular biology experiment guide " commercial law.With pineapple Erwinia 110 chromosomal DNAs is template, carries out the pcr amplification ina gene with high-fidelity DNA Taq plusI DNA polymerase.Experiment is carried out with reference to " modern molecular biology experimental technique ".The primer is as follows:

Primer 1 (+) (forward primer): 5 ' CCCTAATGAAAT TTTAGAC ATG3 ' 27bp

Primer 1 (-) (reverse primer): 5 ' CCTCTG TTATGGCGATTATTCTTCGGG3 ' 27bp

Utilize agarose electrophoresis to detect amplification (Fig. 2).The PCR segment that provides according to TaKaRa Biotech company reclaims test kit (Fragment Recovery Kit), from gel, reclaim 4.0kb PCR product to specifications, PCR is reclaimed product to be connected with the pMD18-T cloning vector, transformed into escherichia coli JM109, detect and enzyme is cut evaluation and obtained positive colony (table 1), this positive colony called after pINA105 according to ice nucleation activity.This recombinant plasmid of alkaline lysis method of extracting carries out single, double enzyme and cuts with being positioned at the EcoRI of both sides, pMD18-T carrier cloning site and SalI, identifies be connected (Fig. 3) of goal gene and cloning vector.Determined dna sequence is finished by Shanghai Bo Ya bio-engineering corporation, and homology analysis shows that with the homology maximum of ina gene inaA, amino acid and nucleotide homology are 94%.This unnamed gene is iceA, logins on Genebank, and accession number is AF387802.

(2.mob-Tn5-iceA being called for short pSZice1) plasmid construction

Extract pINA105 and pSZ21 plasmid (this plasmid is planted living Mr. ShenBing Fu of institute by Chinese Academy of Sciences Shanghai to be provided) DNA, carry out double digestion with BamHI+SalI respectively, reclaim the ina gene fragment of about 4.0Kb and the pSZ21 carrier segments of about 11Kb.Enzyme was cut system in 37 ℃ of incubation 2-4 hours, detected enzyme with 1% agarose electrophoresis and cut result (Fig. 7).Adopt Tataka PCR segment to reclaim test kit and reclaim endonuclease bamhi.Ina gene and carrier pSZ21 fragment that directed ligase enzyme cuts back to close guarantee that pINA105 and the pSZ21 enzyme in the reaction system cuts back to close segmental mol ratio more than 3: 1.Get 5uL connection product and be used to transform the JM109 competent cell, coated plate on the LB/Km+Cm flat board.The several single bacterium colonies of picking extract plasmid at random, carry out double digestion (Fig. 8, Fig. 9) with BamHI+Sa1I respectively, and positive colony is preserved in screening.Picking positive colony JM109 (pSZice1) measures ice nucleation activity (table 1) with Vali small droplets freezing method.

3. the short structure that freezes the killing gene engineering bacteria

Adopt the conjugal transfer method that recombinant plasmid mob-Tn5-iceA is imported among enterobacter cloacae (Enterobactercloacae) Enc1.2.022, the Enc1.181.Vali small droplets freezing method is measured ice nucleation activity.Transposition by the Tn5 transposon is integrated into ina gene on the enterobacter cloacae chromosomal DNA, makes up the stable transgenic engineered bacteria of ice nucleation activity.The recombinant bacterial strain of realization ina gene constitutive expression is named and is Enc181 ^IceAnd Enc2022 ^IceSurvey the stability (table 1) of engineering strain ice nucleation activity checking ina gene with Vali small droplets freezing method.

4. the short desinsection biological activity determination that freezes

Engineering bacteria is made into bacterium liquid after the dull and stereotyped cultivation of LB.The bacterium liquid of step preparation on the abundant dipping of fresh cotton floral leaf, it is standby to dry up the back with fan.Just the hatching larva is used not 23 ± 1 ℃ of raisings of libation at an ancient wedding ceremony bacterium fresh cotton leaves 3 days, continues raising with the cotton leaf of libation at an ancient wedding ceremony bacterium then.Bollworm raise bacterium after 3 days 23 ± 1 ℃ hungry 12 hours down, put 4 ℃ of following precoolings 24 hours again, measure the polypide supercooling point in the artificial white case.Remaining insect continues at 23 ± 1 ℃ of tube feed that place an order with libation at an ancient wedding ceremony bacterium fresh cotton leaves not and supports, and gets 1 time sample after 3 days, and sample is measured supercooling point after hungry and subzero treatment.After this, successively the 5th, 7,9 day sampling and measuring, each every processing is got 10～20 cephalont and is experimentized.Get 5 cephalont from the insect of having measured supercooling point at random, polypide was through 75% ethanol surface sterilization 20 seconds, and dH sterilizes ₂O grinds dH after washing 3 times in mortar ₂Go up coating at LB flat board (adding 20ug/mL Km and 1ug/mL amphotericin) behind the O dilution different multiples, wait to grow behind the bacterium colony to measure and identify and add up engineering bacteria quantity by colonial morphology and ice nucleation activity.The tender stem 1～1.5cm of corn segment, be put into dry up after soaking in the bacterium liquid standby.Just incubate corn borer larvae and do not raise 3 days down for 23 ± 1 ℃, continue to raise with soaking the tender stem of bacterium corn then with soaking the tender stem of bacterium corn.Pyrausta nubilalis (Hubern). raise bacterium after 3 days 23 ± 1 ℃ hungry 12 hours down, again 4 ℃ of following precoolings 24 hours, measure the polypide supercooling point in the artificial white case.Remaining insect does not continue down for 23 ± 1 ℃ to raise with soaking the tender stem of bacterium corn, gets 1 time sample after 3 days, and sample is measured supercooling point after hungry and subzero treatment.After this, successively the 5th, 7,9 day sampling and measuring, every processing is got 10～20 cephalont at every turn and is used for test.With the engineering bacteria quantity in the method test Pyrausta nubilalis (Hubern). enteron aisle of engineering bacteria quantity in the said determination bollworm enteron aisle.Do the subzero treatment experiment with Pyrausta nubilalis (Hubern). and the bollworm raised behind the bacterium 6 days, compare with close worm Pyrausta nubilalis (Hubern). in age and the bollworm of not raising bacterium.To test insect places in the artificial white case, speed cooling by 10 ℃/hour, after waiting to drop to temperature required (5 ℃ ,-7 ℃), keep different time (3 hours, 6 hours, 12 hours) respectively, touching polypide freezes judging whether, treat under relevant temperature, to handle after 12 hours, the insect taking-up is placed on 23 ± 1 ℃ placed 1 hour down, the statistics mortality ratio.

Beneficial effect of the present invention:

The expression vector mob-Tn5-iceA that utilizes the present invention to make up, can import in the enterobacter cloacae bacterial strain insect pest being had short iceA gene transformation of freezing insecticidal action, and be incorporated on the chromosomal DNA, realize efficiently expressing of gene, this gene expression product has the short insecticidal action of freezing to insect; Further, by the present invention the iceA genetic engineering bacterium is frozen and kill Pyrausta nubilalis (Hubern). and bollworm; And cloning process involved in the present invention, iceA gene order, aminoacid sequence, transgenic engineered bacteria, expression vector mob-Tn5-iceA are also peculiar by the present invention.

Description of drawings:

Fig. 1 is the chromosomal DNA of the several bacterial strains of pineapple Erwinia (Erwinia ananas).

Wherein, road M, 1,2,3 and 4 represents λ DNA/HindIII respectively; Bacterial strain 94; Bacterial strain 110; Bacterial strain 1850; Bacterial strain M232A.

Fig. 2 is the pcr amplification result of ina gene.

