CN1369562A - Plant expression carrier with dual insect-resisting genes and its application - Google Patents

Abstract

An artificially synthesized fusion protein gene BtS29K is disclosed, which along the with fusion protease inhibitor gene AP1-BA is used to configure the expression carrier pBS29K-BA of the plant with dual-insect-resisting gene. The said carrier can be used to obtain high-insecticiding transgenic plant, which features broad spectrum, high insecticiding power and delaying the generation of insect resistance.

Description

Plant expression vector and an application thereof with dual insect-resisting genes

The invention belongs to the engineering of insect-resistant plant field, relate to the gene fragment of having synthesized in bacillus thuringiensis (Bt) the crystallin CrylAc gene insecticidal proteins that coding activated fully with chemical process, hereinafter to be referred as the Bt gene; End at the Bt gene adds that synthetic has the polypeptid coding sequence of endoplasmic reticulum positioning function and forms chimeric Bt gene BtS29K, this structure helps improving the stability of Bt insecticidal proteins, also helps making up the dual insect-resisting genes expression vector with protease inhibitor gene; Adopt the DNA recombinant technology that the fusion gene of chimeric Bt gene and arrowhead proteinase inhibitor is built into the dual insect-resisting genes plant expression vector of (or being called divalent insect-resistant gene).Obtain efficient insect-resistant transgenic plants with these carriers by genetic transformation to plant.

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 has been used nearly 50 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, and staple crops such as paddy rice are interior.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, thus can not obtain the most insects not too responsive of poisoning by the render transgenic plant to the Bt toxalbumin, as the main lepidoptera pest of a lot of farm crop.

(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 Cryl Ab 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 Bt gene in plant in the expression regulation many problems still treat further research, also needing significant improvement aspect some in the practical application of Bt transgenic plant:

Often find 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.Reason have following some:

1. may cause somatic variation in the transformation tissue culture process.

2. the position difference inserted on plant chromosome of foreign gene may activate or disturb certain plants expression of gene etc. all may cause the change of economical character.

3. 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.

At present the problems referred to above are not obtained fine solution as yet.The objective of the invention is by signal peptide sequence being introduced the Bt gene so that its expression product can be positioned on the endoplasmic reticulum, improve the stability of insecticidal proteins, alleviate the interference of foreign protein pair cell normal function, help to obtain the good insect-resistant transgenic plant of economical character.

In addition, the insect-resistant transgenic plants of single use Bt gene transformation also has a more serious problem in the present application, is exactly that insect can be to Bt toxalbumin generation tolerance after it uses for some time, thereby the effect of anti-pest crop is greatly lowered.The albumen that the current people of having uses two kinds of insecticidal mechanisms to differ widely transforms plant, two types anti insect gene is expressed in attempt simultaneously in plant, so not only can improve the anti-insect activity of transgenic plant, enlarge pest-resistant spectrum, thereby and produce the effective service life that tolerance prolongs insect-resistant transgenic plants and also can play a significant role for avoiding or delaying targeted insect.Research at present many is that protease inhibitor gene transforms plant with the Bt gene and obtains the divalent insect-resistant transgenic plant.But result of study in this respect also is not very desirable so far, the result who has shows on the contrary and expresses the insect-resistance height (Santos that Bt gene and protease inhibitor gene but do not have single expression Bt gene simultaneously, MO et al, MolecularBreeding 3:183-194,1997).Reason is likely inhibition or the interference that is subjected to proteinase inhibitor owing to the performance of Bt albumen insecticidal activity in insect body, because what the investigator used in these experiments all is the complete Bt gene in 5 ' end coding region, the albumen that is produced still belongs to the precursor protein of desinsection toxalbumin, this albumen still needs the proteolytic enzyme of insect that its N end is processed in insect gut, remove and Bt albumen is activated fully after one section (as being 28 amino acid for CrylAc) to become albumen (the Nagamatsu et al.Agric.Biol.Chem.48:611-619 that insecticidal activity is arranged, 1984), thus the existence of proteinase inhibitor disturb or suppressed this reactivation process and influenced the active performance of Bt insecticidal proteins.

The problems referred to above at present existence, the present invention has synthesized from N and holds the 29th amino-acid residue to begin the CrylAc gene of forming to the 613rd amino acid, this is the shortest activated Bt gene of present synthetic, and add that at this gene 5 ' end 72bp sequence and the 3 ' end of κ light chain signal peptide of coding mouse adds that the coding endoplasmic reticulum locatees the nucleotide sequence of peptide KDEL, the chimeric Bt unnamed gene of this that is built into is BtS29K at last, this expression of gene can make insecticidal proteins rest in the endoplasmic, improve the proteic stability of Bt thus greatly, thereby increased the accumulation volume of this insecticidal proteins in tissue; Simultaneously, the Bt protein accumulation is avoided contacting with other functioning cell devices in endocytoplasmic reticulum, greatly alleviated since foreign protein in cell accumulation and negatively influencing that vegetable cell is produced, the final transfer-gen plant that obtains to have better economical character.

