CN1134540C - Mosaid insecticiding protein gene able to secrete its product to outside of cell - Google Patents

Abstract

The present invention provides a mosaic pesticidal proteins gene Btlm of artificial synthesized bacillus thuringiensis (Bt), which is suitable for high-efficiency expression in advanced plants. A nucleotide sequence for encoding secretory signal peptide is added to the 5' terminal of the gene to form a fusion protein gene BtSlm, and Bt pesticidal protein expressed by the gene in plants can be secreted in cell spaces so as to be favorable to enhancing the stability of the pesticidal protein and relieving the interference of the pesticidal protein to the normal functions of cells.

Description

A kind of its product that makes is secreted into extracellular chimeric insecticidal protein gene

The invention belongs to plant genetic engineering field, relate to 1848 base-pair sequences that synthesized the antigen-4 fusion protein gene Btlm that forms by bacillus thuringiensis (Bt) insecticidal proteins CrylAb (331 amino acid of N end coding) and CrylAc part (284 amino acid of C end coding) with chemical process, can give transgenic plant high insect-resistance after the synthetic gene is expressed in plant.In order to make the Btlm insecticidal proteins of expressing in the plant can be secreted into extracellular intercellular substance to improve the stability of insecticidal proteins, alleviate the interference effect of Bt insecticidal proteins simultaneously to the vegetable cell normal function, help screening economical character and change little insect-resistant transgenic plants, the present invention is synthesizing the nucleotide sequence that has connected a pathogenic associated protein PRlb signal peptide of artificial synthetic encoding nicotiana on the above-mentioned insecticidal protein gene Btlm basis again at its 5 ' end, thereby sets up into the chimeric insecticidal protein gene BtSlm of a 1947bp.With having transformed tobacco and cotton behind Btlm and the BtSlm structure plant expression vector, from the transformation tissue culture plant, can choose efficient insect-resistant transgenic plants, the worm test result of transgene tobacco shows that the plant insect-resistance of expressing BtSlm is higher than expression Btlm's.

The crystallin of bacillus thuringiensis (Bt) can be used as biological pesticide and kills some insect specifically, after this crystallin is absorbed by insect, at the preceding lps molecule about dissolving generation 130KD under the alkaline condition of insect gut.The C of toxin end is degraded before the intestinal protease hydrolysis, produces the toxalbumin of being made up of N end 65-70KD at last.N one section residue of end (being 28 with regard to CrylA) of this toxalbumin also will be removed by ereptase processing could form complete activatory toxalbumin.Receptors bind on activatory toxalbumin and the sensitive insect midgut epithelial cell, be inserted into the little sky or the ionic channel that form 0.5-1.0nm on the midgut epithelial cells film, cause that ionic in the born of the same parents exosmoses and water in ooze, the cell swelling breaks as a result, a large amount of cells are destroyed, and make larva stop feed and death.Proved the Bt crystallin to the human body free of toxic effects, so the Bt preparation used for five more than ten years as a kind of non-harmful natural microbial pesticide at aspects such as agricultural, forestry and environmental healths, the output of Bt preparation is also increasing year by year.But because Bt crystallin poor stability under field conditions (factors), the restriction that can not be penetrated into factors such as organization internal and insecticidal spectrum be narrow makes the development of these biotic pesticide be subjected to very big restriction always, thus at present to agricultural insect management mainly still by chemical insecticide.The eighties mid-term makes people the Bt gene transferred plant might be obtained to express the insect-resistant transgenic plants of insecticidal proteins along with the success of the maturation of plant gene engineering technology, Bt gene clone and to the going deep into of Bt crystallin sterilant Mechanism Study.At present, the existing Bt transgenic plant more than 26 kinds in the whole world comprise cotton, corn, staple crops such as paddy rice, potato.These transgenic plant have all obtained tangible insect-resistance.

The expression amount of wild-type Bt crystal protein gene in transgenic plant is very low.Account for about 0.001% of total soluble protein at its expression amount under the CaMV35S promoters driven, so it is most to the Bt toxalbumin ability of responsive insect not too, as the main lepidoptera pest of a lot of farm crop the render transgenic plant to obtain poisoning.

(Proc.Natl.Acad.Sci.USA 88:3324-3328 such as Perlark, 1991) according to the codon usage frequency of plant gene and the motif that may influence plant mRNA stability under the constant prerequisite of aminoacid sequence that keeps former Bt genes encoding, Bt crystal protein gene CrylAb and CrylAc have been carried out partly transforming or all synthetic again.Part transform and again the insecticidal proteins amount in transgenic plant, expressed of synthetic gene improved about 10 times and 100 times respectively than the expression amount of wild type gene.Yet the expression regulation of Bt gene in plant has many problems still to treat further research, also needing significant improvement aspect some in the practical application of Bt transgenic plant.For example, all be to rest on intracellularly after at present synthetic Bt gene is expressed in plant, foreign protein produces certain interference in the physiological function of the inevitable pair cell self of intracellular high expression level, and the original traits of render transgenic plant is under some influence.As often finding that in insect-resistant transgenic plants seed selection process its agronomy of transgenic plant that insect-resistance is high is relatively poor, influences its yield and quality, and the economical character plant insect-resistance similar to original kind is not ideal enough.The reason that causes these problems is many-sided, as causing somatic variation in the transformation tissue culture process, the position difference that foreign gene inserts on plant chromosome may activate or disturb certain plants expression of gene etc. all may cause the change of economical character.The accumulation of exogenous genes products in transformant, the particularly accumulation of foreign protein in vegetable cell when high level expression, the normal physiological function of possible pair cell causes very big interference, causes the change of some special shape of plant at last.The exogenous gene expression product under the environment, is easy to contact with various proteolytic enzyme in the born of the same parents in born of the same parents very much in addition, makes its stability decreases.So a large amount of accumulation foreign proteins all can have adverse influence aspect plant itself and exogenous genes products two in born of the same parents.

In order to overcome the problem of above-mentioned existence, the present invention considers that the Bt protein excretion can improve its stability outside born of the same parents, thereby improve its accumulation volume in plant, finally cause the raising of transgenic plant insecticidal activity, also can alleviate the interference of expression product simultaneously the vegetable cell function.At present, showing on evidence from the proteic signal peptide of the PMb of tobacco not only to make pathogenesis-related protein secrete (EMBOJ.5:37-40 such as Comelissen to born of the same parents, 1986), and can make foreign protein secrete to born of the same parents (1990 The Plant Cell 2:51-59 such as Denecke).So, based on above existing research background, the present invention has synthesized from N and holds first amino-acid residue to begin CryAb and the CrylAc fusion gene of forming to the 331st and the 332nd to 615 amino acid, and the chimeric Bt unnamed gene of this that is built into is Btlm at last.On the Btlm basis, again a secreting signal peptide PRlb-s of synthetic encoding sequence is connected with Btlm to form a chimeric protein gene Btlm, made up the plant expression vector of these two genes respectively, by the Agrobacterium tumefaciens mediated transformation tobacco and cotton.The worm test result of transgenic plant shows that these two synthetic insecticidal protein genes all can efficiently express the proteic anti-insect activity of Bt in plant, the insect-resistance of BtSlm plant is a little more than the transfer-gen plant of Btlm, so all be the anti insect gene that applying value is arranged very much in the research and development of engineering of insect-resistant plant according to these two genes of various objectives.

