CN1134540C - A chimeric insecticidal protein gene that can secrete its product extracellularly - 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 chimeric insecticidal protein gene that can secrete its product extracellularly

The invention belongs to the field of plant genetic engineering, and relates to a fusion protein gene Btlm composed of a Bacillus thuringiensis (Bt) insecticidal protein CrylAb (331 amino acids encoded at the N-terminal) and a CrylAc part (284 amino acids encoded at the C-terminal) by a chemical method. The 1848 base pair sequence of the synthetic gene can endow the transgenic plant with high insect resistance after being expressed in the plant. In order to enable the Btlm insecticidal protein expressed in plants to be secreted into the extracellular space to improve the stability of the insecticidal protein, and at the same time reduce the interference of the Bt insecticidal protein on the normal function of plant cells, it is beneficial to screen for changes in agronomic traits. Large insect-resistant transgenic plants, the present invention is based on the synthesis of the above-mentioned insecticidal protein gene Btlm, and an artificially synthesized nucleotide sequence encoding the signal peptide of the tobacco pathogenicity-related protein PR1b is connected to its 5' end, thereby forming A 1947bp chimeric insecticidal protein gene BtSlm. After constructing plant expression vectors with Btlm and BtSlm, tobacco and cotton were transformed, and high-efficiency insect-resistant transgenic plants were selected from the transformed regenerated plants. The insect test results of transgenic tobacco showed that the plants expressing BtSlm had higher insect resistance than those expressing Btlm.

The crystal protein of Bacillus thuringiensis (Bt) can be used as a biopesticide to specifically poison certain insects. After the crystal protein is ingested by insects, it dissolves under the alkaline conditions of the insect intestinal tract to produce a protoxin molecule of about 130KD. The C-terminus of the protoxin is degraded by intestinal protease hydrolysis, and finally a toxic protein consisting of 65-70KD of the N-terminus is produced. A segment of N-terminal residues of the toxic protein (28 in terms of CrylA) must also be processed and removed by intestinal protease to form a fully activated toxic protein. The activated toxic protein binds to the receptors on the midgut epithelial cells of sensitive insects, and inserts into the midgut cell membrane to form small holes or ion channels of 0.5-1.0nm, causing the extravasation of intracellular ions and the infiltration of water. Swelling and rupture, a large number of cells are destroyed, so that the larvae stop eating and die. It has been proved that Bt crystal protein has no toxic effect on the human body, so Bt preparation has been used as a pollution-free natural microbial insecticide for more than 50 years in agriculture, forestry and environmental sanitation, etc., and the output of Bt preparation is also increasing year by year Increase. However, due to the poor stability of Bt crystal protein under natural conditions, the inability to penetrate into tissues and the narrow insecticidal spectrum, the development of this biopesticide has been greatly restricted, so the current control of agricultural pests is mainly by chemical pesticides. In the mid-1980s, with the maturity of plant genetic engineering technology, the success of Bt gene cloning and the in-depth research on the mechanism of Bt crystal protein insecticides, it was possible to transfer Bt genes into plants to obtain insect-resistant insecticides that could express insecticidal proteins. transgenic plants. At present, there are more than 26 kinds of Bt transgenic plants in the world, including cotton, corn, rice, potato and other major crops. These transgenic plants have acquired obvious insect resistance.

The expression level of wild-type Bt crystal protein gene in transgenic plants was very low. Driven by the CaMV35S promoter, its expression level accounts for about 0.001% of the total soluble protein, so the transgenic plants cannot obtain the ability to kill most insects that are not sensitive to Bt toxic proteins, such as the main Lepidoptera pests of many crops.