In addition because this Bt albumen has had 28 amino acid of N end, so be a sophisticated insecticidal proteins that has activated fully.This albumen no longer needs the digestive ferment of insect gut to activate, so the BtS29K gene that provides of the present invention is very suitable for and have the protease inhibitor gene of pest-resistant function to cooperate, be built into dual-gene or polygenic insect-resistant transgenic plants, thereby eliminate the influence of proteinase inhibitor, really bring into play the pest-resistant function of the different anti insect gene products of this two class the effect of Bt protein activation.At present, the present invention has obtained the insect-resistant transgenic plants of two valencys, experimental results show that it has better pest-resistant effect than they monovalent transgenic plant.

Having used the light key signals peptide-coding sequence of κ, KEDL encoding sequence and synthetic CrylAc gene to set up chimeric CrylAc gene in the present invention first, also is to use the CrylAc gene of 5 ' end disappearance and the dual insect-resisting genes expression vector that proteinase inhibitor fusion gene API-BA makes up first.We have used these expression carrier to transform plant and have obtained the higher transgenic plant of new insect-resistance, as tobacco, and cotton, Yang Shu etc., experiment confirm the present invention can solve the said problem in front preferably.

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

By synthesizing of Bt CrylAc gene Oligonucleolide primers;

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

The assembling of the CrylAc gene CrylAc activated protein gene clone pBtf29K (accompanying drawing 2) that obtains encoding;

The CrylAc gene order is connected with signal coding sequence, makes up to have signal peptide

The Bt CrylAc gene clone pS29K of encoding sequence;

Chimeric CrylAc gene S29K inserts binary expression vector pBin438 and constitutes plant

Expression vector pBS29K (accompanying drawing 3), and it is transformed into soil Agrobacterium

[Agrobacterium?tumefaciens]LBA4404；

Make up dual insect-resisting genes plant table with protease inhibitor gene with pest-resistant function

Reach carrier pBS29K-BA (accompanying drawing 8).

The intestinal bacteria (Escherichia coli) that contain pBS29K-BA have been transferred to China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.Its registration number is CGMCC NO.0452.

PBS29K and pBS29K-BA or their crown gall soil Agrobacterium LBA4404 transformant are applied to Plant Transformation, can obtain the transgenic plant that economical character is good, have 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.CrylAc gene fragment makes up synoptic diagram

Abridge among the figure: PI-1～PV-9: the numbering of different Oligonucleolide primers, 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; R1:EcoRI; RV:EcoRV; Sc:SacI; SL:SalI.

A. since first section (BamH1-EcoR1) Ya Ke of the 29th amino acid

The structure of grand pBtsI-29

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

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

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

E. the structure of the 5th section (SacI-XhoI) subclone pBtsVK

The structure of accompanying drawing 2.CrylAc activated protein gene clone pBtf29K

The structure of the recombinant plasmid pS29K of accompanying drawing 3. chimeric Bt gene S29K and the plant expression vector pBS29K of S29K

Accompanying drawing 4. synthetic contain the nucleotide sequence and the aminoacid sequence of the active CrylAc mosaic gene (BtS29K) of secreting signal peptide and endoplasmic reticulum location peptide-coding sequence

The comparison of accompanying drawing 5. synthetic CrylAc encoding active protein district's dna sequence dnas and wild-type CrylAc corresponding section dna sequence dna.

W (up) ... wild-type CrylAc gene order (HD-73); M (descending) ... synthetic CrylAc gene order.Base in the square frame is the W base different with the M sequence

The variation of CrylAc encoding active protein district, subordinate list transformation front and back codon usage frequency reaches the comparison with the vegetable codon frequency of utilization.

Codon per-cent: refer to this codon in this gene coding region with the ratio of codon sum of coding same amino acid.

Vegetable codon frequency of utilization %: refer to the frequency of utilization (MurrayEE.et al.Nucleic Acid Res.17:477-499 1989) in dicotyledons

The pcr amplification of accompanying drawing 6.A.API-A gene fragment and the structure of recombinant plasmid pAHA

The structure of the pcr amplification of B.API-B gene fragment and recombinant plasmid pAHB

C. arrowhead proteinase inhibitor fusion gene API-BA recombinant plasmid pAH-BA

Structure

The dna sequence dna and the deduced amino acid of accompanying drawing 7.API-BA gene

The proteinic processing site that haircut expresses possibility among the figure

The structure of accompanying drawing 8. fusion gene API-BA plant expression vector pBBA and dual insect-resisting genes plant expression vector pBS29K-BA

The PCR of accompanying drawing 9a. transformation tissue culture tobacco plant detects

The PCR primer pIIm of Bt gene specific ⁺And PLV-6-

Swimming lane 1. λ-Ecotl4I DNA ladder

Swimming lane 2. non-transformation of tobacco NC89 contrasts

The PCR of swimming lane 3-15.pBS29K-BA transformation tissue culture plant accompanying drawing 9b. transformation tissue culture tobacco plant detects

