CN102634537A - Efficient expressing vector, construction method and applications of Bacillus thuringiensis - Google Patents

Abstract

The invention relates to an 'efficient expressing vector, a construction method and applications of Bacillus thuringiensis', belonging to the technical field of biology. According to the invention, through comparing the transcriptional activities of four cry gene promoters (PcrylA, Pcry3A, Pcry4A and Pcry8E) with different transcription mechanisms of Bacillus thuringiensis, a promoter of a high promoter Pcry8E is screened, and an efficient expression vector pHT315-8E21B is constructed. The Cry protein can be expressed accurately by utilizing the vector, and also has biological activity, and the expression efficiency is higher than a pSXY422b vector instructed by a cry3A promoter widely applied, and the efficient expressing vector of Bacillus thuringiensis has a wide application prospect.

Description

Tribactur efficient expression vector, construction process and application

Technical field

The present invention relates to biological technical field, particularly relate to the construction process and the application of Tribactur efficient expression vector.

Background technology

Tribactur (Bacillus thuringiensis, be called for short Bt) is a kind of bacterium that extensively is present in the soil, and it can form the brood cell stationary phase in growth; And the brood cell contains crystal, the main composition of crystal be insecticidal crystal protein (also being called delta-endotoxin) by cry and cyt genes encoding, have special insecticidal activity [Sanahuja; G.; R.Banakar, R.M.Twyman, et al.; 2011.Bacillus thuringiensis:a century of research, development and commercial applications.Plant Biotechnol is (3) J.9: p.283-300].Multiple Bt product is applied to the control [Bravo of Agricultural pests and sanitary insect pest mosquito; A.; S.Likitvivatanavong; S.S.Gill, et al., 2011.Bacillus thuringiensis:A story of a successful bioinsecticide.Insect Biochem Mol Biol.41 (7): p.423-31].Characteristics according to the cry gene expression regulation can be divided into two types with it; Brood cell's dependent form cry gene such as cry1A, cry2A, cry4A, cry4B and cry11A etc. and do not rely on the cry gene that the brood cell forms are like cry3A [Ibrahim; M.A.; N.Griko, M.Junker, et al.; 2010.Bacillus thuringiensis:a genomics and proteomics perspective.Bioeng Bugs.1 (1): p.31-50.Bravo; A., H.Agaisse, S.Salamitou; Et al.; 1996.Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis.Mol Gen Genet.250 (6): p.734-41.Agaisse, H.and D.Lereclus, 1994.Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxm gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.J Bacteriol.176 (15): p.4734-41.].And the promotor of different mechanisms needs different transcription factors, is eclipsed double-promoter BtI and BtII like the cry1Ac promotor, and the two is respectively by σ ^EAnd σ ^KRegulation and control [Sedlak; M.; T.Walter; And A.Aronson, 2000.Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis delta-endotoxins.J Bacteriol.182 (3): p.734-41]; Cry4A is non-overlapped double-promoter p1 and p2; The regulation and control that receive σ H in the transitional period have than weak expression [Yoshisue; H., T.Fukada, K.Yoshida; Et al., 1993.Transcriptional regulation of Bacillus thuringiensis subsp.israelensis mosquito larvicidal crystal protein gene cryIVA.J Bacteriol.175 (9): p.2750-3]; Cry8E gene and its upper reaches orf1 gene are transcribed with the operon form, and two promotors in the operon receive σ ^HAnd σ ^ERegulation and control (unpublished data); Cry3A promptly expressed since the vegetative phase, received σ ^ARegulation and control [Agaisse; H.and D.Lereclus; 1994.Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.J Bacteriol.176 (15): p.4734-41], and the forfeiture of sigK gene function helps cry3A promotor transcribing in product born of the same parents' later stage.