In order to realize purpose of the present invention, specifically take following technological step:

By synthesizing of the few nuclear of Bt CrykA antigen-4 fusion protein gene (Btlm) former times acid primer;

Synthetic and clone's (accompanying drawing 1) of Btlm gene fragment;

The assembling of the Btlm gene CrylA antigen-4 fusion protein gene clone pSKBtlm (accompanying drawing 2) that obtains encoding;

The Btlm gene order is connected with signal coding sequence, makes up to have the signal peptide volume

The BtSlm gene clone pSlm (accompanying drawing 3) of sign indicating number sequence;

Btlm and chimeric BtSlm gene insert binary expression vector pBin438 and constitute plant

Expression vector pBlm and pBSlm (accompanying drawing 6), and it is transformed into intestinal bacteria

Or Agrobacterium tumefaciens [Agrobacterium tumefaciens] LBA4404;

PBlm and pBSlm or their Agrobacterium tumefaciens LBA4404 transformant are applied to Plant Transformation, and it is good to obtain economical character, the transgenic plant with high insect-resistance.

In order to understand the present invention better, the present invention is described in detail in conjunction with following accompanying drawing:

Synthetic and the subclone of accompanying drawing 1.CrylA gene fragment makes up among the synoptic diagram figure abridges: PI-1～PV-9: the numbering of different few nuclear former times acid primers, and the numeral in the bracket of numbering back is the base number of primer; SK:pBluescriptIISK ⁺KL:Klenow enzyme; The Am:Ampicillin resistant gene; Kb: kilobase is right; Bm:BamHI; H3:HindIII; R1:EcoRI; RV:EcoRV; Sc:SacI; SL:SalI.

A. the structure of first section (BamH1-EcoR1) subclone pBtfsI

B. the structure of second section (EcoR1-EcoRv) subclone pBtsII

C. the structure of the 3rd section (EcoRV-EcoR1) subclone pBtfsIII

D. the structure of the 4th section (EcoRI-SacI) subclone pBtsIV

E. the recombinant plasmid pSlm of the chimeric Bt gene BtSlm of the structure accompanying drawing 3. band secretion signal peptide-coding sequences of the structure accompanying drawing 2.CrylA of the 5th section (SacI-salI) subclone pBtfsV fusion insecticidal protein gene clone pSKBtlm makes up nucleotide sequence and the amino acid that accompanying drawing 4. synthetic contain the chimeric insecticidal protein gene BtSlm of secretion signal peptide-coding sequence

Sequence accompanying drawing 5. synthetic Btlm merge insecticidal proteins coding region dna sequence dna and wild-type corresponding section

The comparison of dna sequence dna.

W (up) ... the wild type gene sequence; M (descending) ... synthetic merges desinsection

Protein gene Btlm gene order.Base in the square frame is the W alkali identical with the M sequence

Before and after transforming, base subordinate list 1. merges the variation of insecticidal protein gene Btlm coding region codon usage frequency

Reach comparison with the vegetable codon frequency of utilization.

※ codon per-cent refer to this codon in this gene coding region with the coding same amino acid

The ratio of codon sum; Vegetable codon frequency of utilization % refers in dicotyledons

Frequency of utilization (Murray EE.et al.Nucleic Acid Res.17:477-499 1989);

Aa-amino acid code.Accompanying drawing 6. merges the structure of the plant expression vector of Bt gene Btlm and chimeric Bt gene BtSlm

Amp: amicillin resistance; NPTII: neomycin phosphotransferase gene; Kn:

Kalamycin resistance gene; DE-35SP: the CaMV35S promotor of the two enhansers of band;

RB and LB are respectively right margin and the left margin sequence of T-DNA.The PCR of accompanying drawing 7. transformation tissue culture tobacco plants detects

Primer: 35S forward primer, Bt reverse primer

1：λ-Ecot 14I Marker

2: positive control (pBlm plasmid)

3: negative control (non-transgenic tobacco)

4-13: swimming lane 4-10 is the PCR result of pBlm transfer-gen plant; Swimming lane 11-13 is

The PCR result of pBSlm transfer-gen plant.Primer is 35S ⁺And Re-Bt-.The Southern blot of accompanying drawing 8. transgenic tobacco plants analyzes (HindIII single endonuclease digestion)

With Btlm gene probe results of hybridization

Swimming lane 1.pSKBtlm plasmid is as positive control

Swimming lane 2. swimming lanes 2,3,4 are three strain pBlm transfer-gen plants; 5,6

Be two strain pBSlm transfer-gen plants

The protein immunization analysis of accompanying drawing 9. transgene tobaccos in contrast of swimming lane 7. non-transgenic tobaccos

The CrylAc albumen (68KD) of swimming lane 1. escherichia coli expressions

The contrast of swimming lane 2. non-transgenic tobaccos

(3-5 is the pBlm transfer-gen plant to the different transgenic tobacco plants of swimming lane 3-9.; 6-8 is

The ELISA detected result of pBSlm transfer-gen plant accompanying drawing 10. transgenic tobacco plants

The plant number that is used to analyze is respectively: contrast 6 strains; 8 strains of pBlm transfer-gen plant;

22 strains of pBSlm transfer-gen plant.Accompanying drawing 11. different genes thaumatropy regeneration plants distribute to the resistance of bollworm

The bracket inner digital that each gene structure back is annotated represents that total plant of participating in the experiment counts the genetic analysis of subordinate list 2. transgenic tobacco plant filial generations

The synthetic 1. Bacillus thuringiensis CrylA of the chimeric insecticidal protein gene BtSlm of embodiment one band signal peptide sequence merge synthesizing synthesizing of 1.1 CrylA antigen-4 fusion protein gene Btlm Oligonucleolide primers of insecticidal activity protein gene Btlm

Oligonucleolide primers is synthetic with the dna synthesizer of Applied Biosystem by institute of microbiology of Chinese Academy of Sciences new technology center, promptly can be used for the synthetic of double-stranded DNA behind OPC (oligonucleotide purification column) or polyacrylamide gel electrophoresis purifying.In order to clone conveniently, according to the restriction endonuclease recognition site that exists in CrylAb and the Cry1Ac gene, the toxicity of this gene is divided into five sections, and to carry out primer synthetic.First section (PI) is totally 6 primers (BamH1-EcoR1), and their sequence is as follows: PIf-1 (+) 5 ' ACGGATCCACC ATG GAC AAC CCAATC AAC GAA TGC ATT CCA TAC AAC TGC TTG