Wherein, road M, 1,2,3 and 4 represents respectively: λ DNA/HindIII; Bacterial strain M232A; Bacterial strain 110; Bacterial strain 94; The amplification of bacterial strain 1850.

Fig. 3 cuts qualification result for the enzyme of recombinant plasmid.

Wherein, road M, 1,2,3,4,5,6,7,8 and 9 represents λ DNA/HindIII respectively; No 1-3transformants/EcoRI; No 1-3/SalI; The enzyme of No 1-3/E+S is cut the result.

The fragment of Fig. 4 for producing behind the exonuclease III digested plasmid pINA105 different time.

Wherein, road M ₁, M ₂, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 and 24 represent λ DNA/HindIII respectively; λ DNA/EcoRI+HindIII; The fragment that produces behind the exonuclease III digested plasmid pINA105 different time.

Fig. 5 cuts qualification result for the enzyme of deletion mutant.

Wherein, road M ₁, M ₂, 1,2,3,4,5 and 6 represent λ DNA/HindIII respectively; λ DNA/EcoRI+HindIII; The EcoRI+HindIII enzyme of different mutant strains is cut.

Fig. 6 is the PCR qualification result of deletion mutant.

Wherein, road M ₁, M ₂, 1,2,3,4,5 and 6 represent λ DNA/HindIII respectively; λ DNA/EcoRI+HindIII; The PCR of different mutant strains.

The enzyme of Fig. 7 plasmid pSZ21 and pINA105 is cut evaluation.

Wherein, road M, 1,2 and 3 represents λ DNA/HindIII respectively; PSZ21 cuts with the BamHI enzyme; PSZ21 cuts with the BamHI+SalI enzyme; PINA105 cuts with the BamHI+SalI enzyme.

Fig. 8 is recombinant plasmid pSZice1～6 electrophoretograms.

Wherein, road M, 1,2,3,4,5 and 6 represents λ DNA/HindIII respectively; PSZice1～6

Fig. 9 is the restriction analysis of recombinant plasmid pSZice1～6.

Wherein, road M, 1,2,3,4,5 and 6 represents λ DNA/HindIII respectively; PSZice1～6 usefulness BamHI+SalI enzymes are cut.

Figure 10 is the structure schema of recombinant plasmid mob-Tn5-iceA (pSZice1).

Electrophoretic analysis after Figure 11 changes enterobacter cloacae over to and swivel base takes place for recombinant plasmid pSZice1 engages.

Wherein, road M, 1,2,3 and 4 represents λ DNA/HindIII, Enc1.181 (pSZice1), Enc1.2022 (pSZice1), Enc181 respectively ^IceAnd Enc2022 ^Ice

Figure 12 is the PCR qualification result of engineering strain.

Wherein, road M, 1,2,3 and 4 represents λ DNA/HindIII, Enc181 respectively ^Ice, Enc2022 ^IceEnc1.181 and Enc2.2022.

Specific embodiments of the present invention:

Below narrate embodiments of the invention.Should be noted that embodiments of the invention have only illustration for the present invention, and effect without limits.

What need particularly point out is, although the detailed in an embodiment clone who describes ina gene, at expression in escherichia coli, be incorporated in the enterobacter cloacae chromosomal DNA and efficiently express, yet this does not mean that gene of the present invention is only limited to transform and produce to have with the enterobacter cloacae bacterial strain insect is had the short engineering bacteria of insecticidal action that freezes.

Therefore, use the short desinsection that freezes of iceA gene described in the invention, use expression vector described in the invention, import in any microorganism, plant or its tissue or the cell with any method that those of ordinary skills were had, and the microorganism with any ice nucleation activity, the plant that obtain therefrom, and such plant offspring's seed, hybridization and transformation offspring, include within interest field of the presently claimed invention.

The clone of iceA gene in embodiment 1, the pineapple Erwinia 110-bacterial strain

With reference to the chromosomal DNA of " fine works molecular biology experiment guide " (Yan Ziying, Wang Hailin are translated, Science Press, Beijing, 1998) extraction pineapple Erwinia 110 bacterial strains, as the pcr amplification template.With pineapple Erwinia 94,110,1850 and M232A chromosomal DNA is template, and according to having cloned ina gene design primer abroad, primer is given birth to worker biotech firm by Shanghai and synthesized.Carry out the pcr amplification ina gene with high-fidelity DNA Taq plusI DNA polymerase.

Primer1 (+) (forward primer): 5 ' CCCTAATGAAAT

TTTAGAC ATG3 ' 27bp

Primer1 (-) (reverse primer): 5 ' CCTCTG TTATGGCGATTATTCTTCGGG3 ' 27bp

Experiment is carried out with reference to " modern molecular biology experimental technique " (Lu Shengdong, Higher Education Publishing House, Beijing, 1993).Detect amplification with 1% agarose electrophoresis.The ice nucleation activity bacterial strain is amplified 1 specific band (Fig. 2) of about 4Kb.PCR reclaims product and is connected with the pMD18-T cloning vector, connect product and transform the JM109 competent cell, conversion is with reference to " the Protocols and ApplicationsGuide " (1996 of Promega company, Promega Corporation) 51-54 page or leaf and document (Jin Dongyan, Li Mengfeng, molecular cloning experiment guide (second edition), Beijing, Science Press, 1993:626～628) carry out.Transforming on the flat board, place respectively to be added with 200uL sterilization dH with toothpick several white colonies of picking at random ₂Make bacteria suspension in the EP pipe of O, and usefulness Vali small droplets freezing method mensuration bacteria suspension ice nucleation activity (Vali, G.J., Atmos.Sci., 1971,28:402-409).

Use the alkaline lysis method of extracting recombinant plasmid, carry out single, double enzyme and cut recombinant plasmid, identify the situation that is connected of goal gene and cloning vector with being positioned at the EcoRI of both sides, pMD18-T carrier cloning site and SalI.Single endonuclease digestion all produces the fragment of 1 about 6.7Kb (carrier and amplified production length sum), and double digestion all produces 2 fragments of 2.6Kb (carrier) and 4Kb (amplified production).Ice nucleation activity is measured and enzyme is cut the result and shown the positive colony that obtains being inserted with purpose fragment (ina gene), with transformant with recombinant plasmid called after pINA105.Adopt the Sanger dideoxy chain termination to identify and ice nucleation activity mensuration is the male recombinant plasmid and carries out sequencing to cutting through enzyme.Order-checking is finished by Shanghai Bo Ya Bioisystech Co., Ltd, gene intermediary highly repetitive sequence (about 600bp) carries out directed sequencing after adopting ExonucleaseIII (Exo III) to handle recombinant plasmid generation nested deletion mutant, experimental implementation is carried out with reference to the Exo III working instructions of " molecular cloning experiment guide " and MBI company (Canada), choose the clone that can produce 1.6～2.3Kb be used for the order-checking (Bo Ya company in Shanghai finishes, adopt M13R (48) primer) (Fig. 4, Fig. 5, Fig. 6).The clone gene coding region total length 3921bp of institute (SEQ ID NO 1), encoded protein matter is 1306 amino acid (SEQ ID NO 2).The corresponding Rankine-Hugoniot relations of the two is listed in SEQ ID NO 3.