The difference of the cry gene promoter mechanism of transcribing has determined it to transcribe characteristics accordingly, and the researchist constructs expression vector according to these characteristics and is used for experiment and production.The Bt expression vector of having reported at present mainly contains pBMB1A, pEMB0557, pSXY-422b and pSTK-3A.PBMB1A is the basis with low copy carrier pHT304; The shuttle expression carrier that the cry1Ac promotor instructs can be used for quick clone and expresses different classes of Cry albumen [Zheng Wen, Ye Weixing; Peng Donghai; Deng the bacillus thuringiensis expression vector establishment of .2012. based on the cry1Ac expression regulation element. hubei agricultural science,, 51 (2): 400-405]; PEMB0557 can hold the dna fragmentation of 70kb at least; Can be used for cloning or expressing bigger gene or gene cluster [Liu; X., D.Peng, Y.Luo; Et al., 2009.Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments.Appl Microbiol Biotechnol.82 (4): p.765-72]; PSXY-422b and pSTK-3A are the basis with high copy vector pHT315; The shuttle expression carrier that the cry3A promotor instructs; Have only resistance different; The pSTK-3A that Wang etc. will carry the cry3Aa7 gene is transformed among the wild strain G03 to the high virulence of lepidoptera pest, obtains Coleoptera and lepidoptera pest are all had engineering bacteria G033A [Wang, the G. of very high virulence; J.Zhang; F.Song, et al., 2006.Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests.Appl Microbiol Biotechnol.72 (5): p.924-30].The pSTK-3A that Yan etc. will carry the cry3Aa7 gene is transformed among the wild strain HBF-1 to the high virulence of grub; Obtain engineering strain 3A-HBF; It not only shows higher virulence to grub, but also obtains insecticidal activity [Yan, G. to the Chrysomelidae insect; F.Song; C.Shu, et al., 2009.An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi) .Biotechnol Lett.31 (5): p.697-703].

The formation of Tribactur insecticidal crystal is the result of protein accumulation, and utilizes a strong promoter to express corresponding gene, can improve proteic output, thereby improve the activity of unit weight fermented liquid.Therefore screen a strong promoter and make up an efficient expression vector, the Tribactur engineering bacteria of high-efficiency broad spectrum will lay the foundation in order to develop more from now on, and the activity that improves the Bt preparation is had boundless application prospect.

Summary of the invention

One of the object of the invention is to make up a kind of analysis carrier, is used for the activity of the cry gene promoter (Pcry1A, Pcry3A, Pcry4A and Pcry8E) of four kinds of differences of comparison mechanism of transcribing, and filters out a strong promoter, instructs the cry expression of gene.

Two of purpose is to use the strong promoter filter out, makes up an efficient expression vector and its efficient is assessed, to be used to develop the more Tribactur engineering bacteria of high-efficiency broad spectrum.

A kind of analysis carrier, its constitutional features is: reporter gene lacZ is inserted between the MCS of the skeleton carrier pHT315 after enzyme is cut.

Said analysis carrier is pHTZ, and its sequence is shown in SEQ ID NO17.

The purposes of above-mentioned analysis carrier in creation analysis transcriptional activity bacterial strain.

A kind of expression vector, its constitutional features is: the overlapping fragments in the non-promoter expression district of cry8E gene promoter and coli expression carrier pET-21b is inserted between the MCS of the skeleton carrier pHT315 after enzyme is cut.

Said expression vector is pHT315-8E21b, and its sequence is shown in SEQ ID NO18.

The construction process of expression vector pHT315-8E21b comprises the steps:

(1) the overlapping fragments 8E-21b in the non-promoter expression district of acquisition cry8E gene promoter and pET-21b,

(2) insert between the MCS of the skeleton carrier pHT315 after enzyme is cut.

The method of said step (1) is: with the Bt185 strain gene group DNA is template, and with the promoter sequence of primer to 8E-up/8E-down (SEQ ID NO11/12) amplification cry8E gene, size is 1352bp; With coli expression carrier pET-21b plasmid is template, and amplification obtains promoterless pET-21b expression district fragment to 21b-up/21b-down (SEQ ID NO13/14) with primer, and size is 213bp, comprising MCS; Reclaim two PCR products; As template, carry out overlapping PCR with primer 8E-up and 21b-down, obtain the overlapping fragments 8E-21b that cry8E gene promoter and pET-21b express the district; Size is 1565bp, and the pHT315 of said skeleton carrier is through the EcoRI/HindIII double digestion.