Perlark et al. (Proc.Natl.Acad.Sci.USA 88:3324-3328, 1991) according to the codon usage frequency of plant genes and the motifs that may affect the stability of plant mRNA in keeping the amino acid sequence encoded by the original Bt gene unchanged Next, the Bt crystal protein genes CrylAb and CrylAc were partially modified or completely resynthesized. The amount of insecticidal protein expressed in transgenic plants by the partially modified and re-synthesized genes was about 10 times and 100 times higher than that of the wild type gene, respectively. However, there are many problems in the regulation of Bt gene expression in plants that still need to be further studied, and some aspects still need to be greatly improved in the practical application of Bt transgenic plants. For example, the currently synthesized Bt gene stays in the cell after being expressed in the plant, and the high expression of the foreign protein in the cell will inevitably interfere with the physiological function of the cell itself, which will affect the original traits of the transgenic plant to a certain extent. For example, in the breeding process of insect-resistant transgenic plants, it is often found that the agronomic performance of transgenic plants with high insect resistance is poor, which affects their yield and quality, while the insect resistance of plants with similar agronomic traits to the original varieties is not ideal. There are many reasons for these problems. For example, the transformation and regeneration process may cause somatic cell variation, and the insertion position of foreign genes on plant chromosomes may activate or interfere with the expression of certain plant genes, which may lead to agronomic traits. change. The accumulation of exogenous gene products in transformed cells, especially the accumulation of exogenous proteins in plant cells when expressed at a high level, may greatly interfere with the normal physiological functions of cells and eventually lead to changes in some plant characteristics. In addition, exogenous gene expression products are easily in contact with various intracellular proteases in the intracellular environment, resulting in a decrease in stability. Therefore, a large amount of exogenous protein accumulated in the cell will have adverse effects on both the plant itself and the exogenous gene product.

In order to overcome the above-mentioned problems, the present invention considers that the Bt protein secreted extracellularly will improve its stability, thereby increasing its accumulation in plants, eventually leading to the improvement of the insecticidal activity of transgenic plants, and also reducing the impact of expression products on Disturbance of plant cell function. At present, existing evidence shows that the signal peptide of PMb protein from tobacco can not only make pathogen-related proteins secreted extracellularly (Comelissen et al. EMBOJ.5: 37-40, 1986), but also can make exogenous proteins secreted extracellularly ( Denecke et al. 1990 The Plant Cell 2:51-59). Therefore, based on the above existing research background, the present invention synthesized a CryAb and CrylAc fusion gene consisting of the first amino acid residue at the N-terminal to the 331st and 332nd to 615th amino acid residues, and finally constructed the chimeric The Bt gene was named Btlm. On the basis of Btlm, a synthetic secretory signal peptide PRlb-s coding sequence was connected with Btlm to form a chimeric protein gene Btlm, and the plant expression vectors of these two genes were respectively constructed, and transformed by Agrobacterium tumefaciens. Tobacco and cotton. The insect test results of transgenic plants show that these two synthetic insecticidal protein genes can efficiently express the insect-resistant activity of Bt protein in plants, and the insect resistance of BtSlm plants is slightly higher than that of Btlm transgenic plants. Therefore, according to different purposes, the two All genes are very valuable insect-resistant genes in the research and development of plant insect-resistant genetic engineering.

In order to realize the purpose of the present invention, specifically take following technical steps:

Through the synthesis of Bt CrykA fusion protein gene (Btlm) oligonucleotide primers;

Synthesis and cloning of Btlm gene fragments (attached Figure 1);

  The assembly of the Btlm gene yielded the gene clone pSKBtlm encoding the CrylA fusion protein (attached Figure 2);

The Btlm gene sequence is connected with the signal peptide coding sequence, and a signal peptide coding sequence is constructed.

The BtSlm gene clone pSlm of coding sequence (accompanying drawing 3);

Btlm and chimeric BtSlm genes were inserted into the binary expression vector pBin438 to form plants

Express vectors pBlm and pBSlm (accompanying drawing 6), and it is transformed into Escherichia coli

Or Agrobacterium tumefaciens [Agrobacterium tumefaciens] LBA4404;

pBlm and pBSlm or their Agrobacterium tumefaciens LBA4404 transformants are applied to plant transformation to obtain transgenic plants with good agronomic properties and high insect resistance.