The structure of embodiment 2, prokaryotic expression carrier mob-Tn5-iceA (being called for short pSZice1)

Extract pINA105 and pSZ21 plasmid DNA, carry out double digestion and BamHI, SalI single endonuclease digestion with BamHI+SalI respectively, reclaim the ina gene fragment of about 4.0Kb and the pSZ21 carrier segments of about 11Kb.Reaction system detected enzyme with 1% agarose electrophoresis and cuts result (Fig. 7) in 37 ℃ of incubation 2-4 hours.Adopt the TatakaPCR segment to reclaim test kit and reclaim endonuclease bamhi.Directed ina gene and the carrier pSZ21 fragment that reclaims that connect guarantees that pINA105 and the pSZ21 enzyme in the reaction system cuts back to close segmental mol ratio more than 3: 1.Reaction system is handled 10 minutes deactivation T for 65 ℃ 22 ℃ of incubations 2～4 hours ₄Dna ligase, get 5uL connection product and be used to transform the JM109 competent cell, coated plate on the LB/Km+Cm flat board, the several single bacterium colonies of picking extract plasmid at random, carry out double digestion (Fig. 8 with BamHI+SalI respectively, Fig. 9), the result shows that selected clone all contains the external source insertion fragment of 11Kb carrier and 4Kb, therefore be the recombinant plasmid of expection, with 1～No. 6 recombinant plasmid difference called after pSZice1-6, pSZice1 called after mob-Tn5-iceA (Figure 10) with wherein can directly find out (the mob fragment of different components the recombinant plasmid from this name, Tn5 transposon and ina gene iceA) between position relation.The single colony inoculation 5mL of picking positive colony JM109 (pSZice1) KB liquid (containing 20ug/mL Km and 50ug/mL Cm), 25 ℃, 200rpm shake training 48 hours, and 4 ℃ of refrigerators were placed 2 hours, were diluted to 10 ⁸CFU/mL measures ice nucleation activity (table 1) with Vali small droplets freezing method.

Embodiment 3, ina gene engineering bacteria make up

Adopt the conjugal transfer method recombinant plasmid pSZice1 (mob-Tn5-iceA) is imported in the enterobacter cloacae (Enterobacter cloacae) (table 1, Figure 11).The single colony inoculation 5mLKB of picking Enc (pSZice1) (containing Km and Cm), 25 ℃, 200rpm shake the training 4 ℃ of refrigerators that spend the night and place that dilution is made into 10 after 2 hours ⁸CFU/mL, Vali small droplets freezing method is measured the recombinant bacterial strain Enc (pSZice1) (table 1) that ice nucleation activity is arranged.In containing the LB nutrient solution of Km and Cm, cultivated 15 hours, change the fresh LB of 5mL (not added with antibiotic) respectively over to 2% inoculum size again, cultivated 12-15 hour.So switching is transferred to 5mL again and contains in the LB nutrient solution of 50ug/mL acridine orange more than 5 times, and after 30 hours, with different extent of dilution coating LB flat boards, 30 ℃ of overnight incubation are duplicated and selected to observe growing state on the substratum, have Km in 30 ℃ of static cultivations ^rCm ^sThe bacterial strain of phenotype should be ina gene and has been incorporated into reorganization bacterium (Figure 12) on the karyomit(e), and the recombinant bacterial strain of realizing the ina gene constitutive expression is named and is Enc ^IceMeasure the ice nucleation activity (table 2) of engineering strain with Vali small droplets freezing method.

The ice nucleation activity of table 1 different strains is measured

Bacterial strain freezes the rate (%) of dripping

Strains?????????????Percentages?of?freezing??droplets(％)

-10℃???????-5℃?????-4℃???????-3℃

JM109(pSZice1)??????????100?????????100??????100????????94

Enc1.181(pSZice1)???????100?????????100??????100????????100

Enc1.2022(pSZice1)??????100?????????100??????100????????87

Enc181 ^ice??????????????100?????????100??????100????????100

Enc2022 ^ice?????????????100?????????100??????100????????92

E.ananas110?????????????100?????????100??????100????????100

JM109???????????????????0???????????0????????0??????????0

Enc1.181????????????????2???????????0????????0??????????0

Enc1.2022???????????????0???????????0????????0??????????0

Table 2 genetic engineering bacterium Enc181 ^IceAnd Enc2022 ^IceIna gene stability measure

Bacterial strain does not have the Km behind the different number of times of transferring continuously under the selective pressure ^rBacterium colony retention (%)

1 time 4 times 8 times 12 times Enc181 ^Ice100 100 100 100 Enc2022 ^Ice100 100 100 100

Embodiment 4, the short desinsection biological activity determination that freezes

Engineering bacteria after the dull and stereotyped activation of LB, picking list colony inoculation LB liquid (containing Km 20ug/mL), 200rpm, 33 ℃ are shaken training 12～15 hours, the centrifugal supernatant of abandoning, thalline is with sterilization dH ₂O is made into 10 ⁸～10 ⁹Cfu/mL bacterium liquid.Adopt back the fresh cotton floral leaf of 5～7 leaf phases from the field, fully the bacterium liquid of step preparation on the dipping is standby after fan dries up.To just hatch larva with writing brush chooses and the Glass tubing of libation at an ancient wedding ceremony bacterium fresh cotton leaves is housed not (in 2.5 * 7.5cm), 1～2 of every pipe seals with tampon, raised 3 days for 23 ± 1 ℃, insect is forwarded in the Glass tubing that the cotton leaf of libation at an ancient wedding ceremony bacterium is housed with writing brush then, 1 of every pipe continues to raise.

Bollworm was raised bacterium after 3 days, the part insect was transferred in the 1.5mL EP pipe of pipe lid punching, place 23 ± 1 ℃ hungry 12 hours down, put 4 ℃ of following precoolings 24 hours again, in artificial white case, measure the polypide supercooling point then, the results are shown in Table 3.Remaining insect forwards in the Glass tubing that libation at an ancient wedding ceremony bacterium fresh cotton leaves not is housed, and continues at 23 ± 1 ℃ of tube feed that place an order and supports, and changes 1 pipe (not libation at an ancient wedding ceremony bacterium fresh cotton leaves is housed) after 3 days, gets sample simultaneously 1 time, and sample is measured supercooling point after hungry and subzero treatment.

After this, successively the 5th, 7,9 day sampling and measuring, each every processing is got 10～20 cephalont and is experimentized.Bollworm is got food engineering bacteria Enc181 ^IceAnd Enc2022 ^IceAfter, supercooling point obviously improved after bollworm was got the food genetic engineering bacterium, about-10 ℃ of polypide brought up to-3 ℃～-4 ℃ from contrast, and increase rate is about 6 ℃, and two kinds of engineering bacterias are handled the raising effect no significant difference (table 3) to the bollworm supercooling point.Get 5 cephalont from the insect of having measured supercooling point at random, polypide was through 70% ethanol surface sterilization 20 seconds, and dH sterilizes ₂O grinds dH after washing 3 times in mortar ₂Go up coating at LB flat board (adding 20ug/mL Km and 1ug/mL amphotericin) behind the O dilution different multiples, wait to grow behind the bacterium colony to measure and identify and add up engineering bacteria quantity by colonial morphology and ice nucleation activity.