AGT AAC CCA GAA GTT 68merPIf-2(-)5′GGA GAT GTC GAT TGG AGT GTA ACC GGT TTC GAT GCG TTC TCC ACC AAG

TAC TTC AAC TTC TGG GTT 66merPIf-3-5′GAA CTC GCT GAG CAG AAA CTG TGT CAA GGA CAA GGA GAT GTC GAT 48merPI-2(-)：5′ACC CCA GAT GAT GTC AAC TAG TCC GAG CAC GAA CCC AGC ACC TGG CAC

GAA CTC GCT GAG 60merPI-3(+)：5′ATC ATC TGG GGT ATC TTT GGT CCA TCC CAA TGG GAC GCA TTC CTG45merPI-4(-)：5′GC GAA TTC TTC GAT CCT CTG GTT GAT GAG CTG TTC AAT TTG AAC CAG GAA

TGC GTC second section (PII) is totally 8 primers (EcoR1-EcoRV), and their sequence is as follows: PII-1 (+): 5 ' AA GAA TTC GCT AGG AAC CAG GCC ATC TCT AGG TTG GAA GGA CTC AGC

AAT CTC TAC CAA ATC TAT 65merPII-2(-)：5′CTC CCT GAG AGC TGG GTT AGT AGG ATC GGC CTC CCA TTC TCT GAA AGA

CTC TC ATA GAT TTG GTA 66merPII-3(+)：5′T CTC AGG GAG GAG ATG CGT ATT CAA TTC AAC GAT ATG AAC AGC GCC

TTG ACC ACT GCT ATC CCA T65merPII-4(-)：5′TT AGC GGC TTG AAC GTA CAC GGA CAA GAG AGG CAC CTG GTA GTT CTG

GAC TGC GAA CAA TGG GAT AGC 68merPII-5(+)：5′CAA GCC GCT AAT GTT CAT CTC AGC GTG GTT CGA GAC GTT TCA GTG TTT

GGA CAG AGG TGG GGA TTC G 67merPII-6(-)：5′C GGT GTA GTT TCC AAT GAG CCT AGT AAG GTC GTT GTA TCT GCT ATT GAT

GGT TGC AGC ATC GAA TCC CCA 70merPII-7(+)：5′AAC TAC ACC GAC CAC GCT GTT CGT TGG TAC AAC ACT GGT TTG GAG CGT

GTC TGG GGT CCT GAT AGC AGA 69merPII-8(-)：5′AC GAT ATC CAA CAC TGT AAG GGT CAA TTC TCT CCT GAACTG GTT GTA

TCT AAT CCA ATC TCT GCT ATC AG 70mer the 3rd section () is totally 6 primers (EcoRV-EcoR1), and their sequence is as follows: PIII-1 (+): 5 ' TTG GAT ATC GTG TCT CTC TTC CCG AAC TAT GAC TCC AGA ACC

TAC CCT ATC CGT ACA GTG 60merPIII-2(-)：5′AAG AAC TGG GTT AGT ATA GAT TTC TCT GGT AAG TTG GGA CAC

TGT ACG GAT AGG 54merPIII-3(+)：5′ACT AAC CCA GTT CTT GAG AAC TTC GAC GGT ACG TTC CGT GGT

TCT GCC CAA GGT ATC GAA GGC 63merPIII-4(-)：5′GAT AGT TAT GCT GTT CAA GAT GTC CAT CAA GTG TGG GCT CCT

GAT GGA GCC TTC GAT ACC 60merPIII-5(+)：5′AAC ACG ATA ACT ATC TAC ACC GAT GCT CAC AGA GGA GAG TAT

TAC TGG TCT GGA CAC CAG ATC 63merPIII-6(-)：5′GGT GAA TTC GGG CCC GCT GAA TCC AAC TGG AGA GGC CAT

GAT CTG GTG TCC AGA the 4th section (PIV) is totally 6 primers (EcoR1-SacI), and its sequence is as follows: PIV-1 (+): 5 ' CC GAA TTC ACC TTC CCT CTC TAT GGA ACT ATG GGT AAC GCC GCT CCA

CAA CAA AGG ATC GTT GCT CA 67merPIV-2(-)：5′GAA TGG CCT TCT GTA CAA AGT GGA AGA CAA GGT TCT GTA GAC ACC CTG

ACC TAG TTG AGC AAC GA 65merPIV-3(+)：5′A AGG CCA TTC AAT ATC GGT ATC AAC AAC CAG CAA CTT TCC GTT CTC

GAT GGA ACA GAG TTC GCC TAT 67merPIV-4(-)：5′A GCT AGC AAC GGT TCC GGA CTT TCT GTA AAC AGC GGA TGG CAA GTT

AGA AGA GGT TCC ATA GGC GGA C 68merPIV-5(+)：5′GTT GAT AGC TTG GAC GAA ATT CCA CCA CAG AAC AAC AAT GTG CCA CCC

AGG CAA GGA TTC AGC CAC AGG T 70merPIV-6(-)：5′AGG AGC TCTGAT GAT GCT CAC GCT ACT GTT GCT GAA ACC GGA ACG GAA

CAT GGA CAC ATG GCT CAA CCT GTG GCT 72mer the 5th section (PV) is totally 9 primers (SacI-SalI), and its sequence is as follows: PV-1 (+): 5 ' TC AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAG TTC AAC

AAT ATC ATT GCA TCC GAT AGC ATC 71merPV-2(-)：5′CC TGA AAT GAC TGA ACC ATT GAA GAG AA GTT TCC CTT AAC TGC AGG

AAT TTG AGT GAT GCT ATC G 66merPV-3(+)：5′TC ATT TCA GGA CCA GGA TTC ACA GGA GGA GAC CTC GTT AGA CTC AAC

AGC AGT GGA AAT AAC ATC 65merPV-4(-)：5′ATA TCT GGT AGA GTT CGT GTG AGG TAT GCT TCT GTG ACT CCT ATT CAT

CTC AAC GTT AAT TGG GGT 67merPV-5(+)：5′T ACC AGA TAT AGA GTT CGT GTG AGG TAT GCT TCT GTG ACT CCT ATT CATCTC

AAC GTT AAT TGG GGT 67merPV-6(-)：5′GAG GTT ATC CAA GGA GGT AGC TGT AGC TGG AAC TGT GTT GCT GAA GAT

AGA TGA ATT ACC CCA ATT A 67merPV-7(+)：5′G GAT AAC CTC CAA TCC AGC GAC TTC GGA TAC TTT GAG AGC GCC AAT GCT

TTC ACA TCT TCA CTC GGC AAC 70merPV-8(-)：5′T CAC ACC TGC AGT TC ACT AA GTT TCT AAC ACC CAC TAT GTT GCC GAG T50merPV-9(-)： 5′CA GTC GAC TCA TTC ATC CTC GAG TGT TGC AGT AAC TGG AAT GAA CTC

The segmental synthetic and clone of AAA TCT GTC TAT GAT CAC ACC TGC AGT 72mer1.2 Cry1A fusion gene