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

Figure 1. Schematic diagram of the synthesis and subcloning of CrylA gene fragments. Abbreviations in the figure: PI-1～PV-9: the numbers of different oligonucleotide primers, and the numbers in parentheses after the numbers are the base numbers of the primers; SK: pBluescriptIISK ⁺ ; KL: Klenow enzyme; Am: Ampicillin resistance gene; Kb: kilobase pairs; Bm: BamHI; H3: HindIII;

A. Construction of the first segment (BamH1-EcoR1) subclone pBtfsI

B. Construction of the second segment (EcoR1-EcoRv) subclone pBtsII

C. Construction of the third segment (EcoRV-EcoR1) subclone pBtfsIII

D. Construction of the fourth segment (EcoRI-SacI) subclone pBtsIV

E. Construction of the fifth segment (SacI-salI) subclone pBtfsV Accompanying drawing 2. Construction of the CrylA fusion insecticidal protein gene clone pSKBtlm Accompanying drawing 3. The recombinant plasmid pSlm of the chimeric Bt gene BtSlm with the secretion signal peptide coding sequence Construction accompanying drawing 4. Nucleotide sequence and amino acid of the chimeric insecticidal protein gene BtSlm containing the secretory signal peptide coding sequence synthesized

Sequence accompanying drawing 5. The DNA sequence of the coding region of the Btlm fusion insecticidal protein synthesized and the corresponding part of the wild type

Comparison of DNA sequences.

W (upper row)...wild-type gene sequence; M (lower row)...synthetic fusion insecticide

Protein gene Btlm gene sequence. The bases in the box are the same bases in the W and M sequences

Table 1. Changes in the frequency of codon usage in the Btlm coding region of the fusion insecticidal protein gene before and after transformation

and comparison with plant codon usage frequency.

※The codon percentage means that the codon is in the coding region of the gene and encodes the same amino acid

The proportion of the total number of codons; plant codon usage frequency % refers to in dicotyledonous plants

The frequency of use (Murray EE. et al. Nucleic Acid Res. 17: 477-499 1989);

aa - amino acid code. Accompanying drawing 6. the construction of the plant expression vector of fusion Bt gene Btlm and chimeric Bt gene BtSlm

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

Kanamycin resistance gene; DE-35SP: CaMV35S promoter with double enhancers;

RB and LB are the right border and left border sequences of T-DNA, respectively. Accompanying drawing 7. PCR detection of transformed regenerated tobacco plants

Primers: 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 lanes 4-10 are PCR results of pBlm transgenic plants; Swimming lanes 11-13 are

PCR results of pBSlm transgenic plants. Primers are 35S ⁺ and Re-Bt-. Accompanying drawing 8. Southern blot analysis of transgenic tobacco plants (HindIII single enzyme digestion)

Hybridization results with Btlm gene probe

Swimming lane 1. pSKBtlm plasmid as positive control

Swimming lane 2. Swimming lanes 2, 3, and 4 are three pBlm transgenic plants; 5, 6

    are two pBSlm transgenic plants

Swimming lane 7. Non-transgenic tobacco as a control Figure 9. Protein immunoassay of transgenic tobacco

Lane 1. CrylAc protein (68KD) expressed in Escherichia coli

Lane 2. Non-transgenic tobacco control

Swimming lane 3-9. Different transgenic tobacco plants (3-5 are pBlm transgenic plants; 6-8 are

   pBSlm transgenic plants Figure 10. ELISA detection results of transgenic tobacco plants

The number of plants used for analysis were: 6 plants for control; 8 plants for pBlm transgenic plants;

22 pBSlm transgenic plants. Accompanying drawing 11. Distribution of resistance to cotton bollworm in transformed and regenerated plants with different gene structures

 The numbers in parentheses behind each gene structure indicate the total number of plants tested

The synthesis of the chimeric insecticidal protein gene BtSlm of embodiment band signal peptide sequence 1. the synthesis of Bacillus thuringiensis CrylA fusion insecticidal activity protein gene Btlm 1.1 Synthesis of CrylA fusion protein gene Btlm oligonucleotide primer