PCR BA gene specific primer B ₁And A ₂

Swimming lane 1,10. λ-Ecotl4I ladder

Swimming lane 8. non-transgenic tobacco NC89 contrast

Swimming lane 9.pBS29K-BA is as the positive control of template

The Southem blot of swimming lane 2-7.pBS29K-BA transformation tissue culture plant accompanying drawing 9c. transgenic tobacco plant analyzes

9c-a: with BA gene probe results of hybridization

Swimming lane 1.BA gene fragment (1.1kb)

Swimming lane 2. non-transgenic tobaccos are as negative control

Swimming lane 3,4,5,6. be respectively the tobacco that pBS29KBA transforms

12#, 18#, 5# and 8#

9c-b: with Bt gene probe results of hybridization

Swimming lane 1. non-transgenic tobaccos are as negative control

Swimming lane 2.Bt fragment is as positive control (1.8kb)

Swimming lane 3,4,5,6. be respectively the tobacco that pBS29KBA transforms

12#, 18#, the protein immunization analysis of 5# and 8# accompanying drawing 10. transgene tobaccos

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

The contrast of swimming lane 2. non-transgenic tobaccos

The different transgenic tobacco plant accompanying drawing of swimming lane 3-9. 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 bollworm resisting test of accompanying drawing 12.pBS29KBA transgene cotton

Up left and right blade is the cotton plant blade of commentaries on classics pBS29KBA bivalent gene,

Intermediate blade is the contrast of non-transgenic plant

Descending left, center, right come rotation pB29K, non-transgenic contrast to reach respectively

The cotton plant blade of the unit price anti insect gene of pBS29K

The PCR of Bt gene detects in the accompanying drawing 13A. insect-resistant transgenic cotton

Swimming lane 1. λ-Ecotl4I ladder.

Swimming lane 2. negative controls, the non-transgenic cotton.

Swimming lane 3. positive controls, the pBS29K plasmid.

The transgenic cotton plant that swimming lane 4-8. is different

The detection of API gene in the accompanying drawing 13B. insect-resistant transgenic cotton

The transgenic cotton plant that swimming lane 1-6. is different

Swimming lane 7. negative controls, the non-transgenic cotton.

Swimming lane 8. positive controls, the pBS29K-BA plasmid.

Swimming lane M. λ-Ecotl 4I ladder.

Embodiment one usefulness Bt gene S29K and protease inhibitor gene make up the synthetic of the synthetic 1.1Bt CrylAc gene Oligonucleolide primers of the proteic gene fragment of dual insect-resisting genes plant expression vector 1 Bacillus thuringiensis CrylAc genes encoding insecticidal activity

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 the CrylAc gene, the toxicity of this gene is divided into five sections, and to carry out primer synthetic.In first primer pI-1 (+) of 5 ' end, except that the encoding sequence of CrylAc, the 29th codon (ATC) upstream at this gene has increased an initiator codon ATG, so that synthetic gene transcription product can correctly begin translation, between ATG and the 29th codon, also added the GCT codon so that form a typical Kozak sequence in plant, for the convenience of cloning, also added the recognition sequence of a BamHI at 5 of this primer ' end.In the primer pV-9K of 3 ' end, the sequence of an encoded K DEL tetrapeptide, a terminator codon and an XhoI recognition sequence except that CrylAc gene 3 ' terminal sequence, have also been added.First section (PI) is totally 4 primers (BamH1-EcoR1), and their sequence is as follows: PI-1 (+): 5 ' GG GAT CCA ACA ATG GCT ATC GAG ACC GGT TAC ACT CCA ATC

GAC?ATC?TCC?TTG?TCC?TTG?ACA?CAG?TTT?CTG?CTC?AGC?GAG?TTC

83merPI-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?TTCCTG 45merPI-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 GCC AGG AAC CAG GCC ATT 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?T?65merPII-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?CTT?CAT?CTC?AGC?GTG?CTT?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?TAT?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?GAA?CTG

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

AGG?TAC?CCA?ATC?CGT?ACT?GTT?TCC?CAA?CTT?ACC?AGA?GAG

77merPIII-2(-)：5′ CTG?GGC?AGA?AAC?ACG?GAA?GCT?ACC?GTC?GGA?ATT?CTC?AAG

AAC?TGG?GTT?AGT?ATA?GAT?CTC?TCT?GGT?A 67merPIII-3(+)：5′ T?TCT?GCC?CAG?GGT?ATA?GAA?AGA?AGC?ATC?AGG?AGC?CCT?CAT

CTC?ATG?GAC?ATC?TTG?AAC?AGC?ATA?ACT?ATC?70merPIII-4(-)：5′ CTG?GTG?TCC?AGA?CCA?ATA?GTA?GTA?TCC?TCT?ATG?AGC?ATC

GGT?GTA?GAT?AGT?TAT 55merPIII-5(-)：5′ GT?GAA?TTC?GGG?ACC?GCT?GAA?TCC?AAC?TGG?GAG?GGC?CAT

GAT CTG GTG TCC A 51mer 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?TCT?GAT?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-Xhol), 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?ACTCCTATT?CAT?CTC