Get two each 20pmol of adjacent primer (10 μ l) mixing in each section, at 70 ℃ of sex change 10min, add 10 * Klenow enzyme buffer liquid (100mmol/L Tris-Cl PH7.5 behind the naturally cooling, 0.5mmol/L NaCl, 1mmol/L EDTA, 50mmol/L DTT) 2 μ l, Klenow archaeal dna polymerase 1 unit, add dNTP to 0.1mmol/L 37 ℃ of insulation 1h in cumulative volume 20 μ l, reaction product and another carry out pcr amplification to produce bigger dna fragmentation in the presence of the primer of two ends after the adjacent big fragment combined degeneration that the reaction of Klenow enzymatic polymerization produces.PCR is reflected in the 20 or 50 μ l volumes by 50mmol/L KCl, 10mmol/L, Tris-HCl pH8.8,1.5mmol/LMgCl2,0.1mg/ml BSA, 0.2mmol/L dNTP, 5 ' end and 3 ' each 1 μ mol/L of end primer, 0.05U/ μ l Taq DNA polymerase, the reaction mixture that each reaction of 1 μ l/ is formed as template from the product of Klenow or PCR reaction is with 94 ℃ then of 94 ℃ of sex change 4min, 1min; 40-50 ℃ (annealing temperature is determined according to primer and template paired base number), 1min; 72 ℃, 1min carries out 30 circulations altogether.The PCR final product of each section behind corresponding restriction endonuclease enzymolysis with cloning vector pBluecriptIISK with same enzymolysis ^{+ [1]}Or pSP71 ^[2]Connect back transformed into escherichia coli DH5 α ^[3]Competent cell, choose white colony containing on the LB flat board of X-gal and IPTG, extract, can choose behind the restriction analysis of plasmid and the dna sequence analysis subclone pBtSI (first section) of CrylA gene fragment through plasmid, pBtSII (second section), pBtSIII (the 3rd section), pBtSIV (the 4th section) and pBtfSV (the 5th section).The building process of these five subclones is seen Figure 1A-1E.Above DNA enzyme is cut, the preparation of the preparation of carrier, ligation, competent cell, conversion, clone's screening is all carried out according to the described method of " molecular cloning " book (Sambrook J.et al. " MolecularCloning " 2nd ed.CSH Laboratory Press, 1989).Dna sequence analysis is pressed the Pharmacia company's T ₇The method that DNA sequencing Kit provides is carried out.1.3 merge the assembling of insecticidal proteins CrylAc gene

After sequential analysis proves that the sequence of Bt gene fragment in each subclone is correct, see Fig. 2 by the restriction enzyme site of each subclone promptly obtain after the connection in order each other the encoding gene clone pSKBtlm building process of CrylA fusion rotein Btlm with conventional recombinant DNA technology.1.4 merge the rectification of insecticidal protein gene sequence

After being carried out sequential analysis, each subclone finds in some clone, to have the disappearance or the displacement of base.In order to obtain right-on gene order, on the subclone basis, use by the method for the rite-directed mutagenesis box specification sheets of Clontech company wrong base place is carried out rite-directed mutagenesis.The selection primer of sudden change is Stu/ScaI:5 ' GTGGACTGGTGAAGTACTCAACCAAGTC or AflIII/BgIII primer: 5 ' CAGGAAAGAAGATCTGAGCAAAAG.Mutant primer is examined sour composition of former times by the base of need change and the widow of a two ends 10-15 based composition.Mutant primer should with select primer on same DNA chain.

The base mistake that occurs about EcoRI in the Bt gene fragment among the subclone pBtfS12 is then corrected by PCR method with two pairs of primers,, simultaneously this EcoRI site is removed for ease of the transformant of screening sudden change.Two pairs of primers are respectively

①P1-1+mI ^-

mI-5’CCTAGCGAACTCTTCGATCCTCTGGTTGATGAG 33mer

②PII-8+mII ⁺

mII+5’ATCGAAGAGTTCGCCAGGAACCAGGCCATCTCTAGG 36mer

To the improved recombinant plasmid of pBtfS12 name into pBtfS12PCR reaction conditions and cloning process ditto described.2. the synthesizing of structure (a) secretion signal peptide-coding sequence that has the chimeric insecticidal protein gene clone pSlm of secretion signal peptide-coding sequence reaches the clone

For make Bt merge insecticidal protein gene in plant, expresses its egg of back from product in born of the same parents collection tire out and be secreted into intercellular substance outside the born of the same parents, the present invention is first with proteic signal coding sequence PRlbS of the tobacco pRlb of synthetic and the chimeric insecticidal protein gene BtSlm that obtains having the secretion signal peptide-coding sequence after above-mentioned synthetic crylA antigen-4 fusion protein gene Btlm is connected.The building process of the recombinant plasmid pSlm of BtSlm gene as shown in Figure 3.Wherein the coding region sequence of synthetic PRlb secreting signal peptide is as follows: above two of Slb-1:5 ' CAT CTA GAT CT ACC ATG GGA TTT TTC CTT TTT TCT CAA ATG CCATCC TIC TTT CTC GTG TCC ACT 51merSlb-2:5 ' TAG TCG ACA CGC GTG AGA GGA GTG AGA GAT AAT CAG GAA AAGGAG AAG AGT GGA CAC GAG 60mer respectively gets 20pmol, after 1h is reacted in (as above described in 1.2) 37 ℃ in 50ml klenow enzyme reaction system, adding benzene volume phenol/chloroform (1: 1) extracts once, get upper water solution and add 2 times of volume ethanol and 5ml 3M NaAc, mixing postposition-70 ℃ deposit D NAlnr, centrifugal 10 minutes collecting precipitations of 12000rpm, 70% ethanol is washed precipitation, be dissolved in after the drying in the 20ml sterilized water, by " molecular cloning " described method above-mentioned DNA and PUC19 plasmid DNA are carried out XbaI and SalI enzymolysis, behind the enzymolysis by 6% polypropylene phenol amine gel electrophoresis separation and purification Slb dna fragmentation, and with pUC19 with sample enzyme enzymolysis ^[4]Connect the recombinant plasmid pUSP that promptly obtains containing S1b, can screen the transformant that contains this recombinant plasmid containing on Xgal and the IPTG flat board behind the transformed into escherichia coli DH5 α, sequence through restriction analysis and dna sequence analysis proof secreting signal peptide S1b is correct, become AGATTC because the mistake in this site can not influence the expression of Slb signal peptide but mistake has taken place when synthetic by AGATCT, see shown in the accompanying drawing 3 so in pUSP, still kept the building process of this sequence pUSP in the BglII restriction enzyme site sequence of opening.In order suitably to improve the GC content in the Slb sequence and to avoid codon rare in some plants when synthetic wherein 15 codons to be changed, but its amino acids coding does not change.(b) have the structure of the chimeric insecticidal protein gene Stlm clone pSlm of secretion signal peptide-coding sequence