A kind of recombinant expression vector inserts goal gene between the MCS of above-mentioned expression vector.

Said recombinant expression vector is pHT-8E-1Ac, and its sequence is shown in SEQ ID NO19.

A kind of engineering bacteria HD-8E1Ac bacterial strain is characterized in that containing recombinant expression vector pHT-8E-1Ac.

The application of above-mentioned expression vector in expressing Cry albumen.

Said albumen is Cry1Ac albumen.

Technical scheme of the present invention is following:

Make up the cry gene promoter (Pcry1A, Pcry3A, Pcry4A and Pcry8E) and reporter gene lacZ fusion expression vector of four kinds of differences mechanism of transcribing, and importing Bt there is not crystal mutant strain HD-73 ^-In, measure through betagalactosidase activity, relatively the activity of four kinds of promotors filters out the promotor with high transcriptional activity.

With the non-promoter expression area overlapping of strong promoter and expression vector pET-21b, be connected among the basic shuttle vectors pHT315, obtain efficient expression vector pHT315-8E21b, and this carrier is carried out protein quantification, the assessment of efficient such as insecticidal activity.

Scheme process of the present invention below is detailed:

The competent preparation of 1 intestinal bacteria JM110 thermal shock

In order to obtain to contain the intestinal bacteria transformant of foreign gene, prepare colibacillary thermal shock transformed competence colibacillus cell [Sambrook, J.; Fritsch; E.F., and Maniatis, T.1989.Molecular cloning:A Laboratory Manual (Cold Spring Harbour Lab.; Cold Spring Harbour, NY)].The method transformed into escherichia coli that adopts thermal shock to transform receives figure's cell, to obtain more to contain the transformant of foreign gene.

2 high copy transcriptional activities are analyzed the structure of carrier pHTZ

With high copy vector pHT315 is carrier is carrier, and lacZ gene fragment among the plasmid pHT304-18Z through pcr amplification, digestion with restriction enzyme, is connected to the MCS of pHT315, obtains high copy transcriptional activity and analyzes carrier pHTZ (Fig. 1).Through the evaluation of cutting and check order of PCR, enzyme, the exactness (Fig. 2) of checking plasmid.

The 3Bt competent preparation of shocking by electricity

In order to improve the transformation efficiency of Bt, electric shock transformed competence colibacillus cell [Sambrook, the J. of preparation Bt; Fritsch, E.F., and Maniatis; T.1989.Molecular cloning:A Laboratory Manual (Cold Spring Harbour Lab., Cold Spring Harbour, NY)]; The method that adopts electric shock to transform transforms Bt recipient bacterium cell, to obtain more to contain the transformant of foreign gene.

4Pcry1A, Pcry3A, Pcry4A and Pcry8E and lacZ gene fusion expression vector construction

Be template with the total DNA of Bt bacterial strain that contains cry1A, cry3A, cry4A and cry8E gene respectively; Carry out the PCR amplification with corresponding special primers, obtain Pcry1A, Pcry3A; Four promoter fragments of Pcry4A and Pcry8E; With being connected with the pHTZ carrier behind the digestion with restriction enzyme, the recombinant plasmid that obtains is through the evaluation of cutting and check order of PCR, enzyme, the exactness (Fig. 4) of checking plasmid.Correct recombinant plasmid is imported Bt do not have crystal mutant strain HD-73 ^-, obtain strain HD LPcry1Ac, HDLPcry3A, HDLPcry4A, HDLPcry8E and HDpHTZ (negative control bacterial strain).

5 four kinds of promoter transcription specific activitys

With the HDLPcry1Ac that contains four kinds of promotors respectively, HDLPcry3A, HDLPcry4A and HDLPcry8E inoculation in SSM substratum [Schaeffer; P.; J.Millet, and J.P.Aubert, 1965.Catabolic repression of bacterial sporulation.Proc Natl Acad Sci U S is (3) A.54: p.704-11]; Take a sample at interval by regular time; The betagalactosidase activity of working sample, relatively the activity of four kinds of promotors filters out the promotor (Fig. 5) with high transcriptional activity.

