CN1083884C - Two kinds of encoding insecticidal protein gene and bivalent fused expression carrier and their application - Google Patents

Abstract

The present invention provides a completely synthetized gene order for encoding bacillus thuringiensis insect-killing protein and a modified gene order for encoding a cowpea trypsin inhibitor and relates to a bivalent fusion plant expression vector including the two kinds of gene orders. After the vector is led in a plant, a transgenic plant and offspring and seeds thereof presenting high insect resistance on insects can be generated. Moreover, the insects are not easy to generate resistance on the transgenic plant containing the two kinds of insect-killing proteins. Thus, the transgenic plant has the characteristic of long insect resistance time effect on use after applied and produced. The present invention especially provides bivalent insect-resistant cotton capable of simultaneously expressing the two kinds of insect-killing proteins.

Description

Two kinds of encoding insecticidal protein genes and two valency fusion expression vectors and application thereof

The dna sequence dna of bacillus thuringiensis insect-killing protein and two kinds of insect-killing proteins of coding Cowpea Trypsin Inhibitor the present invention relates to encode, the two valency combination of plant expression vectors that comprise said dna sequence dna, by said expression vector plant transformed cell, and there are high resistance and insect to be difficult to it tolerific transgenic plant and offspring thereof are comprised plant seed and plant tissue to insect (particularly lepidopterous insects) performance by what said cell produced.

Utilize the biotechnology means can obtain specific insect is had the transgenic plant of toxic action.Wherein, the example of comparatively knowing is a companion cell insecticidal crystalline gene of using bacillus thuringiensis (Bacillusthurigiensis).The known bacillus thuringiensis that belongs to Gram-positive soil genus bacillus, can produce various insects, as lepidopteran (Lepidopterans), Coleoptera (Coleopterans) and the Diptera virose parasporal crystal protein of crop pest (Aronson such as (Dipterans), Microbiol.Rev.50:1-14,1968).This insect-killing protein comprises the protein (Klausner, Bio/Technoloqv 2:408-419) that respectively different insects is had specificity and high selectivity toxic action of many subclass.And this biomolecules sterilant is to plant with comprise that people's animal do not poison, and do not pollute the environment.

Based on the character of bacillus thuringiensis insect-killing protein, just produced many years ago and relevantly contained the spore that bacillus thuringiensis produces and the commodity pesticide preparation (commodity are called DIPEL and THURICIDE) of crystallization of protein in commercial sale.This commodity insecticide can resist the lepidoptera pest (Wilcox, et al., ProteinEngineering, Inouye and Sarma (Eds.) Academic Press, NY, 1968) more than 50 kinds effectively.Yet, a critical limitation using the bacillus thuringiensis pesticide preparation is because said crystallization of protein or delta-endotoxin are degraded in environment, and need duplication of production and this pesticide preparation of repeated application, increase cost greatly, thereby brought difficulty for the practical application in the agriculture production.

For addressing this problem, scientists has begun a series of research (people such as Umbeck, Bio/Technology, 5:262-266,1987 about the insect-resistant transgenic plants that can express the bacillus thuringiensis insect-killing protein; People such as Vaeck, Nature 328:33-37,1987; People such as schhoff, Bio/Technology 5:807-813.1987; People such as Barton, PCT 89004868; People such as Guo's three heaps, CN95119563.8).

Wherein, people such as Willbur (Plant Physiol.92:1-11,1990) studies show that, there are very big-difference in unicellular lower eukaryote body bacterium and higher organism body plant in the use of codon.For example the rate of utilization of codon XXC/G (wherein two X are selected from A, G, C and T separately) in plant is 45-73.5%, and only is 24% in the killing gene of bacillus thuringiensis Kustak HD-1 mutation (B.t.kurstaki HD-1) (Cry IA (b)).In addition, evidence suggests that the tRNA in the plant is difficult to provide the abundant translation of bacterial gene in vegetable cell.And, in bacillus thuringiensis desinsection structure gene, there are sequences such as the unsettled ATTTA be unfavorable in plant, transcribing out complete mRNA and AATAA, the mRNA that transcribes out is because of imperfect, so the protein that translates does not have insecticidal activity.

People such as Perlak (Bio/Technology 8:939-942,1990) according to the dicotyledons optimizing codon, with synthetic bacillus thuringiensis insect-killing protein (kurstaki HD-1, Cry IA (b)) and HD-73, the Cry IA (c) of being derived from of chemical synthesis process) synthetic gene.In the wherein said synthetic gene, 1/2 sequence of 3 ' end is lacked, and has changed the codon of part conserved sequence.Resulting gene order comprises the 1845bp of 615 aminoacid sequences that are equivalent to encode.This sequence is connected to the downstream of cauliflower mosaic virus 35S (CaMV35S) promotor that has 2 enhansers, and (Agrobacteriumtumefaciens) is transformed in the dicotyledons cotton cells by the plant Agrobacterium, and the pest-resistant cotton plants of regenerating.Wherein insect-killing protein expression amount (accounting for the 0.05%-0.1% of soluble protein in the leaf) be about the natural bacillus thuringiensis insecticidal protein gene of encoding expression amount 50-100 doubly.Yet because the ability of its constructed recombinant expression vector expression alien gene is unsatisfactory, thereby pest-resistant rate only be 72-76%, and observed cotton plants is also not satisfactory in the middle and later periods bollworm resisting ability of growing.

People (CN 95119563.8.1995) such as Guo's three heaps adopt segmentation total man worker synthetic method according to the plant optimizing codon, have obtained the bacillus thuringiensis insecticidal protein gene that can efficiently express in plant.And further made up efficient two-way plant expression vector, and change cotton over to, obtained that insect-resistance is stable, pest-resistant rate is the insect-resistant transgenic cotton more than 80%.