By the described recombinant DNA method of " molecular cloning " book with MluI enzymolysis plasmid pUSP then with the Klenow enzyme mend flat end again with the SalI enzymolysis with use the BamH1 enzymolysis, the Klenow enzyme is mended and is put down and with the clone pSlm (seeing accompanying drawing 3 for details) of the chimeric insecticidal protein gene BtSlm that promptly obtains containing the secreting signal peptide numbered sequence after the 1.8kb fusion insecticidal protein gene fragment connection that is separated to behind the SalI enzymolysis pSKBtlm.Sequential analysis shows the sequence of BtSlm gene with original design consistent, its dna sequence dna and amino acid sequence coded see claim 1 and 2 and accompanying drawing 4.The chimeric insecticidal protein gene BtSlm of synthetic total length 1947bp, wherein 1-the 90th nucleotide coding secreting signal peptide Slb; 91-99 is three codons that formed by restriction enzyme site; 100-1092 and 1093-1947 encode respectively CrylAb 1-331 amino acid and CrylAc 332-616 amino acid (comprising whole subcipher), i.e. Btlm gene.Btlm gene and corresponding wild type gene nucleotide sequence relatively see accompanying drawing 5, the GC of Btlm gene is increased to 47.4% than by 37% of wild-type, the variation of its codon usage frequency more helps the expression of this gene in plant.Btlm gene codon frequency of utilization and wild type gene relatively see attached list 1.2, the conversion binary expression vector pBin438 of the structure of the plant expression vector of fusion gene Btlm and mosaic gene BtSlm and Agrobacterium tumefaciens contains the exogenous gene expression framework (Li Taiyuan etc. that are made up of from BamHI-SalI fragment and the Nos transcription termination sequence of pBR322 the Ω fragment one of the CaMV35S promotor (DE35Sp) of being with two enhansers, TMV-RNA cDNA, Chinese science (B collects) 24:276-282,1995).Be inserted into after with BamHI and SalI the fusion gene Btlm among the pSKBtlm being cut out between the BamHI of pBin438 and the SalI site and be built into plant expression vector pBlm.With XbaI enzymolysis pSlm, use the Klenow enzyme with end-filling then, use the SalI enzymolysis again, electrophoresis reclaims the BtSlm gene fragment of 1.9kb and uses the BamH1 enzymolysis on Agarose glue, Klenow mends flat, and the pBin438 behind the SalI enzymolysis builds the plant expression vector pBSlm that connection then is built into BtSlm.After more than connecting product difference transformed into escherichia coli DH5 α, select anti-Kanamycin clone and after the restriction analysis proof makes up correctly, use freeze-thaw method (An et al, Methods inEnzymology 153:239,1987) above expression vector is transformed into respectively among Agrobacterium tumefaciens (A.tumefaciens) LB4404, through the kantlex screening, plasmid analysis proves that its structure structure promptly can be used for Plant Transformation after entirely true.The concrete building process of this plant expression vector is seen accompanying drawing 3.The conversion of acquisition 1. tobaccos of embodiment two insect-resistant transgenic plants

Cross the Ye Panfa (Horch et al.Science 227:1229-1231,1985) that cultivates altogether with Agrobacterium tumefaciens with the blade pass of the tobacco NC89 of sterile culture the pBlm and the T-DNA among the pBSlm (comprising NPTII gene and corresponding anti insect gene) of above structure is transferred to the insect-resistant transgenic tobacco plant that has obtained a collection of anti-kantlex on the tobacco karyomit(e) respectively.2. the PCR of the analysis 2.1 transformation tissue culture plant of transformation tissue culture tobacco plant detects

The PCR of tobacco DNA extraction and transgene tobacco detects and is undertaken by described methods such as Li Taiyuan (Chinese science B collects 24:276-282,1994), and the PCR primer is: 35S ⁺5 ' CTGACGTAAGGGATGACGC 19merRe-Bt, 5 ' TTGAATTGAATACGCATCTCC 21mer

Carry out the about 500bP of PCR products therefrom with these two primers.The PCR of the regeneration plant of the anti-kantlex of part the results are shown in accompanying drawing 7.These PCR results show that Btlm or BtSlm anti insect gene may be incorporated on the karyomit(e) of 7 strains that detected and 3 tobaccos.2.2 the extraction of Southern hybridization analysis (1) DNA of plants

Get and extract nucleus DNA by the method (PlantMol.Bol.Rep, 11 (2): 122-127,1993) of reports such as Paterson after 2 gram blades are pulverized in liquid nitrogen.Quantitative ultraviolet determination method, the i.e. 1OD of using of DNA ₂₆₀=50 μ g/ml or more definite with EB dyeing behind the Agarose electrophoresis.(2) enzymolysis of DNA of plants and electrophoresis

Get HindIII and 20 μ l, 10 * restriction enzyme damping fluid that 20 μ gDNA add 150 units, adding aseptic redistilled water to cumulative volume is 200 μ l, 37 ℃ of incubated overnight.95% ethanol that adds 20 μ l 3M NaAcPH5.5 and 2 times of volumes then, mixing postposition-70 ℃ 15min, the centrifugal 5min deposit D of 12000rpm NA, wash precipitation once with 70% ethanol, be dissolved in an amount of TE (10mmol/L Tris-HCl after DNA precipitated vacuum-drying, 1mmol/L EDTA PH8.0), on 0.8%Agarose glue with 80V electrophoretic separation DNA.(3) commentaries on classics film, primer mark and the hybridization of DNA

All with reference to described the carrying out of molecular cloning one book (Sambrook et a1.CSHL Press, 1989), used probe for α- ³²The EcoRV of P-dCTP and Ready Tb Go dna marker test kit mark and the S29K gene fragment (1.1Kb) of XhoI enzymolysis.Southern results of hybridization (seeing accompanying drawing 8C) to the part plant proves that BT gene and API-BA gene have been incorporated in the tobacco karyomit(e) that is detected, and all is that single copy inserts in the plant that is detected.2.3 the Bt insect-killing protein in the transgene tobacco detects the proteic enzyme linked immunosorbent detection of Bt (ELISA) in (a) transgene tobacco

About tobacco leaf 50mg, add after the liquid nitrogen grinding and extract damping fluid (50mM Na ₂CO ₃, pH9.5, luM Leupeptin, lmM PMSF, 0.05% (v/v)) and Tween-20,0.1% (w/v) NaCl, 0.05% (v/v) mercaptoethanol), 10, the centrifugal 10min of 000rpm.Get supernatant and be used for the ELISA detection.Take the double antibodies sandwich method, with coating buffer (50mM Na ₂CO ₃, 0.02% (w/v) NaN ₃) bag is anti-by one, one anti-ly is the anti-B.t. serum of chicken (1: 2500), and two is anti-with mouse-anti B.t. serum (1: 1000), and the enzyme len antibody is with the IgG of the alkaline phosphate ester enzyme labelling of sheep anti mouse, and antibody dilution is with phosphoric acid buffer PBST (40g/L NaCl, 4.4g/L KH ₂PO ₄, 29.1lg/L Na ₂HPO ₄12H ₂O, 0.5ml/L Tween-20, pH7.2-7.4), usefulness 1mg/mL p-nitrophenyl Di-Sodium Phosphate ( -Nitrophenyl Phoshate Disodium (pNPP)) (Sigma company product) colour developing 15-30min, 405nm surveys the ultraviolet radiation absorption value.Measure protein concentration with Bio-Rad protein assay kit, contrast is made typical curve with the plant leaf of blank conversion with the Bt crystallin, calculates the proteic expression amount of Bt.