The structure of the efficient expression vector that 6 strong promoters instruct

With the non-promoter expression area overlapping of strong promoter and coli expression carrier pET-21b, be connected among the basic shuttle vectors pHT315, obtain efficient expression vector pHT315-8E21b (Fig. 6), cut and the exactness (Fig. 7) of sequence verification recombinant plasmid through enzyme.

The efficiency evaluation of 7 efficient expression vectors

Pcr amplification cry1Ac gene behind digestion with restriction enzyme, imports among the efficient expression vector pHT315-8E21b, obtains recombinant plasmid pHT-8E-1Ac, cuts and checks order through enzyme and identify and correct (Fig. 8) import HD-73 ^-No crystal mutant strain obtains the HD-8E1Ac bacterial strain; Same quadrat method imports the cry1Ac gene among the expression vector pSXY-422b that contains the cry3A gene promoter of widespread use, obtains recombinant plasmid pSXY-422b-1Ac, after evaluation is correct, imports HD-73 ^-No crystal mutant strain obtains the HD-422-1Ac bacterial strain.

Crystal habit is observed: with the HD-8E1Ac inoculation in the SSM substratum, to the most of cracking of cell, with sem observation crystal habit (Fig. 9).Detect efficient expression vector pHT315-8E21b and can correctly express external source Cry albumen.

Protein yield is measured: with no crystal mutant strain HD73 ^-For contrast, utilize SSM culture medium culturing HD-422-1Ac and the complete cracking of HD-8E1Ac bacterial strain to cell, adopt

660nm protein Assay Kit carries out quantitatively the total protein that the three goes up in the appearance mixed solution respectively.The adjustment applied sample amount makes the consistent point sample that carries out of total protein content, utilizes SDS-PAGE to detect the output (Figure 10) of each bacterial strain insecticidal crystal protein.The SDS-PAGE method referring to the molecular cloning experiment guide [J. Sa nurse Brooker. molecular cloning experiment guide (second edition) (Jin Dongyan etc.). Beijing: Science Press, 1992].

Insecticidal activity assay: HD-422-1Ac and HD-8E1Ac bacterial strain are processed pulvis, albumen in the pulvis is carried out quantitatively being diluted to different concentration then, sneak into feed respectively and mix thoroughly, measure activity to small cabbage moth (Plutella xylostella).The result shows that the Cry1Ac albumen that the HD-8E-1Ac bacterial strain produces has activity to small cabbage moth, LC ₅₀Be 0.05561, be about HD-422-1Ac LC ₅₀1/5th, prove biological activity ratio strain HD-422-1Ac active high of strain HD-8E1Ac, so the proteic expression efficiency of Cry is higher than pSXY-422b from bioactive angle explanation pHT315-8E21b carrier.

Description of drawings

Fig. 1: pHTZ vector construction synoptic diagram,

Fig. 2: pHTZ carrier PCR and enzyme are cut evaluation,

1: λ/Eco130I Marker, 2:Pst I single endonuclease digestion product, 3:Hind III+Pst I double digestion product, the 4:PCR product,

Fig. 3: Pcry1A, Pcry3A, Pcry4A and Pcry8E and lacZ gene fusion expression vector construction synoptic diagram,

Fig. 4: recombinant plasmid pHTZPcry1Ac, pHTZPcry3A, pHTZPcry4A and pHTZLPcry8E enzyme are cut with PCR and are identified,

1: λ/Eco130I Marker, 2:pHTZPcry1Ac/Xba I single endonuclease digestion product, 3:pHTZPcryIAc/KpnI+Xba I double digestion product; 4:Pcry1Ac PCR product, 5:pHTZPcry3A/Xba I single endonuclease digestion product, 6:pHTZPcry3A/KpnI+Xba I double digestion product; 7:Pcry3A PCR product, 8:pHTZPcry4A/Xba I single endonuclease digestion product, 9:pHTZPcry4A/KpnI+Xba I double digestion product; The 10:Pcry4APCR product, 11:pHTZLPcry8E/Xba I single endonuclease digestion product, 12:pHTZLPcry8E/KpnI+Xba I double digestion product; 13:LPcry8E PCR product, 14:DL2000Marker