The oligonucleotide primers were synthesized by Applied Biosystem's DNA synthesizer at the New Technology Center of the Institute of Microbiology, Chinese Academy of Sciences, and purified by OPC (oligonucleotide purification column) or polyacrylamide gel electrophoresis before being used for the synthesis of double-stranded DNA. For the convenience of cloning, according to the endonuclease recognition sites in the Cry1Ab and Cry1Ac genes, the toxicity of the gene was divided into five segments for primer synthesis. There are 6 primers (BamH1-EcoR1) in the first segment (PI), and their sequences are 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 TTC TTC TTC GTT 66merPif-3-5′GAA CTC GCT GAG CAG CAG AAA CTG TGT CAA GGA GGA GATGTC GATG 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

There are 8 primers (EcoR1-EcoRV) in the second segment of TGC GTC (PII), and their sequences are 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 segment 3 () has 6 primers (EcoRV-EcoR1), and their sequences are 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

There are 6 primers (EcoR1-SacI) for the fourth segment of GAT CTG GTG TCC AGA (PIV), the sequence of which 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 fifth segment (PV) a total of 9 primers (SacI-SalI), the 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

Synthesis and cloning of AAA TCT GTC TAT GAT CAC ACC TGC AGT 72mer1.2 Cry1A fusion gene fragment

Mix 20pmol (10μl) of two adjacent primers in each segment, denature at 70°C for 10min, add 10×Klenow enzyme buffer (100mmol/L Tris-Cl pH7.5, 0.5mmol/L NaCl, 1mmol/L EDTA, 50mmol/L DTT) 2μl, Klenow DNA polymerase 1 unit, add dNTP to 0.1mmol/L, incubate at 37°C for 1h in a total volume of 20μl, the reaction product is adjacent to another polymerase produced by Klenow enzyme polymerization After the large fragments are mixed and denatured, PCR amplification is performed in the presence of primers at both ends to generate larger DNA fragments. PCR reaction in 20 or 50 μl volume with 50mmol/L KCl, 10mmol/L, Tris-HCl pH8.8, 1.5mmol/LMgCl2, 0.1mg/ml BSA, 0.2mmol/L dNTP, 5' end and 3' end primers Each 1μmol/L, 0.05U/μl Taq DNA polymerase, 1μl/each reaction The product from the Klenow or PCR reaction was used as a template to denature the reaction mixture at 94°C for 4min, then 94°C for 1min; 40-50°C (annealing temperature according to The number of bases paired between the primer and the template is determined), 1 min; 72°C, 1 min for a total of 30 cycles. The final PCR product of each segment was digested by the corresponding endonuclease and then ligated with the cloning vector pBluecriptIISK ^+[1] or pSP71 ^[2] with the same digestion, and then transformed into Escherichia coli DH5α ^[3] competent cells. White colonies were selected on the LB plates of X-gal and IPTG, and after plasmid extraction, plasmid restriction analysis and DNA sequence analysis, the subclones pBtSI (first section) and pBtSII (second section) of the CrylA gene fragment were selected. segment), pBtSIII (third segment), pBtSIV (fourth segment) and pBtfSV (fifth segment). The construction process of these five subclones is shown in Figures 1A-1E. The above DNA digestion, preparation of vectors, ligation reaction, preparation of competent cells, transformation, and screening of clones were all performed in accordance with the book "Molecular Cloning" (Sambrook J. et al. "Molecular Cloning" 2nd ed. CSH Laboratory Press, 1989 ) The method is carried out. DNA sequence analysis was carried out according to the method provided by Pharmacia Company T ₇ DNA sequencing Kit. 1.3 Assembly of fusion insecticidal protein CrylAc gene

After the sequence analysis proves that the sequence of the Bt gene fragment in each subclone is correct, the gene clone pSKBtlm encoding the CrylA fusion protein Btlm is obtained after the conventional recombinant DNA technology is used to connect the restriction sites of each subclone in sequence. Figure 2. 1.4 Correction of fusion insecticidal protein gene sequence

After sequence analysis of each subclone, deletions or substitutions of bases were found in some clones. In order to obtain a completely correct gene sequence, on the basis of subcloning, site-directed mutagenesis was performed on the wrong bases by following the instructions of Clontech's site-directed mutagenesis box. The selection primer for mutation is Stu/ScaI: 5'GTGGACTGGTGAAGTACTCAACCAAGTC or AflIII/BgIII primer: 5'CAGGAAAGAAGATCTGAGCAAAAG. The mutation primer consists of the base to be changed and oligonucleotides consisting of 10-15 bases at both ends. The mutation primer should be on the same DNA strand as the selection primer.