Table 3 bollworm is got food genetic engineering bacterium (Enc181 ^IceAnd Enc2022 ^Ice) after supercooling point (SCPs) change

The mistake that bacterial strain is raised different time polypide behind the bacterium is point (X ± S but _X, ℃)

0 day 3 days 5 days 7 days 9 days Enc181 ^Ice-3.54 ± 0.18-3.40 ± 0.17-4.01 ± 0.27-4.01 ± 0.38-4.7 ± 0.49Enc2022 ^Ice-4.07 ± 0.19-3.74 ± 0.23-3.70 ± 0.26-4.08 ± 0.34-4.83 ± 0.31CK-9.83 ± 0.87-9.53 ± 0.43-10.04 ± 0.43-9.39 ± 0.79-9.23 ± 0.82

Get the tender stem of trumpet period corn (top 1～2 joint) from the field, be cut into 1～1.5cm segment, be put into and soaked in the bacterium liquid 15～20 minutes, take out the back and dry up standby with fan with scissors.With writing brush newly hatched larvae is chosen and to be equipped with in the Cans that do not soak the tender stem of bacterium corn, 100～150 every bottle, seal with the bottle cap that has added thin wire gauze and one deck medicated napkin, raised 3 days down for 23 ± 1 ℃, then insect is forwarded to and be equipped with in the Cans that soak the tender stem of bacterium corn, 50～100 every bottle, continue to raise.Pyrausta nubilalis (Hubern). was raised bacterium after 3 days, the part insect was transferred in the 1.5mL EP pipe of pipe lid punching, place 23 ± 1 ℃ hungry 12 hours down, again 4 ℃ of following precoolings 24 hours, in artificial white case, measure the polypide supercooling point then.Remaining insect forwards to is equipped with in the Cans that do not soak the tender stem of bacterium corn, continues down to raise in 23 ± 1 ℃, after 3 days) change 1 bottle (be equipped with and do not soak the tender stem of bacterium corn), get sample simultaneously 1 time, sample is measured supercooling point after hungry and subzero treatment.After this, successively the 5th, 7,9 day sampling and measuring, every processing is got 10～20 cephalont at every turn and is used for test.After Pyrausta nubilalis (Hubern). was got the food engineering bacteria, its supercooling point significantly improved compared with the control, brought up to-3 ℃～-4 ℃ from-11 ℃～-17 ℃, and increase rate is about 10 ℃ (table 4).Get 5 cephalont from the insect of having measured supercooling point at random, polypide was through 70% ethanol surface sterilization 20 seconds, and dH sterilizes ₂O grinds dH after washing 3 times in mortar ₂Go up coating at LB flat board (adding 20ug/mL Km and 1ug/mL amphotericin) behind the O dilution different multiples, wait to grow behind the bacterium colony to measure and identify and add up engineering bacteria quantity by colonial morphology and ice nucleation activity.

Table 4 Pyrausta nubilalis (Hubern). is got food genetic engineering bacterium (Enc181 ^IceAnd Enc2022 ^Ice) after supercooling point (SCPs) change

The mistake that bacterial strain is raised different time polypide behind the bacterium is point (X ± S but _X, ℃)

0 day 3 days 5 days 7 days 9 days Enc181 ^Ice-3.89 ± 0.15-3.77 ± 0.20-4.24 ± 0.18-3.68 ± 0.35-4.42 ± 0.28Enc2022 ^Ice-3.81 ± 0.17-3.96 ± 0.51-3.61 ± 0.38-3.91 ± 0.19-4.51 ± 0.35ck-16.82 ± 1.30-12.2 ± 0.95-10.78 ± 0.81-11.23 ± 0.77-11.56 ± 1.26

Engineering strain Enc181 ^IceAnd Enc2022 ^IceIn Pyrausta nubilalis (Hubern). and bollworm enteron aisle, all can stablize surely and grow.In 7 days, the quantity of engineering strain in insect gut is up to 10 after raising bacterium ⁴～10 ⁵The cfu/ polypide was raised behind the bacterium 9 days, and two kinds of engineering bacterias all descend to some extent at the colonization ability of polypide enteron aisle, but quantity is still 10 ³Cfu/ polypide above (table 5).

Table 5 genetic engineering bacterium (Enc181 ^IceAnd Enc2022 ^Ice) Pyrausta nubilalis (Hubern). and bollworm enteron aisle colonization ability measure raise bacterium after different time bacterial number (log cfu/ polypide)

(my god) the Pyrausta nubilalis (Hubern). bollworm

Enc181 ^ice???Enc2022 ^ice?????????Enc181 ^ice???Enc2022 ^ice

0?????????????4.8??????????4.6?????????????????4.7??????????4.4

3?????????????5.0??????????4.9?????????????????5.1??????????4.9

5?????????????5.3??????????5.2?????????????????5.2??????????5.4

7?????????????4.7??????????5.0?????????????????4.8??????????4.6

9?????????????3.5??????????3.8?????????????????3.4??????????3.6

The raising of Pyrausta nubilalis (Hubern). and bollworm and to raise the bacterium method the same.Do the subzero treatment experiment with Pyrausta nubilalis (Hubern). and the bollworm raised behind the bacterium 6 days, compare with close worm Pyrausta nubilalis (Hubern). in age and the bollworm of not raising bacterium.To test insect and place in the artificial white case,, wait to drop to temperature required (5 ℃ by 10 ℃/hour speed cooling,-7 ℃) after, keep different time (3 hours, 6 hours, 12 hours) respectively, touching polypide freezes judging whether, treat under relevant temperature, to handle after 12 hours, the insect taking-up is placed on 23 ± 1 ℃ placed 1 hour down, the statistics mortality ratio, engineering bacteria is handled and has been significantly improved the freeze polypide rate of Pyrausta nubilalis (Hubern). after subzero treatment, and causes insect icing and dead.Handled 3 hours down at-5 ℃, the Pyrausta nubilalis (Hubern). that two kinds of engineering bacterias are handled is freezed the polypide rate and all reaches more than 80%, and then none freezes without contrast polypide that engineering bacteria is handled; After handling in 12 hours, the polypide freezing rate that 2 engineering bacterias are handled all reaches more than 90%, and the contrast insect does not still have and freezes; Handled 3 hours for-7 ℃, the Pyrausta nubilalis (Hubern). that engineering bacteria is handled is freezed the polypide rate and reaches more than 90%, handles that freezing rate reaches 100% after 6 hours, and without the contrast Pyrausta nubilalis (Hubern). of engineering bacteria processing; Handled 12 hours down at-7 ℃, its polypide freezing rate only is 7.4%.After 12 hours, most polypides (about 96%) are freezed to death the Pyrausta nubilalis (Hubern). that engineering bacteria is handled-5 ℃ of processing, and the contrast insect then all survives; Handled 12 hours for-7 ℃, it is all dead that engineering bacteria is handled polypide, and the mortality ratio of the polypide of contrast only is 7.4% (table 6).Handled 3 hours for-5 ℃, the polypide rate of freezing that engineering bacteria is handled bollworm promptly reaches about 80%, continues to handle 6～12 hours, have 92.3～93.7% polypides to freeze and freezed to death, and the contrast insect only has 7.1% to be freezed to death.Handled 3 hours for-7 ℃, handling in the insect has 93.7～100% to freeze, and continues to handle 6～12 hours, tests all that insect is freezed and dead, and the contrast insect only has 14.3% to freeze death (table 7).

Table 6 engineering bacteria (Enc181 ^IceAnd Enc2022 ^Ice) low temperature of Pyrausta nubilalis (Hubern). is frozen effect measuring extremely

Freeze polypide rate (%)

Bacterial strain-5 ℃-7 ℃ of cold mortalities (%) test worm number/

12h-5 ℃ of-7 ℃ of Temperature Treatment Enc181 of 3h 6h 12h 3h 6h ^Ice84.6 96.1 96.1 92.3 100 100 96.1 100 26Enc2022 ^Ice87.5 91.7 91.7 95.8 100 100 95.8 100 24CK 0.0 0.0 0.0 3.7 7.4 7.4 0.0 7.4 28

Table 7 engineering bacteria (Enc181 ^IceAnd Enc2022 ^Ice) to the short effect measuring that kills of the low temperature of bollworm

Freeze polypide rate (%)

Bacterial strain-5 ℃-7 ℃ of cold mortalities (%) test worm number

12h-5 ℃ of-7 ℃/Temperature Treatment Enc181 of 3h 6h 12h 3h 6h ^Ice76.9 92.3 92.3 100 100 100 92.3 100 13Enc2022 ^Ice87.5 93.7 93.7 93.7 100 100 93.7 100 16CK 0.0 7.1 7.1 7.1 14.3 14.3 7.1 14.3 14 are attached: dna sequence dna involved in the present invention and protein sequence (1) SEQ ID NO 1 (nucleotide sequence of iceA gene):