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?AACCTC?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?GTTGCC?GAGT 50merPV-9K(-)：5′?CA?CTC?GAG?ATC?TCA?AAG?CTC?GTC?CTT?TTC?GAG?TGT?TGC

AGT?AAC?TGG?AAT?GAA?CTC?AAA?TCT?GTC?TAT?GAT?CAC?ACC

TGC 80merPV-9(-)：5′ CA?GTC?GAC?TCA?TTC?GAG?TGT?TGC?AGT?AAC?TGG?AAT?GAA

Synthetic and the clone of CTC AA TCT GTC TAT GAT CAC 65mer1.2CrylAc gene fragment

Get two each 20pmol of adjacent primer (10 μ l) mixing in each section, at 70 ℃ of sex change 10min, add 10 X 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/LMgCl ₂, 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 reacts 30 circulations.The PCR final product of each section behind corresponding restriction endonuclease enzymolysis with cloning vector pBluecriptIISK with same enzymolysis ⁺Or pSP71 connects back transformed into escherichia coli DH5 α 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 Cry1Ac gene fragment through plasmid, pBtSII (second section), pBtSIII (the 3rd section), pBtSIV (the 4th section) and pBtSVK (the 5th section).The building process of these five subclones is seen accompanying drawing 1:A-E.Above DNA enzyme is cut, the preparation of carrier, ligation, the preparation of competent cell, DNA transform, clone's screening all can be carried out according to the described method of " molecular cloning " book (Sambrook J.et al. " Molecular Cloning " 2nd ed.CSH Laboratory Press, 1989).Dna sequence analysis is undertaken by the method that Pharmacia company's T 7DNA sequencing Kit provides.1.3 the assembling of CrylAc gene

After sequential analysis proves that the sequence of Bt gene fragment in each subclone is correct, with conventional recombinant DNA technology by the restriction enzyme site of each subclone promptly obtain after the connection in order each other the encoding gene clone pBtf29K of CrylAc activated protein.The pBtf29K building process see accompanying drawing 2.1.4 CrylAc gene order rectification and with being connected of signal coding sequence

(a), 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/BglII primer: 5 ' CAGGAAAGAAGATCTGAGCAAAAG.Mutant primer is made up of the base of need change and the oligonucleotide 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 pBts12 (29) 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

①PI-1+mI ^-

mI-?5′ CCTGGCGAACTCTTCGATCCTCTGGTTGATGAG 33mer

②PII-8+mII ⁺

mII+5′ ATCGAAGAGTTCGCCAGGAACCAGGCCATTTCTAGG 36mer

To the improved recombinant plasmid of pBts12 (29) name into pBts12 (29m) PCR reaction conditions and cloning process ditto described.

(b), the structure that has the Bt CrylAc gene clone pS29K of signal coding sequence

For the Bt gene product can be positioned on the endoplasmic reticulum of cell effectively, see accompanying drawing 3 with the concrete building process of recombinant plasmid pS29K that is built into chimeric Bt gene S29K after mouse single-chain antibody signal coding sequence is connected with the gene of coding CrylAc 29-613 amino acid and KDEL.

The primer of single-chain antibody signal coding sequence is as follows: AT31 (+): 5 ' CTGGATCCAACAATGGGCATCAAGATGGAGACCCACTCTCAGGTGTTCGTGTAC 54merAT32 (-): the DNA total length 1857bp of 5 ' TGAGATCTGTGTCCACACCAGACAACCAAAGCAACATGTACACGAACACCTG 52mer synthetic (chimeric Cry1Ac gene) S29K, its sequence is seen accompanying drawing 4.Coding CrylAc insecticidal activity protein part is total to the 1755bp coding from 29-613 amino acid in the synthetic S29K gene.Change the change that 342 bases relate to 311 codons in synthetic altogether, accounted for 53.2% of total codon.But total aminoacid sequence does not change.GC brings up to 47.4% than by 37.6% of wild-type, and the nucleotide sequence homology before and after transforming is 80%.Relatively reaching of the sub-frequency of utilization of amino acid code and relatively seeing attached list of plant optimizing codon before and after it is transformed, with wild-type CrylAc gene nucleotide series relatively see accompanying drawing 5.S29K full length gene 1857bp except that comprising above-mentioned 1755bp Bt gene order, comprises that also 72bp coded signal peptide, 12bp encoded K DEL and 15bp connect the combining site sequence (seeing accompanying drawing 4) of signal peptide and Bt gene.2, the structure of the plant expression vector of chimeric CrylAc gene S29K and the conversion of Agrobacterium

Binary expression vector pBin438 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 CaMV 35S promoter (DE35Sp) of the two enhansers of band, TMV-RNA cDNA, Chinese science (B collects) 24:276-282,1995).Be inserted into after with BamHI and XhoI the chimeric CrylAc gene S29K among the pS29K being cut out between the BamHI of pBin438 and the SalI site and be built into plant expression vector pBS29K.After making up correctly, the restriction analysis proof uses freeze-thaw method (An et al, Methodsin Enzymology 153:293,1987) it is transformed among soil Agrobacterium (A.tumefaciens) LB4404, through the kantlex screening, promptly can be used for Plant Transformation after plasmid analysis proof pBS29K structure is entirely true.The concrete building process of this plant expression vector is seen accompanying drawing 3.3. the structure of arrowhead proteinase inhibitor (API) antigen-4 fusion protein gene (BA)