The base errors around EcoRI in the Bt gene fragment in the subclone pBtfS12 were corrected by PCR with two pairs of primers, and the EcoRI site was removed at the same time for the convenience of screening mutant transformants. The two primers are

①P1-1+ ^mI-

mI-5'CCTAGCGAACTCTTCGATCCTCTGGTTGATGAG 33mer

②PII-8+mII ⁺

mII+5'ATCGAAGAGTTCGCCAGGAACCAGGCCATCTCTAGG 36mer

The pBtfS12 transformed recombinant plasmid was named pBtfS12. The PCR reaction conditions and cloning methods were the same as those described above. 2. Construction of chimeric insecticidal protein gene clone pSlm with secretion signal peptide coding sequence (a) Synthesis and cloning of secretion signal peptide coding sequence

In order to make the Bt fusion insecticidal protein gene expressed in plants, its protein product does not accumulate in the cell and is secreted into the extracellular intercellular space, the present invention combines the signal peptide coding sequence PRlbS of the artificially synthesized tobacco pRlb protein with the above-mentioned synthesis for the first time. A chimeric insecticidal protein gene BtSlm with a secretory signal peptide coding sequence was obtained after joining the crylA fusion protein gene Btlm. The construction process of the recombinant plasmid pSlm of the BtSlm gene is shown in Figure 3. The sequence of the coding region of the synthetic PRlb secretion signal peptide is as follows: 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 20pmol of each of the above two 60mers, react in 50ml klenow enzyme reaction system (as described in 1.2 above) at 37°C for 1h, add benzene volume phenol/chloroform ( 1:1) extract once, take the upper layer aqueous solution and add 2 times the volume of ethanol and 5ml 3M NaAc, mix well and place at -70°C to precipitate DNAlnr, centrifuge at 12000rpm for 10 minutes to collect the precipitate, wash the precipitate with 70% ethanol, dry and dissolve in 20ml sterile In water, carry out XbaI and SalI enzymatic hydrolysis on the above-mentioned DNA and PUC19 plasmid DNA according to the method described in "Molecular Cloning". The recombinant plasmid pUSP containing S1b was obtained by ligation with the pUC19 ^[4] that was decomposed. After transforming Escherichia coli DH5α, the transformant containing the recombinant plasmid could be screened on the Xgal and IPTG plates, and the secretion was proved by enzyme digestion analysis and DNA sequence analysis. The sequence of the signal peptide S1b is correct, but the sequence of Zhang’s BglII restriction site is incorrectly synthesized from AGATCT to AGATTC. Since the error at this site will not affect the expression of the Slb signal peptide, it is still retained in pUSP. The construction process of this sequence pUSP is shown in Figure 3. In order to properly increase the GC content in the Slb sequence and avoid rare codons in some plants, 15 codons were changed during synthesis, but the encoded amino acids were not changed. (b) Construction of chimeric insecticidal protein gene Stlm clone pSlm with secretion signal peptide coding sequence