The pest-resistant plant of Btlm and pest-resistant plant that BtSlm is changeed in 22 strains are changeed in 8 strains to carry out the ELISA detected result and reduces accompanying drawing 10, the result shows that tentatively Bc insecticidal proteins average expression amount is that the Bt insecticidal proteins average expression amount that 0.07% of blade total soluble protein changes in the BtSlm gene plant can reach 0.23% in the plant that changes Btlm, though the reaction of the background of contrast is higher in detecting, but total expression amount of relatively seeing the latter is the former three times, this sign shows may change the proteic cellular localization of Bt after having added secreting signal peptide, and to help the proteic collection of Bt tired thereby increased the proteic stability of Bt.(b) Westemblot analyzes

Get the fresh blade of 100mg, add 100 μ l, 2 * sample-loading buffer after the liquid nitrogen grinding, 100 ℃ are boiled 3-5min, and behind the centrifugal 5min of 12000rpm, the gained supernatant promptly can be used for electrophoretic analysis.Get above-mentioned protein extract 20 μ l, on 10%SDS-PAGE glue, with Tris-glycine buffer system (PH8.3), voltage 60V, electrophoresis 4-6hr.After electrophoresis finished, with 9 volts of the half-dried transfer instrument of Bio-Rad constant voltages, 30min was transferred to albumen with on the pretreated pvdf membrane of methyl alcohol.Then film is placed the not protein-bonded position of solution III sealing, shake 1hr or 4 ℃ under the room temperature and spend the night; Film is changed in the anti-reaction solution (solution I+1%BSA, ProteinA Spharose6B column purification is crossed exempts from anti-CrylAc antibody, 1: 500 times of dilution), and room temperature is shaken 1hr; Wash 3 times each 3-5min with solution I; Film is placed colour developing liquid (10ml solution II+33 μ lNBT+66 μ lBCIP) colour developing.The used solution of Western blot is as follows: 2 * sample-loading buffer: 125mM Tris-HCl PH6.8,20% glycerine, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, the half-dried transfering buffering liquid of 2% mercaptoethanol: 48mmol/L Tris, 39mmol/L glycine, 0.0375%SDS, 20% methanol solution I:20mmol/L Tris-HCl PH7.4,0.15mol/L NaCl, 1mmol/LEDTA, 0.1%Tween-20 solution II: 0.1mol/L Tris, 0.1mol/L NaCl, 5mmol/L MgCl ₂(PH9.5) solution III: the solution I that contains 5% skimmed milk powder.The qualitative Western blot analytical results of part plant is seen accompanying drawing 9, and the result shows that improved Btlm or BtSlm gene have obvious expression in transfer-gen plant.The molecular weight of albumen of specific immune reaction is consistent in expression in escherichia coli product (swimming lane 1) size with the 1.8KbBtlm gene fragment, show that these two Bt genes can both correctly express insecticidal proteins in plant, and the signal peptide on the BtSlm chimeric insecticidal proteins of expressing is removed by correct processing.But whether relevant signal peptide has been brought into play its due function and processing situation thereof is still needed and further experimental results show that.2.4 the fresh blade that transgene tobacco or non-transgenic tobacco are got in the pest-resistant experiment of transgene tobacco and filial generation genetic analysis with aseptic water washing clean and with gauze the water on the leaf is blotted after put into small plastic box, every box is put the bollworm (Heliothisarmigera H ü bner) that a tribal chief worker raises, and every strain tobacco is cooked 6～12 cephalonts.After worm examination box is added a cover, add up larval mortality after 3 days or 5 days 25 ℃ of placements.Pest-resistant test triplicate.Transformation tissue culture tobacco worm test result reduces accompanying drawing 11.

Worm test result result shows the plant that can choose high insect-resistance in the plant that changes Btlm and change the BtSlm gene, plant with the examination worm mortality ratio more than 50% accounts for 76% and 85% of the plant number of always participating in the experiment respectively, this result is consistent with the proteic expression level of Bt, and another shows that the transfer-gen plant that changes the BtSlm gene that has signal coding sequence may be easier to choose high insect-resistance plant.

In order to understand Btlm and the genetic stability of BtSlm gene in transfer-gen plant, kalamycin resistance and the insect-resistance of the selfing generation plant of part plant detected.Getting changes the tired plant selfing monobasic seed (T1) of base by the working method of doing aseptic seedling, and the seed after the sterilization is transferred to the MS that contains the 200mg/ml kantlex ₀On the plate culture medium, put in the illumination box and cultivate, the plant of anti-kantlex becomes white very soon to treat to grow behind a pair of true leaf not by seedling, and the seedling of anti-kantlex still keeps green, count the green seedling number and the white seedling number of each strain system, the anti-separation ratio of statistics kantlex.For carrying out pest-resistant test for plant, method is same as above simultaneously, and the statistics insect-resistance is the separation case in the generation.The statistical meter of kalamycin resistance the results are shown in shown in the subordinate list 2: the progeny analysis result shows that the NPTII gene (kalamycin resistance gene) that is taken on the plant chromosome by expression vector is isolating by single-gene in the transfer-gen plant filial generation substantially, so should be inheritance stability, can choose transgenosis from filial generation isozygotys and is, and also demonstrate 3: 1 separation characteristic with the insect-resistance of the closely linked Bt gene of NPTII, show that tentatively synthetic Bt base is stranded Btlm and BtSlm also can genetic stability the transfer-gen plant offspring.

The production kind Ji that has transformed cotton with the Agrobacterium tumefaciens transformant of pBlm and pBSlm closes 321 and check and efficient insect-resistant transgenic plant has tentatively been chosen in the bollworm resisting test through PCR, so Bt provided by the invention merges anti insect gene engineering research and exploitation that insecticidal protein gene Btlm and BtSlm can be used for plant.Reference [1] Stratagene company product, Cat No.212207[2] Promega company product, Cat No.P2191[3] GibcoBRL company product, Cat No18258-012[4] GibcoBRL company product, Cat No15364-011[5] GibcoBRL company product, Cat No18313-015

The variation of subordinate list 1 transformation front and back fusion insecticidal protein gene Btlm coding region codon usage frequency reaches the comparison with the vegetable codon frequency of utilization