1 ATG AAA GAA GAT AAG GTT TTA ATA TTA CGT ACC TGT GCT AAT AAT ATG GCC GAT CAC GGC GGA ATC ATC 69 70 TGG CCG TTA AGC GGT ATC GTA GAG TGT AAA TAC TGG AAG CCG GTT AAA GGC TTT GAG AAC GGA CTA ACG 138 139 GGG CTA ATC TGG GGA AAA GGA TCG GAT TCA CCG CTG AGC CTG CAC GCT GAT GCC AGG TGG GTT GTC GCT 207 208 GAA GTG GAT GCC GAT GAG TGT ATC GCT ATT GAA ACT CAT GGC TGG ATT AAA TTT CCC CGT GCT GAG GTT 276 277 CTT CAC GTT GGA ACG AAA ACC AGT GCG ATG CAA TTT ATC CTG CAC CAT CGG GCT GAT TAC GTT GCC TCT 345 346 ACG GAA ATG CAG GCC GGT CCT GGA GCG CCT GAT GTC ACT TCA GAG GTA AAG GCA GGC AAT CGA TCG CTC 414 415 CCT GTA ACA GAT GAT ATT GAT GCG ACC ATC GAA TCA GGC AGT ACG CAG CCG ACA CAA ACG ATT GAA ATC 483 484 GCA ACC TAT GGC AGT ACG CTC AGC GGC ACG CAT CAG AGT CAG CTG ATT GCC GGA TAT GGC AGT ACT GAG 552 553 ACG GCG GGT GAT AGC AGC ACA TTA ATT GCC GGT TAT GGT AGC ACC GGT ACA GCG GGA TCA GAC AGC ACA 621 622 TTA GTC GCG GGC TAC GGC AGT ACC CAA ACC GCA GGT GAA GAG AGC AGC CAG ATG GCG GGT TAC GGC AGC 690 691 ACG CAG ACC GGC ATG AAA GGC AGC GAT CTC ACC GCC GGT TAC GGC AGT ACC GGC ACG GCG GGC GCC GAC 759 760 AGC TCC CTG ATC GCC GGT TAC GGC AGT ACC CAG ACG GCG GGC GAA GAC AGC TCG CTG ACA GCC GGT TAC 828 829 GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTT ACG GCC GGT TAT GGC AGT ACC GGC ACG GCG GGT 897 898 GCC GAC AGT TCA TTA ATC GCG GGC TAC GGC AGC ACC CAG ACG GCC GGG GAA GAA AGC ACC CAG ACA GCC 966 967 GGT TAT GGC AGC ACC CAG ACC GCG CAG AAG GGC AGC GAC CTT ACG GCC GGT TAC GGC AGT ACC GGT ACG 1035 1036 GCC GGT GAC GAC AGC TCC CTG ATC GCC GGT TAC GGC AGT ACC CAG ACG GCG GGC GAA GAC AGC TCG CTG 1104 1105 ACA GCC GGT TAC GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTC ACT GCA GGT TAT GGC AGT ACC 1173 1174 GGC ACG GCG GGT GCC GAC AGT TCA TTA ATT GCG GGC TAC GGC AGC ACC CAG ACG GCC GGG GAA GAG AGC 1242 1243 ACC CAG ACA GCC GGT TAT GGC AGC ACC CAG ACC GCG CAG AAG GGC AGC GAC CTT ACG GCC GGT TAC GGC 1311 1312 AGT ACC GGT ACG GCG GGT GAC GAC AGC TCC CTG ATC GCC GGT TAC GGT AGT ACC CAG ACG GCG GGC GAA 1380 1381 GAC AGC TCG CTG ACA GCC GGT TAC GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTT ACT GCA GGT 1449 1450 TAT GGC AGT ACC TCT ACG GCA GGC TAT GAA AGT TCA TTG ATC TCG GGC TAT GGC AGT ACC CAG ACA GCG 1518 1519 GGT TAC GGT AGC ACG CTG ACA GCG GGT TAC GGC AGT ACG CAG ACC GCG CAG AAC GAA AGC GAT CTC ATC 1587 1588 ACC GGC TAT GGC AGT ACG TCT ACA GCC GGG GCA AAT AGT TCA CTG ATC GCA GGC TAT GGC AGC ACG CAG l656 1657 ACA GCC AGC TAC AAC AGT GTG CTA ACG GCG GGC TAC GGC AGT ACC CAG ACG GCG AGA GAA GGC AGT GAC 1725 1726 CTC ACT GCC GGG TAC GGC AGC ACC GGC ACG GCA GGC TCG GAC AGC TCA ATC ATT GCA GGT TAC GGC AGC 1794 1795 ACC CAG ACC GCC AGT TAT CAC AGT AGC CTG ACG GCG GGC TAC GGC AGT ACG CAG ACG GCG CGC GAA CAG 1863 1864 AGT GTG CTG ACA ACC GGC TAC GGC AGC ACC TCA ACC GCC GGC GCC GAC AGT TCC CTG ATT GCA GGC TAT 1932 1933 GGC AGC ACG CAG ACC GCC GGT TAC AAC AGT ATT CTG ACG GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG 2001 2002 GAG GGC AGC GAT CTC ACC GCA GGT TAT GGC AGT ACC TCA ACC GCC GGT GCC GAC AGC TCC CTG ATT GCG 2070 2071 GGC TAC GGC AGC ACC CAG ACC GCC AGC TAT CAC AGT AGC CTG ACG GCG GGT TAC GGC AGT ACG CAG ACG 2139 2140 GCG CGC GAA CAG AGT GTG CTG ACG ACC GGC TAC GGC AGC ACC TCA ACC GCC GGT GCC GAC AGC TCC CTG 2208 2209 ATT GCA GGC TAC GGC AGC ACG CAG ACA GCG GGT TAC AAC AGC ATC CTG ACG GCC GGT TAC GGC AGC ACC 2277 2278 CAG ACG GCG CAG GAG CGC AGC GAT CTC ACC GCA GGT TAT GGC AGC ACC TCA ACC GCC GGT GCC GAC AGC 2346 2347 TCT CTG ATT GCA GGC TAC GGC AGC ACG CAG ACA GCG GGT TAT CAC AGT ATT CTG ACG GCC GGT TAC GGC 2415 2416 AGC ACC CAG ACG GCG CAG GAG CGC AGC GAT CTG ACC ACC GGC TAC GGC AGT ACC TCG ACG GCA GGC TCC 2484 2485 GAC AGC TCC CTG ATC GCG GGC TAC GGC AGC ACG CAG ACA GCG GGT TAC AAC AGC ATC CTG ACG GCC GGT 2553 2554 TAC GGC AGC ACC CAG ACG GCG CAG GAG AAT AGC GAT CTG ACC ACG GGC TAC GGC AGT ACC TCG ACG GCG 2622 2623 GGT TAC GAC AGT TCG TTG ATT GCA GGC TAC GGC AGC ACG CAG ACC GCC GGT TAT CAC AGT ATT CTG ACG 2691 2692 GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG GAG CGC AGC GAT CTG ACC ACC GGC TAC GGC AGT ACC TCG 2760 2761 ACG GCA GGC CCC GAC AGT TCC CTG ATC GCG GGC TAC GGC AGC ACG CAG ACC GCC GGT TAC AAC AGT ATT 2829 2830 CTG ACG GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG GAG AAT AGC GAT CTG ACG ACC GGC TAC GGC AGT 2898 2899 ACT TCT ACG GCA GGT TAT GAG AGT TCA CTG ATC GCA GGC TAC GGC AGC ACC CAG ACC GCC AGT TTT AAA 2967 2968 AGT ACG TTG ATG GCT GGT TAC GGG AGT TCG CAG GCT GCC AGA GAA CAG AGT TCG TTG ACT GCC GGA TAC 3036 3037 GGC AGT ACC TCA ATG GCC GGT TAT GAC AGC TCA TTG ATT GCC GGT TAC GGT AGT ACC CAG ACG GCG GGT 3105 3106 TAT CAA AGT ACG CTG ACA GCC GGT TAC GGC AGC ACG CAA ACG GCC GAG CAC AGT AGT ACG CTA ACG GCA 3174 3175 GGT TAC GGC AGT ACC GCA ACG GCG GGC GCC GAC AGC TCC CTG ATC GCA GGC TAC GGC AGT TCG CTG ACC 3243 3244 AGC GGT ATT CGC AGC TTC CTG ACG GCG GGT TAT GGC AGT ACG TTG ATC AGC GGA CTT CGC AGC GTA CTC 3312 3313 ACC GCC GGT TAC GGA AGC AGC CTG ATT TCG GGC AGA CGC AGT AGC CTG ACG GCG GGA TAT GGC AGT AAT 3381 3382 CAG ATC GCC AGC CAC CGA AGC TCG TTG ATT GCT GGC CCG GAA AGC ACC CAG ATC ACC GGC AAC CGC AGC 3450 3451 ATG CTG ATT GCG GGA AAG GGC AGC TCA CAA ACG GCG GGT TAT CGC AGC ACA TTG ATC TCC GGG GCA GAC 3519 3520 AGC GTG CAA ATG GCC GGA GAG CGC GGC AAG CTG ATT GCC GGA GCG GAC AGC ACC CAA ACC GCC GGG GAT 3588 3589 CGC AGC AAG CTT CTG GCA GGC AAC AAC AGC TAC CTC ACC GCT GGC GAT CGC AGC AAA CTC ACG GCA GGC 3657 3658 AAT GAC TGC ATC CTC ATG GCG GGA GAC CGA AGC AAG CTG ACG GCG GGC ATC