According to the recombinant DNA method described in the book "Molecular Cloning", the plasmid pUSP was digested with MluI, then the end was bluntly filled with Klenow enzyme, and then the end was digested with SalI and BamH1. After the 1.8kb fusion insecticidal protein gene fragments were ligated, the clone pSlm of the chimeric insecticidal protein gene BtSlm containing the secretory signal peptide numbering sequence was obtained (see Figure 3 for details). Sequence analysis shows that the sequence of the BtSlm gene is consistent with the original design, and its DNA sequence and encoded amino acid sequence are shown in claims 1 and 2 and accompanying drawing 4 . The synthetic chimeric insecticidal protein gene BtSlm has a full length of 1947bp, in which the 1st to 90th nucleotides encode the secretion signal peptide Slb; 91-99 are three codons formed by restriction sites; 100-1092 and 1093 -1947 encodes the 1-331 amino acids of CrylAb and the 332-616 amino acids of CrylAc (including a terminal codon), namely the Btlm gene. The comparison of Btlm gene and corresponding wild-type gene nucleotide sequence is shown in accompanying drawing 5, and the GC of Btlm gene increases to 47.4% by wild-type 37% than, and the change of its codon usage frequency is more conducive to this gene in plant Express. The comparison between the codon usage frequency of the Btlm gene and the wild-type gene is shown in Table 1. 2. Construction of plant expression vectors for the fusion gene Btlm and the chimeric gene BtSlm and transformation of Agrobacterium tumefaciens. CaMV35S promoter (DE35Sp), TMV-RNA cDNA Ω fragment, a BamHI-SalI fragment from pBR322 and a foreign gene expression framework composed of the Nos transcription termination sequence (Li Taiyuan et al., Chinese Science (B Series) 24: 276- 282, 1995). The fusion gene Btlm in pSKBtlm was cut out with BamHI and SalI and inserted between the BamHI and SalI sites of pBin438 to construct the plant expression vector pBlm. Digested pSlm with XbaI, then filled in the end with Klenow enzyme, then digested with SalI, recovered the 1.9kb BtSlm gene fragment by electrophoresis on Agarose gel, and constructed pBin438 after enzymatic digestion with BamH1, Klenow filling, and SalI enzymatic digestion The connection was then constructed into the plant expression vector pBSlm of BtSlm. After the above ligation products were transformed into Escherichia coli DH5α, the anti-Kanamycin clones were selected and analyzed by enzyme digestion to prove that the construction was correct, and then the above expression vectors were transformed into Agrobacterium tumefaciens by the freeze-thaw method (An et al, Methods in Enzymology 153:239, 1987). (A. tumefaciens) LB4404 can be used for plant transformation after selection by kanamycin and plasmid analysis proves that its structure is completely correct. The specific construction process of this plant expression vector is shown in Figure 3. The acquisition of embodiment two insect-resistant transgenic plants 1. Transformation of tobacco

The T-DNA (including NPTII Genes and corresponding insect-resistant genes) were transferred to tobacco chromosomes to obtain a batch of kanamycin-resistant insect-resistant transgenic tobacco plants. 2. Analysis of Transformed and Regenerated Tobacco Plants 2.1 PCR Detection of Transformed and Regenerated Plants

Tobacco DNA extraction and PCR detection of transgenic tobacco were carried out according to the method described by Li Taiyuan et al. (Chinese Science B Series 24: 276-282, 1994). PCR primers were: 35S ⁺ 5' CTGACGTAAGGGATGACGC 19merRe-Bt 5' TTGAATTGAATACGCATCTCC 21mer

The product obtained by PCR with these two primers is about 500bP. The PCR results of some regenerated plants resistant to kanamycin are shown in Figure 7. These PCR results indicated that the Btlm or BtSlm insect resistance gene may have been integrated into the chromosomes of 7 and 3 tobacco strains tested. 2.2 Southern hybridization analysis (1) Extraction of plant DNA

Take 2 grams of leaves and grind them into powder in liquid nitrogen, then extract the nuclear DNA according to the method reported by Paterson et al. (PlantMol.Bol.Rep, 11(2):122-127, 1993). Quantification of DNA was determined by UV assay, ie 1OD ₂₆₀ =50 μg/ml or comparatively by EB staining after Agarose electrophoresis. (2) Enzyme hydrolysis and electrophoresis of plant DNA

Take 20 μg of DNA, add 150 units of HindIII and 20 μl of 10× restriction endonuclease buffer, add sterile redistilled water to a total volume of 200 μl, and incubate overnight at 37°C. Then add 20 μl of 3M NaAcPH5.5 and 2 times the volume of 95% ethanol, mix well and place at -70°C for 15 min, centrifuge at 12000 rpm for 5 min to precipitate DNA, wash the precipitate once with 70% ethanol, vacuum dry the DNA precipitate and dissolve it in an appropriate amount of TE ( 10mmol/L Tris-HCl, 1mmol/L EDTA PH8.0), separated DNA by 80V electrophoresis on 0.8% Agarose gel. (3) DNA transfer, primer labeling and hybridization reaction