Password	aa	Btlm				The frequent code check % of plant	Password	aa	Btlm				Vegetable codon frequency %	Password	aa	Btlm				Vegetable codon frequency %	Password	aa	Btlm				Vegetable codon frequency %
																												The password subnumber		*Codon %		The password subnumber		*Codon %		The password subnumber		*Codon %		The password subnumber		*Codon %
		Before changing	After changing	Before changing	After changing				Before changing	After changing	Before changing	After changing				Before changing	After changing	Before changing	After changing				Before changing	After changing	Before changing	After changing
																												TTT F 31 7 86.1 19.4 45 TTC F 5 29 13.9 80.6 55 TTA L 22 0 44.9 0 10 TTG L 5 18 10.2 36.7 26							TCT S 12 15 19.4 24.2 25 TCC S 7 17 11.3 27.4 18 TCA S 12 4 19.3 6.5 19 TCG S 7 0 11.3 0 6							TAT Y 21 9 84.0 36.0 43 TAC Y 4 16 16.0 64.0 57 TAA End 0 0 - - 48 TAG End 0 0 - - 19							TGT C 1 0 50.0 0 44 TGC C 1 2 50.0 100 56 TGA End 0 0 - - 33 TGG W 10 10 100 100 100
CTT L 10 8 20.4 16.3 28 CTC L 2 19 4.1 38.8 19 CTA L 9 2 18.4 4.1 8 CTG L 1 2 2.0 4.1 9							CCT P 10 9 30.3 27.3 32 CCC P 2 2 6.1 6.1 17 CCA P 15 21 45.5 63.6 42 CCG P 6 1 18.2 3.0 9							CAT H 8 5 80.0 50.0 54 CAC H 2 5 20.0 50.0 46 CAA Q 22 14 81.5 51.9 59 CAG Q 5 13 18.5 48.1 41							CGT R 7 8 17.1 19.5 12 CGC R 1 1 2.4 2.5 36 CGA R 4 1 9.8 2.4 4 CGG R 1 0 2.4 0 13
																												ATT I 23 13 47.9 27.1 45 ATC I 6 31 12.5 64.6 37 ATA I 19 4 39.6 8.3 37 ATG M 8 8 100 100 100							ACT T 11 13 29.7 35.1 35 ACC T 7 14 18.9 37.8 30 ACA T 13 10 35.1 27.0 27 ACG T 6 0 16.2 0 8							AAT N 34 12 70.8 25.0 - AAC N 14 36 29.2 75.0 55 AAA K 1 0 50.0 0 39 AAG K 1 2 50.0 100 61							AGT S 21 5 33.9 8.0 14 AGC S 3 21 4.8 33.9 18 AGA R 23 18 56.1 43.9 30 AGG R 5 13 12.2 31.7 25
GTT V 18 20 43.9 48.8 34 GTC V 0 4 0 9.8 24 GTA V 17 1 41.5 2.4 11 GTG V 6 16 14.6 39.0 31							GCT A 19 18 54.3 51.4 42 GCC A 4 9 11.4 25.7 27 GCA A 11 8 31.4 22.9 25 GCG A 1 0 2.9 0 6							GAT D 20 11 80.0 44.0 58 GAC D 5 14 20.0 56.0 42 GAA E 26 16 86.7 53.3 49 GAG E 4 14 13.3 46.7 51							GGT G 12 19 26.1 41.3 34 GGC G 5 2 10.9 4.3 16 GGA G 22 21 47.8 45.7 38 GGG G 7 4 15.2 8.7 12

The genetic analysis of subordinate list 2. transgenic tobacco plant filial generations

H ₀：O-T＝0，α＝0.05，df＝1，x ² _0.05＝3.841

Work as x ²＜x ² _0.05, the resistance ratio of kantlex meets typical 3: 1 separates ratio

Expression vecror	Transgenic plant No.	Green plant number/White plant number	x 2	3∶1 segregation
					pBlm	17	174/59	0.0014	Yes
	424	107/33	0.1523	Yes
						410	190/25	33.876	No
	407	372/115	0.4989	Yes
						1mQ	143/43	0.9316	Yes
	413	90/31	0.0248	Yes
					pBSlm	7m-3	223/70	0.1923	yes
NC89，Non-transformed control		0/198	-	-

Claims (11)

1. chimeric protein gene BtSlm who constitutes by the nucleotide sequence of secretion signal peptide-coding sequence and bacillus thuringiensis insecticidal proteins, total length 1947bp, its nucleotide sequence is as follows:

9 18 27 36 45 545′ATG GGA TTT TTC CTT TTT TCT CAA ATG CCA TCC TTC TTT CTC GTG TCC ACT CTT

63 72 81 90 99 108 CTC CTT TTC CTC ATT ATC TCT CAC TCC TCT CAC GCG GAT CCG ACC ATG GAC AAC

117 126 135 144 153 162 AAC CCA AAC ATC AAC GAA TGC ATT CCA TAC AAC TGC TTG AGT AAC CCA GAAGTT

171 180 189 198 207 216 GAA GTA CTT GGT GGA GAA CGC ATC GAA ACC GGT TAC ACT CCA ATC GAC ATCTCC

225 234 243 252 261 270 TTG TCC TTG ACA CAG TTT CTG CTC AGC GAG TTC GTG CCA GGT GCT GGG TTCGTG

279 288 297 306 315 324 CTC GGA CTA GTT GAC ATC ATC TGG GGT ATC TTT GGT CCA TCC CAA TGG GACGCA

333 342 351 360 369 378 TTC CTG GTT CAA ATT GAA CAG CTC ATC AAC CAG AGG ATC GAA GAG TTC GCTAGG

387 396 405 414 423 432 AAC CAG GCC ATC TCT AGG TTG GAA GGA CTC AGC AAT CTC TAC CAA ATC TATGCA

441 450 459 468 477 486 GAG TCT TTC AGA GAA TGG GAG GCC GAT CCT ACT AAC CCA GCT CTC AGG GAGGAG

495 504 513 522 531 540 ATG CGT ATT CAA TTC AAC GAT ATG AAC AGC GCC TTG ACC ACT GCT ATC CCA TTG

549 558 567 576 585 594 TTC GCA GTC CAG AAC TAC CAG GTG CCT CTC TTG TCC GTG TAC GTT CAA GCCGCT

603 612 621 630 639 648 AAT CTT CAT CTC AGC GTG CTT CGA GAC GTT TCA GTG TTT GGA CAG AGG TGGGGA

657 666 675 684 693 702 TTC GAT GCT GCA ACC ATC AAT AGC AGA TAC AAC GAC CTT ACT AGG CTC ATTGGA

711 720 729 738 747 756

AAC TAC ACC GAC CAC GCT GTT CGT TGG TAC AAC ACT GGT TTG GAG CGT GTCTGG

765 774 783 792 801 810

GGT CCT GAT AGC AGA GAT TGG ATT AGA TAC AAC CAG TTC AGG AGA GAA TTGACC

819 828 837 846 855 864

CTT ACA GTG TTG GAT ATC GTG TCT CTC TTC CCG AAC TAT GAC TCC AGA ACC TAC

873 882 891 900 909 918

CCT ATC CGT ACA GTG TCC CAA CTT ACC AGA GAA ATC TAT ACT AAC CCA GTTCTT

927 936 945 954 963 972

GAG AAC TTC GAC GGT AGC TTC CGT GGT TCT GCC CAA GGT ATC GAA GGC TCCATC

981 990 999 1008 1017 1026

AGG AGC CCA CAC TTG ATG GAC ATC TTG AAC AGC ATA ACT ATC TAC ACC GATGCT

1035 1044 1053 1062 1071 1080

CAC AGA GGA GAG TAT TAC TGG TCT GGA CAC CAG ATC ATG GCC TCT CCA GTTGGA

1089 1098 1107 1116 1125 1134

TTC AGC GGG CCC GAA TTC ACC TTC CCT CTC TAT GGA ACT ATG GGT AAC GCCGCT

1143 1152 1161 1170 1179 1188

CCA CAA CAA AGG ATC GTT GCT CAA CTA GGT CAG GGT GTC TAC AGA ACC TTGTCT

1197 1206 1215 1224 1233 1242

TCC ACT TTG TAC AGA AGG CCA TTC AAT ATC GGT ATC AAC AAC CAG CAA CTTTCC

1251 1260 1269 1278 1287 1296

GTT CTC GAT GGA ACA GAG TTC GCC TAT GGG ACC TCT TCT AAC TTG CCA TCCGCT

1305 1314 1323 1332 1341 1350

GTT TAC AGA AAG TCC GGA ACC GTT GAT AGC TTG GAC GAA ATT CCA CCA CAGAAC

1359 1368 1377 1386 1395 1404

AAC AAT GTG CCA CCC AGG CAA GGA TTC AGC CAC AGG TTG AGC CAT GTG TCCATG

1413 1422 1431 1440 1449 1458

TTC CGT TCC GGT TTC AGC AAC AGT AGC GTG AGC ATC ATC AGA GCT CCT ATGTTC

1467 1476 1485 1494 1503 1512

TCT TGG ATA CAT CGT AGT GCT GAG TTC AAC AAT ATC ATT GCA TCC GAT AGC ATC

1521 1530 1539 1548 1557 1566

ACT CAA ATT CCT GCA GTT AAG GGA AAC TTT CTC TTC AAT GGT TCT GTC ATT TCA

1575 1584 1593 1602 1611 1620

GGA CCA GGA TTC ACA GGA GGA GAC CTC GTT AGA CTC AAC AGC AGT GGA AATAAC

1629 1638 1647 1656 1665 1674

ATC CAG AAT AGA GGG TAT ATT GAA GTT CCA ATT CAT TTC CCT TCC ACA TCT ACC

1683 1692 1701 1710 1719 1728

AGA TAT AGA GTT CGT GTG AGG TAT GCT TCT GTG ACT CCT ATT CAT CTC AAC GTT

1737 1746 1755 1764 1773 1782

AAT TGG GGT AAT TCA TCT ATC TTC AGC AAC ACA GTT CCA GCT ACA GCT ACCTCC

1791 1800 1809 1818 1827 1836

TTG GAT AAC CTC CAA TCC AGC GAC TTC GGA TAC TTT GAG AGC GCC AAT GCTTTC

1845 1854 1863 1872 1881 1890

ACA TCT TCA CTC GGC AAC ATA GTG GGT GTT AGA AAC TTT AGT GGA ACT GCAGGT

1899 1908 1917 1926 1935 1944

GTG ATC ATA GAC AGA TTT GAG TTC ATT CCA GTT ACT GCA ACA CTC GAG GCTGAA

TGA3′

2. chimeric protein gene BtSlm according to claim 1, the antigen-4 fusion protein gene Btlm that it is made up of 284 amino acid of a part by 331 amino acid of a part of bacillus thuringiensis insecticidal proteins CrylAb and CrylAc of synthetic and merge the chimeric protein gene BtSlm that forms behind the nucleotide sequence of the secreting signal peptide that one section coding is made up of 30 amino acid at its N end.

3. chimeric protein gene BtSlm according to claim 1, its amino acid sequence coded is:

1 18

M G F F L F S Q M P S F F L V S T L

37 54

N P N I N E C I P Y N C L S N P E V 55 72 E V L G G E R I E T G Y T P I D I S 73 90 L S L T Q F L L S E F V P G A G F V 91 108 L G L V D I I W G I F G P S ^-Q W D A109 126 F L V Q I E Q L I N Q R I E E F A R127 144 N Q A I S R L E G L S N L Y Q I Y A145 162 E S F R E W E A D P T N P A L R E E163 180 M R I Q F N D M N S A L T T A I P L181 198 F A V Q N Y Q V P L L S V Y V Q A A199 216 N L H L S V L R D V S V F G Q R W G217 234 F D A A T I N S R Y N D L T R L I G236 252 N Y T D H A V R W Y N T G L E R V W254 270 G P D S R D W I R Y N Q F R R E L T271 288 L T V L D I V S L F P N Y D S R T Y289 306 P I R T V S Q L T R E I Y T N P V L307 324 E N F D G S F R G S A Q G I E G S I325 342 R S P H L M D I L N S I T I Y T D A342 360 H R G E Y Y W S G H Q I M A S P V G361 378

379 396 P Q Q R I V A Q L G Q G V Y R T L S397 414 S T L Y R R P F N I G I N N Q Q L S415 432 V L D G T E F A Y G T S S N L P S A433 450 V Y R K S G T V D S L D E I P P Q N451 468 N N V P P R Q G F S H R L S H V S M469 486 F R S G F S N S S V S I I R A P M F487 504 S W I H R S A E F N N I I A S D S I505 522 T Q I P A V K G N F L F N G S V I S523 540 G P G F T G G D L V R L N S S G N N541 558 I Q N R G Y I E V P I H F P S T S T559 576 R Y R V R V R Y A S V T P I H L N V 577 594

N W G N S S I F S N T V P A T A T S 595 612

L D N L Q S S D F G Y F E S A N A F 613 630

T S S L G N I V G V R N F S- G T A G 631 648

V I I D R F E F I P V T A T L E A E

4. a recombinant plasmid of naming to pSlm is characterized in that inserting the described chimeric protein gene of claim 1 BtSlm and forms on a kind of cloning vector.

5. a recombinant microorganism is characterized in that containing the described recombinant plasmid pSlm of claim 4.

6. a kind of recombinant microorganism according to claim 5 is characterized in that the bacillus coli DH 5 alpha that has been transformed by recombinant plasmid pSlm.

7. the expression vector of energy constitutive expression external source anti insect gene in plant is characterized in that it contains the described chimeric protein gene of claim 1 BtSlm, and this plant expression vector is named and is pBSlm.

8. recombinant microorganism is characterized in that it contains the described plant expression vector pBSlm of claim 7.

9. recombinant microorganism according to claim 8, it is bacillus coli DH 5 alpha or the Agrobacterium tumefaciens LBA4404 that plant expression vector pBSlm has transformed.

10. the application of expression vector pBSlm according to claim 7 in plant genetic engineering.

11. according to Claim 8 or the application of the recombinant microorganism described in 9 in plant genetic engineering.

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