AAC AGC ATT CTC ACC GCC 3726 3727 GGA TGC CGT AGC AAG CTG ATA GGA AGC AAT GGT TCA ACC CTC ACC GCC GGG GAA AAC TCA GTT CTG ATT 3795 3796 TTT CGC TGC TGG GAT GGA AAG CGC TAC ACT AAC GTG GTC GCT AAA ACC GGA AAG GGG GGA ATA GAA GCG 3864 3865 GAT ATG CCT TAC CAG ATG GAT GAG GAC AAT AAT ATC GTG AAT AAA CCC GAA GAA TAA 3921 (2) SEQ ID NO 2 (iceA gene encoding the amino acid sequence of the protein) 1 MKEDKVLILRTCANNMADHGGIIWPLSGIVECKYWKPVKGFENGLTGLIWGKGSDSPLSL 60 61 HADARWVVAE VDADECIAIETHGWIKFPRAEVLHVGTKTSAMQFILHHRADYVASTEMQA 120 121 GPGAPDVTSE VKAGNRSLPVTDDIDATIESGSTQPTQTIEI 161 162 ATYGSTLSGT HQSQLI AGYGSTETAGDSSTLI AGYGSTGTAGSDSTLV 209 210 AGYGSTQTAG EESSQM AGYGSTQTGMKGSDLT AGYGSTGTAGADSSLI 257 258 AGYGSTQTAG EDSSLT AGYGSTQTAQKGSDLT AGYGSTGTAGADSSLI 305 306 AGYGSTQTAG EESTQT AGYGSTQTAQKGSDLT AGYGSTGTAGDDSSLI 353 354 AGYGSTQTAG EDSSLT AGYGSTQTAQKGSDLT AGYGSTGTAGADSSLI 401 402 AGYGSTQTAG EESTQT AGYGSTQTAQKGSDLT AGYGSTGTAGDDSSLI 449 450 AGYGSTQTAG EDSSLT AGYGSTQTAQKGSDLT AGYGSTSTAGYESSLI 497 498 SGYGSTQTAG YGSTLT AGYGSTQTAQNESDLI TGYGSTSTAGANSSLI 545 546 AGYGSTQTAS YNSVLT AGYGSTQTAREGSDLT AGYGSTGTAGSDSSII 593 594 AGYGSTQTAS YHSSLT AGYGSTQTAREQSVLT TGYGSTSTAGADSSLI 641 642 AGYGSTQTAG YNSILT AGYGSTQTAQEGSDLT AGYGSTSTAGADSSLI 689 690 AGYGSTQTAS YHSSLT AGYGSTQTAREQSVLT TGYGSTSTAGADSSLI 737 738 AGYGSTQTAG YNSILT AGYGSTQTAQERSDLT AGYGSTSTAGADSSLI 785 786 AGYGSTQTAG YHSILT AGYGSTQTAQERSDLT TGYGSTSTAGSDSSLI 833 834 AGYGSTQTAG YNSILT AGYGSTQTAQENSDLT TGYGSTSTAGYDSSLI 881 882 AGYGSTQTAG YHSILT AGYGSTQTAQERSDLT TGYGSTSTAGPDSSLI 929 930 AGYGSTQTAG YNSILT AGYGSTQTAQENSDLT TGYGSTSTAGYESSLI 977 978 AGYGSTQTAS FKSTLM AGYGSSQAAREQSSLT AGYGSTSMAGYDSSLI 1025 1026 AGYGSTQTAGYQSTLT AGYGSTQTAEHSSTLT AGYGSTATAGADSSLI 1073 1074 AGYGSSLTSGIRSFLT AGYGSTLISGLRSVLT AGYGSSLISGRRSSLT 1121 1122 AGYGSNQIASHRSSLI AGPESTQITGNRSMLI AGKGSSQTAGYRSTLI 1169 1170 SGADSVQMAGERGKLI AGADSTQTAGDRSKLL AGNNSYLTAGDRSKLT 1217 1218 AGNDCILMAGDRSKLT AGINSILTAGCRSKLI GSNGSTLTAGENSVLI 1265 1266 FRCWDGKRYTNVVAKTGKGGIEADMPYQMDEDNNIVNKPEE 1306 (3) SEQ ID NO 3 (iceA nucleotide sequence and its encoded amino acid sequence corresponding to the arrangement) 1 M K E D K V L I L R T C A N N M A D H G G I I 23 1 ATG AAA GAA GAT AAG GTT TTA ATA TTA CGT ACC TGT GCT AAT AAT ATG GCC GAT CAC GGC GGA ATC ATC 69 24 W P L S G I V E C K Y W K P V K G F E N G L T 46 70 TGG CCG TTA AGC GGT ATC GTA GAG TGT AAA TAC TGG AAG CCG GTT AAA GGC TTT GAG AAC GGA CTA ACG 138 47 G L I W G K G S D S P L S L H A D A R W V V A 69 139 GGG CTA ATC TGG GGA AAA GGA TCG GAT TCA CCG CTG AGC CTG CAC GCT GAT GCC AGG TGG GTT GTC GCT 207 70 E V D A D E C I A I E T H G W I K F P R A E V 92 208 GAA GTG GAT GCC GAT GAG TGT ATC GCT ATT GAA ACT CAT GGC TGG ATT AAA TTT CCC CGT GCT GAG GTT 276 93 L H V G T K T S A M Q F I L H H R A D Y V A S 115 277 CTT CAC GTT GGA ACG AAA ACC AGT GCG ATG CAA TTT ATC CTG CAC CAT CGG GCT GAT TAC GTT GCC TCT 345 116 T E M Q A G P G A P D V T S E V K A G N R S L 138 346 ACG GAA ATG CAG GCC GGT CCT GGA GCG CCT GAT GTC ACT TCA GAG GTA AAG GCA GGC AAT CGA TCG CTC 414 139 P V T D D I D A T I E S G S T Q P T Q T I E I 161 415 CCT GTA ACA GAT GAT ATT GAT GCG ACC ATC GAA TCA GGC AGT ACG CAG CCG ACA CAA ACG ATT GAA ATC 483 162 A T Y G S T L S G T H Q S Q L I A G Y G S T E 184 484 GCA ACC TAT GGC AGT ACG CTC AGC GGC ACG CAT CAG AGT CAG CTG ATT GCC GGA TAT GGC AGT ACT GAG 552 185 T A G D S S T L I A G Y G S T G T A G S D S T 207 553 ACG GCG GGT GAT AGC AGC ACA TTA ATT GCC GGT TAT GGT AGC ACC GGT ACA GCG GGA TCA GAC AGC ACA 621 208 L V A G Y G S T Q T A G E E S S Q M A G Y G S 230 622 TTA GTC GCG GGC TAC GGC AGT ACC CAA ACC GCA GGT GAA GAG AGC AGC CAG ATG GCG GGT TAC GGC AGC 690 231 T Q T G M K G S D L T A G Y G S T G T A G A D 253 691 ACG CAG ACC GGC ATG AAA GGC AGC GAT CTC ACC GCC GGT TAC GGC AGT ACC GGC ACG GCG GGC GCC GAC 759 254 S S L I A G Y G S T Q T A G E D S S L T A G Y 276 760 AGC TCC CTG ATC GCC GGT TAC GGC AGT ACC CAG ACG GCG GGC GAA GAC AGC TCG CTG ACA GCC GGT TAC 828 277 G S T Q T A Q K G S D L T A G Y G S T G T A G 299 829 GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTT ACG GCC GGT TAT GGC AGT ACC GGC ACG GCG GGT 897 300 A D S S L I A G Y G S T Q T A G E E S T Q T A 322 898 GCC GAC AGT TCA TTA ATC GCG GGC TAC GGC AGC ACC CAG ACG GCC GGG GAA GAA AGC ACC CAG ACA GCC 966 323 G Y G S T Q T A Q K G S D L T A G Y G S T G T 345 967 GGT TAT GGC AGC ACC CAG ACC GCG CAG AAG GGC AGC GAC CTT ACG GCC GGT TAC GGC AGT ACC GGT ACG 1035 346 A G D D S S L I A G Y G S T Q T A G E D S S L 368 1036 GCC GGT GAC GAC AGC TCC CTG ATC GCC GGT TAC GGC AGT ACC CAG ACG GCG GGC GAA GAC AGC TCG CTG 1104 369 T A G Y G S T Q T A Q K G S D L T A G Y G S T 391 1105 ACA GCC GGT TAC GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTC ACT GCA GGT TAT GGC AGT ACC 1173 392 G T A G A D S S L I A G Y G S T Q T A G E E S 414 1174 GGC ACG GCG GGT GCC GAC AGT TCA TTA ATT GCG GGC TAC GGC AGC ACC CAG ACG GCC GGG GAA GAG AGC 1242 415 T Q T A G Y G S T Q T A Q K G S D L T A G Y G 437 1243 ACC CAG ACA GCC GGT TAT GGC AGC ACC CAG ACC GCG CAG AAG GGC AGC GAC CTT ACG GCC GGT TAC GGC 1311 438 S T G T A G D D S S L I A G Y G S T Q T A G E 460 1312 AGT ACC GGT ACG GCG GGT GAC GAC AGC TCC CTG ATC GCC GGT TAC GGT AGT ACC CAG ACG GCG GGC GAA 1380 461 D S S L T A G Y G S T Q T A Q K G S D L T A G 483 1381 GAC AGC TCG CTG ACA GCC GGT TAC GGC AGT ACC CAG ACC GCG CAG AAG GGC AGC GAT CTT ACT GCA GGT 1449 484 Y G S T S T A G Y E S S L I S G Y G S T Q T A 506 1450 TAT GGC AGT ACC TCT ACG GCA GGC TAT GAA AGT TCA TTG ATC TCG GGC TAT GGC AGT