Fig. 5: different cry gene promoter transcriptional activities compare,

Fig. 6: pHT315-8E21b vector construction collection of illustrative plates,

Fig. 7: the evaluation of pHT315-8E21b single endonuclease digestion,

1:Wide range Marker, 2:BamH I single endonuclease digestion product, 3:Sal I single endonuclease digestion product, 4:EcoR I single endonuclease digestion product, 5:Sac I single endonuclease digestion product, 6:Hind III single endonuclease digestion product, 7:Xho I single endonuclease digestion product,

Fig. 8: the evaluation of recombinant plasmid pHT-8E-1Ac double digestion,

1:1kb DNALadder, 2:BamHI+Sal I double digestion product,

Fig. 9: strain HD 73 ^-With the scanning electron microscopic observation result of HD-8E1Ac,

Figure 10: HD-422-1Ac, the proteic SDS-PAGE of HD-8E-1Ac bacterial strain Cry1Ac analyze,

1:HD-73 ^-Bacterial strain, 2:HD-422-1Ac bacterial strain, 3:HD-8E1Ac bacterial strain.

Embodiment

Below in conjunction with embodiment the present invention is done further detailed description.

Following used carrier and bacterial strain are the commercially available prod.All there is preservation in the applicant's laboratory, can externally openly provide.

Embodiment 1 competent preparation of intestinal bacteria JM110 thermal shock and conversion

Picking list bacterium colony is in 5ml LB liquid nutrient medium, and 37 ℃ shake overnight cultures; Be inoculated in the 100ml LB liquid nutrient medium by 1% inoculum size, 37 ℃, 220rpm cultivates 2-2.5hr, (OD ₆₀₀=0.5-0.6); 4 ℃, the centrifugal 10min of 4000rpm; Abandon supernatant, in deposition, add the 0.1M CaCl of 50ml precooling ₂Suspension cell places 30min on ice; 4 ℃, the centrifugal 10min of 4000rpm reclaims cell; 0.1M CaCl with the precooling of 2-4ml ice ₂Re-suspended cell is distributed in the 200 μ L/0.5mL centrifuge tubes, in 4 ℃ of preservations, uses up in the week.With 200 μ L competent cells and plasmid mixing, ice bath 30min, 42 ℃ of thermal shock 1.5min, ice bath 3min adds 800 μ L LB substratum, cultivates 60min, gets 200 μ L coated plates, 37 ℃ of cultivations for 37 ℃.

Embodiment 2 high copy transcriptional activities are analyzed the structure of carrier pHTZ

With plasmid pHT304-18Z is template, and amplification obtains lacZ gene fragment (3115bp) to LacZ5 '/LacZ3 ' (SEQ ID NO1/2) with primer, behind restriction enzyme HindIII and Pst I double digestion; Insert corresponding site among the high copy vector pHT315, obtain recombinant plasmid pHTZ (Fig. 1), recombinant plasmid is carried out PCR and enzyme is cut evaluation; The result shows (Fig. 2), and pcr amplification obtains the specific band of 3115bp, with Pst I carrier is carried out enzyme and cuts; Obtain the band of about 9.5kb size; With HindIII and Pst I carrier is carried out double digestion and identify, obtain the band of 6.5kb and 3.0kb, prove that construction of recombinant plasmid is correct.Sequence is seen (SEQ ID NO17).

The embodiment 3Bt competent preparation of shocking by electricity

Picking list bacterium colony is in 5ml LB liquid nutrient medium, and 30 ℃ shake overnight cultures.Be inoculated in the 100ml BH liquid nutrient medium by 1% inoculum size, 37 ℃, 220rpm cultivates 3-4hr (OD ₆₀₀=3.0).4 ℃, the centrifugal 10min of 4000rpm.Abandon supernatant, add the ultrapure water suspension cell of precooling, 4 ℃, the centrifugal 10min of 4000rpm repeats twice, abandons supernatant, in deposition, adds 1ml 40%PEG re-suspended cell, packing, and-70 ℃ of preservations are subsequent use.