Arrowhead is a kind of aquatic vegetable that rushes down damp section, contains abundant proteinase inhibitor, and various pests is had stronger inhibition and lethal effect.API has two components, API-A and API-B, and A and B are the multifunctional dual-head proteinase inhibitor.The result of study of (Chinese science B collect, 12:1271-1276,1990) such as the biochemical Yang Hui of the institute bells in Chinese Academy of Sciences Shanghai shows that API-A equivalent simultaneously suppresses trypsinase and Quimotrase, to the restraining effect of kininase a little less than; API-B can equivalent suppress 2 molar trypsinase, and is similar to API-A to the inhibition vigor of kininase, but to the restraining effect of Quimotrase more than inhibitor A a little less than.Xu Wenfeng etc. (Acta Biochimica et Biophysica Sinica 25:207-215,1993) have at first cloned API-A and API-B cDNA, thereby lay a good foundation for utilizing these two genes to carry out insect-resistant transgenic research.

For thereby the effect of bringing into play A and two inhibitor of B simultaneously obtains better pest-resistant effect, we are built into antigen-4 fusion protein gene with API-A and API-B, and concrete steps are as follows: the synthetic structure that reaches the API-BA gene of primer

API-B primer: B ₁5 ' GCTGGATCCACCATGGCGGCCTCCAACGCT 27mer

B ₂5′TGCCTGCAGAGATCTCATTGCGAGTGCGTCGAA 33mer

API-A primer: A ₁5 ' GTCGGATCCTGCCACGGAGATCCCGTC 27mer

A ₂5′TGCAAGCTTCTCGAGCTACTGCGGTGCAGTTTTC 34mer

It is just military to use marine life chemistry institute relative, API-A that the luxuriant Mr. of Gong provides and API-B cDNAM13 phage clone are made template and are carried out PCR respectively with above-mentioned primer, the PCR product is connected clone pAHA and the pAHB (seeing accompanying drawing 6-A and 6-B) that promptly gets these two genes with the carrier pUC19 of same enzymolysis after suitably enzyme is cut, after sequential analysis is justified, the API-A gene is cut out the pAHB that with HindIII-BglII enzyme cut with BamHI from pAHA with HindIII and be connected the fusion gene API-BA that promptly gets B and A, contain the recombinant plasmid called after pAH-BA (seeing accompanying drawing 6-C) of API-BA.The dna sequence dna of API-BA antigen-4 fusion protein gene and deduced amino acid are seen shown in the accompanying drawing 7.PCR, restriction analysis, clone and sequence measurement are with mentioned above consistent.

The encoding sequence of the N end signal peptide of API-B and API-A gene C end 8 peptides all is retained down in the API-BA gene, the junction of B and A has kept 2 amino-acid residues of B precursor protein C end, 3 amino-acid residues of C end of A precursor protein N end signal peptide and two amino-acid residues that are connected generation by BglII with BamHI.Such structure should be unable to influence the processing to precursor protein.Still might be processed to API-A and two maturation proteins of API-B after in plant, expressing.4.API-BA the structure of gene plant expression vector and chimeric CrylAc gene BtS29K and API-BA dual insect-resisting genes plant expression vector

Cutting pAH-BA with BamHI with the XhoI enzyme comes out the API-BA gene isolation then to be connected the plant expression vector pBBA that promptly gets the API-BA gene with the pBin438 of BamHI-SalI enzymolysis; After connecting, the pD12 of above-mentioned API-BA gene fragment and BamHI-SalI enzymolysis (structure of pD12 is seen Yingchuan, field etc., Botany Gazette 42:263-268,2000) gets recombinant plasmid pDBA.The API-BA gene is in DE35SP (35S promoters of two enhansers) in pDBA---TMV Ω fragment downstream, Nos transcription termination sequence upstream, with HindIII this is expressed framework and downcut back and HindIII enzymolysis, the pBS29K that shrimp alkaline phosphotase is handled connects and promptly gets the dual insect-resisting genes plant expression vector pBS29K-BA that has API-BA and S29K (the chimeric CrylAc gene of being made up of CrylAc and signal peptide sequence).The building process of PBBA and pBS29K-BA is seen accompanying drawing 8.The intestinal bacteria transformant of pBS29K-BA is China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and its registration number is: CGMCC No.0452.

The API-BA gene can be used as a representative of protease inhibitor gene in pBS29K-BA, and any protease inhibitor gene with anti-insect activity can replace that API-BA and BtS29K are gene constructed to become the dual insect-resisting genes expression vector.5. the conversion of Agrobacterium tumefaciens

PBBA and pBS29K-BA are transformed into the method for Agrobacterium tumefaciens LBA4404 with described in the present embodiment one.Recombinant plasmid in the gained transformant promptly can be used for Plant Transformation after restriction analysis is justified.