All were carried out with reference to the book Molecular Cloning (Sambrook et al. CSHL Press, 1989), and the probes used were S29K gene fragments digested by EcoRV and XhoI labeled with α- ^32P -dCTP and Ready Tb Go DNA Labeling Kit (1.1Kb). The results of Southern hybridization of some plants (see Figure 8C) prove that the BT gene and API-BA gene have been integrated into the detected tobacco chromosomes, and all of them are single-copy insertions in the detected plants. 2.3 Detection of Bt insecticidal protein in transgenic tobacco (a) Enzyme-linked immunoassay (ELISA) of Bt protein in transgenic tobacco

About 50mg of tobacco leaves, ground with liquid nitrogen and added extraction buffer (50mM Na ₂ CO ₃ , pH9.5, luM Leupeptin, lmM PMSF, 0.05% (v/v)) Tween-20, 0.1% (w/v) NaCl , 0.05% (v/v) mercaptoethanol), centrifuged at 10,000rpm for 10min. Take the supernatant for ELISA detection. The double-antibody sandwich method was adopted, and the primary antibody was coated with the coating solution (50mM Na ₂ CO ₃ , 0.02% (w/v) NaN ₃ ), the primary antibody was chicken anti-Bt serum (1:2500), and the secondary antibody was mouse anti- Bt serum (1:1000), the enzyme-linked antibody was labeled with goat anti-mouse alkaline phosphatase IgG, and the antibody was diluted with phosphate buffer PBST (40g/L NaCl, 4.4g/L KH ₂ PO ₄ , 29.1lg/ L Na ₂ HPO ₄ 12H ₂ O, 0.5ml/L Tween-20, pH7.2-7.4), with 1mg/mL disodium p-nitrophenylphosphate ( -Nitrophenyl Phoshate Disodium (pNPP)) (product of Sigma Company) develops color for 15-30min, and measures ultraviolet absorption value at 405nm. The protein concentration was measured with Bio-Rad protein assay kit, and the Bt crystal protein was used as a standard curve to calculate the expression level of Bt protein as compared with the blank transformed plant leaves.

The ELISA detection results of 8 Btlm-transformed insect-resistant plants and 22 BtSlm-transformed insect-resistant plants are summarized in Figure 10. The results preliminarily show that the average expression of Bc insecticidal protein in the Btlm-transferred plants is 0.07 of the total soluble protein in leaves. % and the average expression of Bt insecticidal protein in BtSlm gene plants can reach 0.23%. Although the background reaction of the control in the detection is relatively high, the expression of the latter is three times that of the former in general comparison. This sign It indicated that the cell localization of Bt protein might be changed after adding the secretion signal peptide, thus increasing the stability of Bt protein and benefiting the accumulation of Bt protein. (b) Western blot analysis

Take 100 mg of fresh leaves, grind with liquid nitrogen, add 100 μl of 2× loading buffer, cook at 100°C for 3-5 minutes, centrifuge at 12,000 rpm for 5 minutes, and the supernatant obtained can be used for electrophoresis analysis. Take 20 μl of the above protein extract, run it on a 10% SDS-PAGE gel, use Tris-glycine buffer system (PH8.3), voltage 60V, and electrophoresis for 4-6 hours. After the electrophoresis, the protein was transferred to the PVDF membrane pretreated with methanol using a Bio-Rad semi-dry transfer instrument with a constant voltage of 9 volts for 30 min. Then place the membrane in solution III to block unbound proteins, shake at room temperature for 1 hr or overnight at 4°C; transfer the membrane to the primary antibody reaction solution (solution I+1% BSA, ProteinA Spharose6B column-purified immune anti-CrylAc antibody , 1:500 dilution), shake at room temperature for 1 hr; wash 3 times with solution I, 3-5 min each time; place the membrane in color developing solution (10 ml solution II + 33 μl NBT + 66 μl BCIP) for color development. The solutions used for Western blot are as follows: 2× Loading buffer: 125mM Tris-HCl PH6.8, 20% glycerol, 4% SDS, 0.2% bromophenol blue, 2% mercaptoethanol Semi-dry transfer buffer: 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, 0.1 mol/L NaCl, 5mmol/L MgCl ₂ (PH9.5) solution III: solution I containing 5% skimmed milk powder. The qualitative Western blot analysis results of some plants are shown in Figure 9, and the results show that the transformed Btlm or BtSlm gene is significantly expressed in the transgenic plants. The protein molecular weight of the specific immune response is consistent with the size of the expression product (lane 1) of the 1.8KbBtlm gene fragment in Escherichia coli, indicating that the two Bt genes can correctly express insecticidal proteins in plants, and the chimeric insecticidal protein expressed by BtSlm The signal peptide on has been correctly processed and removed. However, further experiments are needed to prove whether the relevant signal peptide has exerted its due function and its processing condition. 2.4 Insect resistance experiment of transgenic tobacco and genetic analysis of progeny Take fresh leaves of transgenic tobacco or non-transgenic tobacco, rinse them with sterile water, blot the water on the leaves with gauze, put them into small plastic boxes, and put one artificial head in each box. For the raised cotton bollworm (Heliothis armigera Hübner), 6 to 12 head worms were prepared for each tobacco plant. After the worm test box was covered, it was placed at 25°C for 3 days or 5 days, and then the larval mortality was counted. The insect resistance test was repeated three times. The insect test results of transformed and regenerated tobacco are summarized in accompanying drawing 11.