ACC CAG ACA GCG 1518 507 G Y G S T L T A G Y G S T Q T A Q N E S D L I 529 1519 GGT TAC GGT AGC ACG CTG ACA GCG GGT TAC GGC AGT ACG CAG ACC GCG CAG AAC GAA AGC GAT CTC ATC 1587 530 T G Y G S T S T A G A N S S L I A G Y G S T Q 552 1588 ACC GGC TAT GGC AGT ACG TCT ACA GCC GGG GCA AAT AGT TCA CTG ATC GCA GGC TAT GGC AGC ACG CAG 1656 553 T A S Y N S V L T A G Y G S T Q T A R E G S D 575 1657 ACA GCC AGC TAC AAC AGT GTG CTA ACG GCG GGC TAC GGC AGT ACC CAG ACG GCG AGA GAA GGC AGT GAC 1725 576 L T A G Y G S T G T A G S D S S I I A G Y G S 598 1726 CTC ACT GCC GGG TAC GGC AGC ACC GGC ACG GCA GGC TCG GAC AGC TCA ATC ATT GCA GGT TAC GGC AGC 1794 599 T Q T A S Y H S S L T A G Y G S T Q T A R E Q 621 1795 ACC CAG ACC GCC AGT TAT CAC AGT AGC CTG ACG GCG GGC TAC GGC AGT ACG CAG ACG GCG CGC GAA CAG 1863 622 S V L T T G Y G S T S T A G A D S S L I A G Y 644 1864 AGT GTG CTG ACA ACC GGC TAC GGC AGC ACC TCA ACC GCC GGC GCC GAC AGT TCC CTG ATT GCA GGC TAT 1932 645 G S T Q T A G Y N S I L T A G Y G S T Q T A Q 667 1933 GGC AGC ACG CAG ACC GCC GGT TAC AAC AGT ATT CTG ACG GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG 2001 668 E G S D L T A G Y G S T S T A G A D S S L I A 690 2002 GAG GGC AGC GAT CTC ACC GCA GGT TAT GGC AGT ACC TCA ACC GCC GGT GCC GAC AGC TCC CTG ATT GCG 2070 691 G Y G S T Q T A S Y H S S L T A G Y G S T Q T 713 2071 GGC TAC GGC AGC ACC CAG ACC GCC AGC TAT CAC AGT AGC CTG ACG GCG GGT TAC GGC AGT ACG CAG ACG 2139 714 A R E Q S V L T T G Y G S T S T A G A D S S L 736 2140 GCG CGC GAA CAG AGT GTG CTG ACG ACC GGC TAC GGC AGC ACC TCA ACC GCC GGT GCC GAC AGC TCC CTG 2208 737 I A G Y G S T Q T A G Y N S I L T A G Y G S T 759 2209 ATT GCA GGC TAC GGC AGC ACG CAG ACA GCG GGT TAC AAC AGC ATC CTG ACG GCC GGT TAC GGC AGC ACC 2277 760 Q T A Q E R S D L T A G Y G S T S T A G A D S 782 2278 CAG ACG GCG CAG GAG CGC AGC GAT CTC ACC GCA GGT TAT GGC AGC ACC TCA ACC GCC GGT GCC GAC AGC 2346 783 S L I A G Y G S T Q T A G Y H S I L T A G Y G 805 2347 TCT CTG ATT GCA GGC TAC GGC AGC ACG CAG ACA GCG GGT TAT CAC AGT ATT CTG ACG GCC GGT TAC GGC 2415 806 S T Q T A Q E R S D L T T G Y G S T S T A G S 828 2416 AGC ACC CAG ACG GCG CAG GAG CGC AGC GAT CTG ACC ACC GGC TAC GGC AGT ACC TCG ACG GCA GGC TCC 2484 829 D S S L I A G Y G S T Q T A G Y N S I L T A G 851 2485 GAC AGC TCC CTG ATC GCG GGC TAC GGC AGC ACG CAC ACA GCG GGT TAC AAC AGC ATC CTG ACG GCC GGT 2553 852 Y G S T Q T A Q E N S D L T T G Y G S T S T A 874 2554 TAC GGC AGC ACC CAG ACG GCG CAG GAG AAT AGC GAT CTG ACC ACG GGC TAC GGC AGT ACC TCG ACG GCG 2622 875 G Y D S S L I A G Y G S T Q T A G Y H S I L T 897 2623 GGT TAC GAC AGT TCG TTG ATT GCA GGC TAC GGC AGC ACG CAG ACC GCC GGT TAT CAC AGT ATT CTG ACG 2691 898 A G Y G S T Q T A Q E R S D L T T G Y G S T S 920 2692 GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG GAG CGC AGC GAT CTG ACC ACC GGC TAC GGC AGT ACC TCG 2760 921 T A G P D S S L I A G Y G S T Q T A G Y N S I 943 2761 ACG GCA GGC CCC GAC AGT TCC CTG ATC GCG GGC TAC GGC AGC ACG CAG ACC GCC GGT TAC AAC AGT ATT 2829 944 L T A G Y G S T Q T A Q E N S D L T T G Y G S 966 2830 CTG ACG GCC GGT TAC GGC AGC ACC CAG ACG GCG CAG GAG AAT AGC GAT CTG ACG ACC GGC TAC GGC AGT 2898 967 T S T A G Y E S S L I A G Y G S T Q T A S F K 989 2899 ACT TCT ACG GCA GGT TAT GAG AGT TCA CTG ATC GCA GGC TAC GGC AGC ACC CAG ACC GCC AGT TTT AAA 2967 990 S T L M A G Y G S S Q A A R E Q S S L T A G Y 1012 2968 AGT ACG TTG ATG GCT GGT TAC GGG AGT TCG CAG GCT GCC AGA GAA CAG AGT TCG TTG ACT GCC GGA TAC 3036 1013 G S T S M A G Y D S S L I A G Y G S T Q T A G 1035 3037 GGC AGT ACC TCA ATG GCC GGT TAT GAC AGC TCA TTG ATT GCC GGT TAC GGT AGT ACC CAG ACG GCG GGT 3105 1036 Y Q S T L T A G Y G S T Q T A E H S S T L T A 1058 3106 TAT CAA AGT ACG CTG ACA GCC GGT TAC GGC AGC ACG CAA ACG GCC GAG CAC AGT AGT ACG CTA ACG GCA 3174 1059 G Y G S T A T A G A D S S L I A G Y G S S L T 1081 3175 GGT TAC GGC AGT ACC GCA ACG GCG GGC GCC GAC AGC TCC CTG ATC GCA GGC TAC GGC AGT TCG CTG ACC 3243 1082 S G I R S F L T A G Y G S T L I S G L R S V L 1104 3244 AGC GGT ATT CGC AGC TTC CTG ACG GCG GGT TAT GGC AGT ACG TTG ATC AGC GGA CTT CGC AGC GTA CTC 3312 1105 T A G Y G S S L I S G R R S S L T A G Y G S N 1127 3313 ACC GCC GGT TAC GGA AGC AGC CTG ATT TCG GGC AGA CGC AGT AGC CTG ACG GCG GGA TAT GGC AGT AAT 3381 1128 Q I A S H R S S L I A G P E S T Q I T G N R S l150 3382 CAG ATC GCC AGC CAC CGA AGC TCG TTG ATT GCT GGC CCG GAA AGC ACC CAG ATC ACC GGC AAC CGC AGC 3450 1151 M L I A G K G S S Q T A G Y R S T L I S G A D 1173 3451 ATG CTG ATT GCG GGA AAG GGC AGC TCA CAA ACG GCG GGT TAT CGC AGC ACA TTG ATC TCC GGG GCA GAC 3519 1174 S V Q M A G E R G K L I A G A D S T Q T A G D 1196 3520 AGC GTG CAA ATG GCC GGA GAG CGC GGC AAG CTG ATT GCC GGA GCG GAC AGC ACC CAA ACC GCC GGG GAT 3588 1197 R S K L L A G N N S Y L T A G D R S K L T A G 1219 3589 CGC AGC AAG CTT CTG GCA GGC AAC AAC AGC TAC CTC ACC GCT GGC GAT CGC AGC AAA CTC ACG GCA GGC 3657 1220 N D C I L M A G D R S K L T A G I N S I L T A 1242 3658 AAT GAC TGC ATC CTC ATG GCG GGA GAC CGA AGC AAG CTG ACG GCG GGC ATC AAC AGC ATT CTC ACC GCC 3726 1243 G C R S K L I G S N G S T L T A G E N S V L I 1265 3727 GGA TGC CGT AGC AAG CTG ATA GGA AGC AAT GGT TCA ACC CTC ACC GCC GGG GAA AAC TCA GTT CTG ATT 3795 1266 F R C W D G K R Y T N V V A K T G K G G I E A 1288 3796 TTT CGC TGC TGG GAT GGA AAG CGC TAC ACT AAC GTG GTC GCT AAA ACC GGA AAG GGG GGA ATA GAA GCG 3864 1289 D M P Y Q M D E D N N I V N K P E E * 1307 3865 GAT ATG CCT TAC CAG ATG GAT GAG GAC AAT AAT ATC GTG AAT AAA CCC GAA GAA TAA 3921 ...