The conversion of embodiment two insect-resistant transgenic plants 1. tobaccos

Blade pass with the tobacco NC89 of sterile culture is crossed Ye Panfa (the Horch et al.Science 227:1229-1231 that cultivates altogether with Agrobacterium tumefaciens, 1985) with the pBS29K of above structure, pBBA, the T-DNA among the pBS29K-BA (comprising NPTII gene and corresponding anti insect gene) 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 CrylAc gene specific primer is:

PIIm+ 5′ ATCTATGCAGAGTCTTTCAGA

PLV-6 ^-?5′ GAGGTTATCCAAGGAGGT

Carrying out the PCR products therefrom with these two primers should be 1370bP.The API-BA gene specific primer is: B ₁: GGATCCACCATGGCGGCCTCCAACGCT

A ₂：CTCGAGCTACTGCGGTGCAGTTTTC

Carrying out the pcr amplification products therefrom with this a pair of primer should be about 1.2kb.The PCR of the regeneration plant of the anti-kantlex of part the results are shown in accompanying drawing 9a and 9b.These PCR results show that S29K or S29K-BA dual insect-resisting genes may be incorporated on the karyomit(e) of tobacco.2.2 the extraction of Southern hybridization analysis (1) DNA of plants

Get 2 gram blades in liquid nitrogen, pulverize the back by the method for report such as Paterson (PlantMol.Bol.Rep, 11 (2):, 1993) extract nucleus DNA.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 3MNaAc PH5.5 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 be all with reference to described the carrying out of molecular cloning one book (Sambrook etal.CSHL Press, 1989), used probe for α- ³²The EcoRV of P-dCTP and Ready To GoDNA labelling kit mark and the S29K gene fragment (1.1Kb) of XhoI enzymolysis.Southern results of hybridization (seeing accompanying drawing 9C) 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

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 Westem blot analytical results of part plant is seen accompanying drawing 10, and the result shows that improved Bt gene has 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 1.8KbCrylAc gene 5 ' end fragment.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.Expression and the processing situation of API gene in transfer-gen plant also still needed and determined by further detection.2.4 the pest-resistant experiment of transgene tobacco

The fresh blade of getting transgene tobacco or non-transgenic tobacco 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 (Heliothis armigera H ü bner) that a tribal chief worker raises, and the blade of every strain tobacco connects 12 cephalonts.Worm examination box is added a cover in case bollworm escapes and the evaporation of moisture, adds up larval mortality 25 ℃ of placements after 3 days and 5 days.Pest-resistant test triplicate.The transformation tissue culture tobacco worm test result of each anti insect gene structure reduces accompanying drawing 11.The worm test result shows that the ratio of high resistance helicoverpa armigera plant (mortality ratio is at 80-100%) accounts for total worm respectively in the transformed plant of the Bt gene (BtS29K) of plus signal peptide and endoplasmic reticulum location peptide and API-A and B antigen-4 fusion protein gene (BA) and tries 54% and 39% of plant number, and the high anti-plant of dual insect-resisting genes (S29K-BA) transformed plant accounts for 63.5%.The ratio of high anti-plant is apparently higher than only transforming single anti insect gene, than only transforming the high by 9.5% of BtS29K, than only changeing the high by 25.2% of API-BA.Have single-chain antibody signal peptide and C end and have endoplasmic reticulum location peptide, the N end is all higher significantly than the insect-resistance of single expression Bt gene or proteinase inhibitor fusion gene (API-BA) plant since the insect-resistance of the commentaries on classics dual insect-resisting genes plant that the Bt gene (BtS29K) of the 29th amino acid and protease inhibitor gene transform plant jointly and produced.The structure that shows the chimeric Bt gene of institute's synthetic of the present invention helps cultivating the zoophobous that changes dual insect-resisting genes.Thereby it is higher to make the anti insect gene that utilizes different pest-resistant mechanism obtain insect-resistance, and the transgenic plant that pest-resistant spectrum is wideer are delayed or avoid the tolerific strategy of targeted insect to become possibility.3. insect-resistant transgenic cotton

Utilize the gene constructed dual insect-resisting genes expression vector of BtS29K or Bt29K and API to transform cotton by the soil Agrobacterium mediated method, it is (seeing accompanying drawing 12) that the transgene cotton that has obtained high resistance helicoverpa armigera isozygotys.The PCR of divalent insect-resistant cotton the results are shown in accompanying drawing 13A and 13B.Method for transformation is described with reference to (cotton journal 10:237-243,1998) such as Li Yane.

BtS29K provided by the invention and BtS29K-BA gene can be used for Plant Transformation, obtain to have accordingly high insect-resistance transgenic plant.