Embodiment 4Pcry1A, Pcry3A, Pcry4A and Pcry8E and lacZ gene fusion expression vector construction

Be that template is with the promoter sequence of primer to LP1Ac5 '/LP1Ac3 ' (SEQ ID NO3/4) amplification cry1Ac Gene A TG upper reaches 382bp with the total DNA of HD-73 bacterial strain respectively; With the total DNA of Bt22 bacterial strain is template, and LP3A5 '/LP3A3 ' (SEQ ID NO5/6) is the promoter sequence of primer amplification cry3A Gene A TG upper reaches 570bp; With the total DNA of Bti bacterial strain is template, and LP4A5 '/LP4A3 ' (SEQ ID NO7/8) is the promoter sequence of primer amplification cry4A Gene A TG upper reaches 826bp; With the total DNA of Bt185 bacterial strain is template, and LPorf15 '/LP8E3 ' (SEQ ID NO9/10) is the promoter sequence of primer amplification cry8E Gene A TG upper reaches 1352bp.Above promoter fragment is respectively behind restriction enzyme Kpn I and Xba I double digestion; Insert the corresponding site of plasmid pHTZ; Transformed into escherichia coli JM110 bacterial strain; Obtain recombinant plasmid pHTZPcry1Ac, pHTZPcry3A, pHTZPcry4A and pHTZLPcry8E, Fig. 3 is the construction of recombinant plasmid synoptic diagram.Four recombinant plasmids are identified through pcr amplification, double digestion and order-checking, prove to make up correct (Fig. 4).With identifying that correct plasmid and control vector pHTZ import no crystal mutant strain HD-73 ^-, acquisition contains strain HD LPcry1Ac, HDLPcry3A, HDLPcry4A, HDLPcry8E and the HDpHTZ (negative control bacterial strain) of different promoters respectively.

5 four kinds of promoter transcription specific activitys of embodiment

The activatory strain HD of will spending the night LPcry1Ac, HDLPcry3A, HDLPcry4A and HDLPcry8E insert and contain in the SSM substratum of Oxacyclotetradecane,erythromycin deriv, and 30 ℃, the 220rpm shaking culture is from T ₀(thalline logarithmic phase will finish to be about to get into stationary phase time point) is sampled to T ₁₂(T ₀Back 12 hours), per hour take a sample once, the 2ml that at every turn takes a sample, the centrifugal supernatant of abandoning of 12000 * g, deposition places-20 ℃ of preservations rapidly.Betagalactosidase activity is measured: in deposition, add 500 μ L Buffer Z (0.06M Na ₂HPO ₄2H ₂O, 0.04M NaH ₂PO ₄H ₂O, 0.01M KCl, 0.001M MgSO ₄7H ₂O) and the 0.2g steel ball, smudge cells 1min; 4 ℃, the centrifugal 5min of 12000 * g gets 20 μ l supernatants and is used to measure protein concentration, reads the OD595 value; Get 100 μ l supernatants and mix, add 200 μ l ONPG (4mg/ml) with 700 μ l Buffer Z, mixing, 37 ℃ of reactions, timing presents yellow up to reaction solution, adds 500 μ l, 1M Na ₂CO ₃, read the OD420 value.Calculate Miller Units [Miller JH.1972.Experiments in Molecular Genetics.New York:Cold Spring Harbor Laboratory Press, 352-355] according to formula.Every group of data are carried out independent revision test three times, average.The result shows (Fig. 5), and in the SSM substratum, Pcry1A and Pcry4A promotor are from T ₂Beginning has activity, and Pcry1A is from T ₂To T ₉Active continuing raises, and Pcry4A is at T ₈The time activity reach the highest; Pcry8E and Pcry3A are from T ₀Beginning has activity, T ₀To T ₁Between, the Pcry3A activity is a little more than Pcry8E, T ₁Back Pcry8E activity begins to be higher than Pcry3A, gets into brood cell's after date, and Pcry8E, Pcry1A and Pcry4A activity all have raising by a relatively large margin, and the Pcry3A variation is comparatively mild.The transcriptional activity of empty carrier pHTZ is very low.Above presentation of results promoter transcription activity is followed successively by Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A from high to low, proves that Pcry8E is a strong promoter.