Claims (11)

1. the gene order of an ina gene iceA is characterized in that this sequence has the nucleotide sequence shown in SEQ ID NO 1, and coding has the protein of the aminoacid sequence shown in SEQ ID NO 2, and has ice nucleation activity and urge to freeze insect effect extremely;

2. the gene order of claim 1, wherein said gene order comprises the partial sequence of this gene order;

3. the gene order of claim 1, wherein said gene order comprises the homologous sequence of this gene order;

4. the aminoacid sequence of an ice nucleation protein matter is characterized in that this aminoacid sequence is coded by the gene order of claim 1, has the aminoacid sequence shown in SEQ ID NO 2, and has ice nucleation activity and urge to freeze insect effect extremely;

5. the aminoacid sequence of claim 4, wherein said aminoacid sequence comprises the partial sequence of this aminoacid sequence;

6. the aminoacid sequence of claim 4, wherein said aminoacid sequence comprises the homologous sequence of this aminoacid sequence;

7. an expression vector is characterized in that this expression vector is constructed by the gene order of claim 1-3, and can import ina gene iceA in the host bacterium and be incorporated on the host bacterium karyomit(e);

8. the expression vector of claim 7, wherein said host bacterium is an enterobacter cloacae;

9. one kind transforms the method that the gram negative bacterium cell obtains engineering bacteria, it is characterized in that this method application rights requires 7 expression vector;

10. transgenic engineering bacterial strain with ice nucleation activity, it is characterized in that this bacterial strain to be the expression vector that utilizes claim 7 with the gene order of claim 1-3 be incorporated into to stablize in polypide growing surely and on the chromosomal DNA of the host bacterium of colonization ability difference on the plant materials, making up and form, and have the short insect effect extremely of freezing;

11. the transgenic engineering bacterial strain of claim 10, wherein said insect is Pyrausta nubilalis (Hubern). or bollworm.

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