Claims (16)

1. the anti insect gene of an efficient localization and expression in plant is characterized in that it is a kind of mosaic gene of being made up of 1857 Nucleotide shown in following nucleotide sequence.ATGGGCATCAAGATGGAGACCCACTCTCAGGTGTTCGTGTACATGTTGCTTTGGTTGTCTGGTGTGGACACAGATCCAACAATGGCTATCGAGACCGGTTACACTCCAATCGACATCTCCTTGTCCTTGACACAGTTTCTGCTCAGCGAGTTCGTGCCAGGTGCTGGGTTCGTGCTCGGACTAGTTGACATCATCTGGGGTATCTTTGGTCCATCCCAATGGGACGCATTCCTGGTTCAAATTGAACAGCTCATCAACCAGAGGATCGAAGAGTTCGCTAGGAACCAGGCCATCTCTAGGTTGGAAGGACTCAGCAATCTCTACCAAATCTATGCAGAGTCTTTCAGAGAATGGGAGGCCGATCCTACTAACCCAGCTCTCAGGGAGGAGATGCGTATTCAATTCAACGATATGAACAGCGCCTTGACCACTGCTATCCCATTGTTCGCAGTCCAGAACTACCAGGTGCCTCTCTTGTCCGTGTACGTTCAAGCCGCTAATCTTCATCTCAGCGTGCTTCGAGACGTTTCAGTGTTTGGACAGAGGTGGGGATTCGATGCTGCAACCATCAATAGCAGATACAACGACCTTACTAGGCTCATTGGAAACTACACCGACTATGCTGTTCGTTGGTACAACACTGGTTTGGAGCGTGTCTGGGGTCCTGATAGCAGAGATTGGGTGAGATACAACCAGTTCAGGAGAGAATTGACCCTTACAGTGTTGGATATCGTGGCTCTCTTCCCGAACTATGACAGCAGAAGGTACCCAATCCGTACTGTTTCCCAACTTACCAGAGAGATCTATACTAACCCAGTTCTTGAGAATTTCGACGGTAGCTTCCGTGGTTCTGCCCAGGGTATAGAAAGAAGCATCAGGAGCCCTCATCTCATGGACATCTTGAACAGCATAACTATCTACACCGATGCTCATAGAGGATACTACTATTGGTCTGGACACCAGATCATGGCCTCTCCAGTTGGATTCAGCGGGCCCGAATTCACCTTCCCTCTCTATGGAACTATGGGTAACGCCGCTCCACAACAAAGGATCGTTGCTCAACTAGGTCAGGGTGTCTACAGAACCTTGTCTTCCACTTTGTACAGAAGGCCATTCAATATCGGTATCAACAACCAGCAACTTTCCGTTCTCGATGGAACAGAGTTCGCCTATGGGACCTCTTCTAACTTGCCATCCGCTGTTTACAGAAAGTCCGGAACCGTTGATAGCTTGGACGAAATTCCACCACAGAACAACAATGTGCCACCCAGGCAAGGATTCAGCCACAGGTTGAGCCATGTGTCCATGTTCCGTTCCGGTTTCAGCAACAGTAGCGTGAGCATCATCAGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCTGAGTTCAACAATATCATTGCATCCGATAGCATCACTCAAATTCCTGCAGTTAAGGGAAACTTTCTCTTCAATGGTTCAGTCATTTCAGGACCAGGATTCACSGGAGGAGACCTCGTTAGACTCAACAGCAGTGGAAATAACATCCAGAATAGAGGGTATATTGAAGTTCCAATTCATTTCCCTTCCACATCTACCAGATATAGAGTTCGTGTGAGGTATGCTTCTGTAACTCCTATTCATCTCAACGTTAATTGGGGTAATTCATCTATCTTCAGCAACACAGTTCCAGCTACAGCTACCTCCTTGGATAACCTCCAATCCAGCGACTTCGGATACTTTGAGAGCGCCAATGCTTTCACATCTTCACTCGGCAACTATGTGGGTGTTAGAAACTTTAGTGGAACTGCAGGTGTGATCATAGACAGATTTGAGTTCATTCCAGTTACTGCAACACTCGAAAAGGACGAGCTTTGA

1857

2. mosaic gene according to claim 1, it comprises the 1st to the 72nd nucleotide sequence of the sequence of coding mouse single-chain antibody signal peptide; The the 88th to the 1842nd nucleotide sequence of the sequence of coding bacillus thuringiensis (Bacillusthuringiensis) Bt insecticidal proteins CrylAc the 29th to the 613rd amino acids; And the 1843rd to the 1854th nucleotide sequence of the sequence of an endoplasmic reticulum location of coding peptide; The the 73rd to the 87th Nucleotide is signal coding sequence sequence by the five amino acid of the nucleotide coding of connecting portion when being connected with the CrylAc gene.