The structure of the efficient expression vector that embodiment 6 strong promoters instruct

Through the promoter transcription activation analysis, find that the promoter activity of brood cell's cry8E gene during the phase is the strongest, therefore choose this promotor and make up shuttle expression carrier.With the Bt185 strain gene group DNA is template, and with the promoter sequence of primer to 8E-up/8E-down (SEQ ID NO11/12) amplification cry8E gene, size is 1352bp; With coli expression carrier pET-21b plasmid is template, and amplification obtains promoterless pET-21b expression district fragment to 21b-up/21b-down (SEQ ID NO13/14) with primer, and size is 213bp, comprising MCS; Reclaim above-mentioned PCR product, as template, carry out overlapping PCR with primer 8E-up and 21b-down, obtain the overlapping fragments 8E-21b that cry8E gene promoter and pET-21b express the district, size is 1565bp.8E-21b cuts through the SmaI enzyme, inserts shuttle vectors pHT315 and also mends the site after putting down through the EcoRI/HindIII double digestion, obtains recombinant plasmid pHT315-8E21b.This plasmid contains cry8E gene promoter, MCS and T7 terminator, physical map such as Fig. 6.Recombinant plasmid is identified through multiple restriction enzyme single endonuclease digestion and order-checking, proves to make up correct (Fig. 7), and sequence is seen (SEQ ID NO18).

The efficiency evaluation of embodiment 7 efficient expression vectors

With the HD-73 strain gene group DNA is template, and primer 1Ac-up and 1Ac-down (SEQ ID NO15/16) pairing amplification size is the cry1Ac gene of 3537bp, behind BamHI and SalI double digestion; Import between the BamHI and SalI site of pHT315-8E21b; Transformed into escherichia coli JM110 bacterial strain obtains recombinant plasmid pHT-8E-1Ac, identifies (Fig. 8) through double digestion and order-checking; Proof makes up correct, and sequence is seen (SEQ ID NO19).Recombinant plasmid is imported HD-73 ^-No crystal mutant strain obtains strain HD-8E1Ac.Same quadrat method imports the cry1Ac gene among the expression vector pSXY-422b that contains the cry3A gene promoter of widespread use, obtains recombinant plasmid pSXY-422b-1Ac, behind the demethylation, imports HD-73 ^-No crystal mutant strain obtains strain HD-422-1Ac.

Crystal habit is observed: with HD-8E1Ac bacterial strain and control strain HD-73 ^-No crystal mutant strain is inoculated in the SSM substratum, and is centrifugal to the most of cracking of cell, abandons supernatant, and deposition is resuspended in the sterilized water; Simultaneously deckglass is bonded on the Stage microscope, then the brilliant mixture of born of the same parents is coated on the deckglass, with absolute ethyl alcohol dewater step by step, drying, osmic acid is fixed, metal spraying; Observe the crystalline form under the sem.The result proves (Fig. 9), and the HD-8E1Ac bacterial strain can form rhomboidan, and HD-73 ^-Do not observe the existence of rhomboidan in the bacterial strain, explain that the pHT315-8E21b carrier can correctly express the cry1Ac gene.