The results of the insect test showed that the plants with high insect resistance could be selected from the plants of the Btlm and BtSlm genes, and the plants with more than 50% mortality of the test insects accounted for 76% and 85% of the total number of test plants respectively. This result is consistent with the expression level of Bt protein, which once again indicates that transgenic plants transgenic with BtSlm gene coding sequence of signal peptide may be more likely to select high insect-resistant plants.

In order to understand the genetic stability of the Btlm and BtSlm genes in the transgenic plants, the kanamycin resistance and insect resistance of the first selfed plants of some plants were tested. Get the seeds (T1) of the self-crossing first generation of transgenic trapped plants, and transfer the sterilized seeds to the MS ₀ flat medium containing 200mg/ml kanamycin according to the operation method of aseptic seedlings, and put them in a light incubator After the seedlings grow a pair of true leaves, the plants that are not resistant to kanamycin will soon turn white, while the seedlings that are resistant to kanamycin will still remain green. The number of green seedlings and white seedlings of each line is counted. , the segregation ratio of kanamycin resistance was counted. Simultaneously, the insect resistance test was carried out for the generation plants, the method was the same as above, and the insect resistance was counted according to the segregation situation in the first generation. The statistical score result of kanamycin resistance is shown in attached table 2: progeny analysis result shows that the NPTII gene (kanamycin resistance gene) that is brought on the plant chromosome by the expression vector is basically in transgenic plant progeny It is segregated by a single gene, so it should be genetically stable, and transgenic homozygous lines can be selected from the offspring, and the insect resistance of the Bt gene closely linked to NPTII also shows a 3:1 segregation characteristic, which preliminarily indicates that the synthetic Bt genes, Btlm and BtSlm can also be stably inherited in the offspring of transgenic plants.

The Agrobacterium tumefaciens transformants of pBlm and pBSlm have transformed the production variety Jihe 321 of cotton, and the high-efficiency insect-resistant transgenic plants have been preliminarily selected through PCR inspection and cotton bollworm resistance test, so the Bt fusion insecticidal protein gene provided by the invention Btlm and BtSlm can be used in the research and development of insect-resistant genetic engineering of plants. References [1] Stratagene products, Cat No.212207[2]Promega products, Cat No.P2191[3]GibcoBRL products, Cat No18258-012[4]GibcoBRL products, Cat No15364-011[5]GibcoBRL Company product, Cat No18313-015

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

Supplementary Table 2. Genetic Analysis of Progeny of Transgenic Tobacco Plants

H ₀ : OT=0, α=0.05, df=1, x ² _0.05 =3.841

When x ² < x ² _0.05 , the kanamycin resistance ratio conforms to the typical 3:1 segregation ratio Expressionvecror Transgenic Plant No. Green plant number/White plant number ^x2 3:1 segregation pB 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 PPML 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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