3.1,： 1M G I K M E T H S Q V F V Y M LL W L S G V D T D P T M A I E TG Y T P I D I S L S L T Q F L LS E F V P G A G F V L G L V D II W G I F G P S Q W D A F L V QI E Q L I N Q R I E E F A R N QA I S R L E G L S N L V Q I Y AE S F R E W E A D P T N P A L RE E M R I Q F N D M N S A L T TA I P L F A V Q N Y Q V P L L SV Y V Q A A N L H L S V L R D VS V F G Q R W G F D A A T I N SR Y N D L T R L I G N Y T D Y AV R W Y N T G L E R V W G P D SR D W V R Y N Q F R R E L T L TV L D I V A L F P N Y D S R R YP I R T V S Q L T R E I Y T N PV L E N F D G S F R G S A Q G IE R S I R S P H L M D I L N S IT I Y T D A H R G Y Y Y W S G HQ I M A S P V G F S G P E F T FP L Y G T M G N A A P Q Q R I VA Q L G Q G V Y R T L S S T L YR R P F N I G I N N Q Q L S V LD G T E F A Y G T S S N L P S AV Y R K S G T V D S L D E I P PQ N N N V P P R Q G F S H R L SH V S M F R S G F S N S S V S II R A P M F S W I H R S A E F NN I I A S D S I T Q I P A V K GN F L F N G S V I S G P G F T GG D L V R L N S S G N N I Q N RG Y I E V P I H F P S T S T R YR V R V R Y A S V T P I H L N VN W G N S S I F S N T V P A T AT S L D N L Q S S D F G Y F E SA N A F T S S L G N Y V G V R NF S G T A G V I I D R F E F I P

618V T A T L E K D E L

4. mosaic gene according to claim 1 is meant the BtS29K gene.

5.2BtCrylAc330614,：30I E T G Y T P I D I S L S L T QF L L S E F V P G A G F V L G LV D I I W G I F G P S Q W D A FL V Q I E Q L I N Q R I E E F AR N Q A I S R L E G L S N L Y QI Y A E S F R E W E A D P T N PA L R E E M R I Q F N D M N S AL T T A I P L F A V Q N Y Q V PL L S V Y V Q A A N L H L S V LR D V S V F G Q R W G F D A A TI N S R Y N D L T R L I G N Y TD Y A V R W Y N T G L E R V W GP D S R D W V R Y N Q F R R E LT L T V L D I V A L F P N Y D SR R Y P I R T V S Q L T R E I YT N P V L E N F D G S F R G S AQ G I E R S I R S P H L M D I LN S I T I Y T D A H R G Y Y Y WS G H Q I M A S P V G F S G P EF T F P L Y G T M G N A A P Q QR I V A Q L G Q G V Y R T L S ST L Y R R P F N I G I N N Q Q LS V L D G T E F A Y G T S S N LP S A V Y R K S G T V D S L D EI P P Q N N N V P P R Q G F S HR L S H V S M F R S G F S N S SV S I I R A P M F S W I H R S AE F N N I I A S D S I T Q I P AV K G N F L F N G S V I S G P GF T G G D L V R L N S S G N N IQ N R G Y I E V P I H F P S T ST R Y R V R V R Y A S V T P I HL N V N W G N S S I F S N T V PA T A T S L D N L Q S S D F G YF E S A N A F T S S L G N Y V GV R N F S G T A G V I I D R F E

614F I P V T A T L

6. recombinant plasmid, it is by described chimeric Bt insecticidal protein gene BtS29K of claim 4 and cloning vector pBluescript II SK ⁺Be built into, name a kind of recombinant plasmid into pSKBt29K.

7. a recombinant microorganism contains the described recombinant plasmid pSKBt29K of claim 6.

8. the plant expression vector of energy constitutive expression external source anti insect gene in plant is characterized in that comprising described chimeric Bt insecticidal protein gene BtS29K of claim 4 and binary expression vector pBin438, and this plant expression vector is named and is pBS29K.

9. plant expression vector that contains two kinds of anti insect genes, it is characterized in that comprising a kind of protease inhibitor gene, the expression cassette that CaMV35S promotor and Nos transcription termination sequence are formed is inserted among the described expression vector pBS29K of claim 8 and constitutes.

10. the plant expression vector that contains two kinds of anti insect genes according to claim 9, it is characterized in that protease inhibitor gene wherein is that two genes of arrowhead proteinase inhibitor B and A are connected a kind of antigen-4 fusion protein gene that the back forms from beginning to end, this gene is named and is API-BA.

11. a kind of plant expression vector with two kinds of anti insect genes according to claim 9 is characterized in that containing the plant expression vector that chimeric Bt gene BtS29K and a kind of proteolytic enzyme suppress antigen-4 fusion protein gene API-BA, is called pBS29K-BA.

12. a recombinant microorganism, it contains the described plant expression vector of claim 8.

13. a recombinant microorganism, it also contains the described plant expression vector of claim 11.

14. recombinant microorganism according to claim 13, it is intestinal bacteria [Escherichiacoli (Migula)]

It number is CGMCCNO.0452 that DH5 α, bacterial classification preserve.

15. recombinant microorganism according to claim 13 is Agrobacterium tumefaciens [Agrobacterium tumefaciens (Smith et Townsend) Conn] LBA4404.

16. the plant expression vector of 11,12,13,14 described insecticidal protein genes and the recombinant microorganism application in engineering of insect-resistant plant according to Claim 8.

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