Protein yield is measured: with no crystal mutant strain HD73 ^-For contrast, utilize SSM culture medium culturing HD-422-1Ac and the complete cracking of HD-8E1Ac bacterial strain to cell, the HD-422-1Ac bacterial strain needs the complete cracking of 24h, and HD-8E1Ac needs 30h.Adopt

660nm protein Assay Kit respectively the total protein that the three goes up in the appearance mixed solution to be carried out quantitatively.The adjustment applied sample amount makes the consistent point sample that carries out of total protein content, utilizes SDS-PAGE to detect the output (Figure 10) of each bacterial strain insecticidal crystal protein.The SDS-PAGE method referring to the molecular cloning experiment guide [J. Sa nurse Brooker. molecular cloning experiment guide (second edition) (Jin Dongyan etc.). Beijing: Science Press, 1992].Can know that by Figure 10 strain HD-422-1Ac and HD-8E1Ac all can produce the Cry1Ac albumen of about 130KDa; And under the situation of total protein content unanimity; Quantitatively learn through Quantity One software; The expressed Cry1Ac albumen of HD-8E1Ac bacterial strain is about 4 times of HD-422-1Ac, explain the pHT315-8E21b carrier to the proteic expression efficiency of Cry apparently higher than pSXY-422b.

Insecticidal activity assay: utilize SSM culture medium culturing HD-422-1Ac and the complete cracking of HD-8E1Ac bacterial strain to cell; Bacterial strain is processed pulvis; Albumen in the pulvis is carried out quantitatively; Be diluted to different concentration then, sneak into feed respectively and mix thoroughly, measure activity small cabbage moth (Plutella xylostella).The result shows (table 1), and the Cry1Ac albumen that the HD-8E-1Ac bacterial strain produces has activity to small cabbage moth, LC ₅₀Be 0.05561, be about HD-422-1Ac LC ₅₀1/5th, prove biological activity ratio strain HD-422-1Ac active high of strain HD-8E1Ac, so the proteic expression efficiency of Cry is higher than pSXY-422b from bioactive angle explanation pHT315-8E21b carrier.

Table 1 strain HD-422-1Ac and HD-8E1Ac are to the toxicity test of small cabbage moth

。

Claims (12)

1. analyze carrier for one kind, its constitutional features is: reporter gene lacZ is inserted between the MCS of the skeleton carrier pHT315 after enzyme is cut.

2. be pHTZ according to the said analysis carrier of claim 1, its sequence is shown in SEQ ID NO17.

3. claim 1 or the purposes of 2 said analysis carriers in creation analysis transcriptional activity bacterial strain.

4. expression vector, its constitutional features is: the overlapping fragments in the non-promoter expression district of cry8E gene promoter and coli expression carrier pET-21b is inserted between the MCS of the skeleton carrier pHT315 after enzyme is cut.

5. be pHT315-8E21b according to the said expression vector of claim 4, its sequence is shown in SEQ ID NO18.

6. the construction process of claim 4 or 5 said expression vectors comprises the steps:

(1) the overlapping fragments 8E-21b in the non-promoter expression district of acquisition cry8E gene promoter and pET-21b,

(2) insert between the MCS of the skeleton carrier pHT315 after enzyme is cut.

7. the described construction process of claim 6, the method for said step (1) is: with the Bt185 strain gene group DNA is template, with the promoter sequence of primer to 8E-up/8E-down amplification cry8E gene, size is 1352bp; With coli expression carrier pET-21b plasmid is template, and amplification obtains promoterless pET-21b expression district fragment to 21b-up/21b-down with primer, and size is 213bp, comprising MCS; Reclaim two PCR products,, carry out overlapping PCR with primer 8E-up and 21b-down as template; Obtain the overlapping fragments 8E-21b that cry8E gene promoter and pET-21b express the district; Size is 1565bp, and the pHT315 of said skeleton carrier is through the EcoRI/HindIII double digestion

Said 8E-up sequence shown in SEQ ID NO11, said 8E-down sequence shown in SEQ ID NO12,

Said 21b-up sequence is shown in SEQ ID NO13, and said 21b-down sequence is shown in SEQ ID NO14.

8. a recombinant expression vector inserts goal gene between the MCS of claim 4 or 5 said expression vectors.

9. said according to Claim 8 recombinant expression vector is pHT-8E-1Ac, and its sequence is shown in SEQ ID NO19.

10. strain HD-8E1Ac is characterized in that containing the described recombinant expression vector pHT-8E-1Ac of claim 9.

11. claim 8 or the 9 said expression vectors application in expressing Cry albumen.

12. application according to claim 11, said albumen are Cry1Ac albumen.

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