In addition, the biomolecules that also has a class to can be used for insect-resistant transgenic plants research is proteinase inhibitor (Proteinase inhibitor).The proteinase inhibitor that occurring in nature exists has four big classes: serpin, thiol protease inhibitor, inhibitors of metalloproteinase and aspartyl protease preparation.In plant, also do not find aspartyl protease inhibitor, it and inhibitors of metalloproteinase all do not have insect-resistance basically, and in the insect-resisting plant gene engineering field, serpin and thiol protease inhibitor are proved to be to get a good eye and using value.Wherein, one comparatively common example be the Cowpea Trypsin Inhibitor that belongs to serpin (Cowpwa typsininhibior, CpTI).1987, Hilder etc. at first reported the CpTI gene have been imported the pest-resistant plant that tobacco has obtained to efficiently express CpTI.Do insecticidal test with cigarette beetle, the result is to cigarette beetle resistance remarkable (Hilder, V.A.etc.Nature 330,160-163,1987).

As biotic pesticide, the insecticidal activity of single bacillus thuringiensis insect-killing protein is comparatively single-minded, and the target insect more easily to generation resistance (McGaughey W.H., Science.229:193-195,1985).Proteinase inhibitor is the desinsection spectrum width then, and insect is difficult to directly produce resistance.But its killing ability is not as the bacillus thuringiensis insect-killing protein.Thereby, for obtain insect be difficult to generations resistance, the transgenic plant with strong killing ability, the present invention attempted making the expression simultaneously in vegetable cell of bacillus thuringiensis insect-killing protein and two kinds of killing genes of Cowpea Trypsin Inhibitor.This will make insect may reduce greatly transgenic plant are tolerific when improving the transgenic plant insect resistance capacity.

The present invention relates to the bacillus thuringiensis insecticidal protein gene, particularly at the bacillus thuringiensis insecticidal protein gene of the toxic effect of lepidoptera pest.According to an aspect of the present invention, bacillus thuringiensis insecticidal protein gene provided by the invention is the dna sequence dna of coding B.t.kurstaki HD-1 and HD-73 insect-killing protein.

In particularly preferred embodiment of the present invention, in this nucleotide sequence in all coding bacillus thuringiensis insect-killing proteins each amino acid whose codon all be to be suitable for optimizing codon and the combination thereof that vegetable cell is expressed.According to a preferred embodiment more specifically of the present invention, in the adorned synthetic bacillus thuringiensis insecticidal protein gene sequence of the present invention, the associating rate of utilization of codon that with XXC and XXG (wherein X represents A, G, C, T separately) is representative is greater than 53.5%, and its G+C content is more than 48.1%.

According to one embodiment of the invention, the bacillus thuringiensis insecticidal protein gene that is adopted is described in the Chinese patent 95119563.8, adopts the plant optimizing codon, and the segmentation synthetic obtains.According to one embodiment of the invention, this gene has Nucleotide 64-1824 shown in the SEQ ID NO:2 or 1-1824 Nucleotide.

The dna sequence dna of coding bacillus thuringiensis insect-killing protein of the present invention is based on above-mentioned codon optimized principle substantially and the needs of corresponding restriction enzyme site is provided, according to known dna sequence dna synthetic method, use suitable DNA synthesizer synthetic, but it will be appreciated by those skilled in the art that, be purpose of the present invention, also can make dna sequence dna or its functional equivalent sequence of coding bacillus thuringiensis insect-killing protein related among the present invention with technology such as nucleotide site directed mutagenesis technology PCR enzymatic synthesize based on the wild sequence of natural bacillus thuringiensis insecticidal protein gene.

The invention still further relates to cowpea trypsin inhibitor gene (CpTI gene).Particularly through this gene of modification to achieve the object of the present invention.

According to a preferred implementing method of the present invention, CpTI gene provided by the present invention is that the method by PCR obtains.According to an implementation step of the present invention, a remarkable difference of this gene and natural CpTI gene is that the EcoRI site is not contained in gene inside.

Knew already that 5 ' end of natural CpTI gene had one section non-translational region sequence about about 50 base pairs, the disappearance of this sequence fragment can not influence the biosynthesizing of the enzyme inhibitors molecule with natural radioactivity.Thereby according to a preferred implementing method of the present invention, CpTI gene provided by the present invention does not contain said non-translational region sequence.

According to a preferred implementing method of the present invention, 5 ' and 3 ' end of CpTI gene provided by the present invention has suitable restriction enzyme site.Wherein, the gene 5 ' end has the PstI site, and 3 ' end has the SacI site.

According to one embodiment of the invention, the CpTI gene that is adopted has the encoding sequence of Nucleotide 11-271 shown in the SEQ IDNO:4 or 53-271 Nucleotide.Its encoded protein matter belongs to T/T type double end trypsin inhibitor.Be that each proteinase inhibitor molecule contains two tryptic inhibition active centre.

According to another aspect of the present invention, the present invention relates to be required adjusting and the controlling elements of described two kinds of insect-killing proteins above in recipient plant, expressing effectively.

For make external source killing gene product in vegetable cell transcribe with translation skill on obtain effective expression, must be connected with suitable expression adjusting and controlling elements at the exogenous DNA array flank that comprises the coding region.These controlling elementss comprise promotor, enhanser, terminator, the untranslated part of transcription product and polyadenylic acid sequence and be convenient in suitable substratum screening by the selectable marker gene of transformant etc.Vegetable cell transcripting promoter commonly used comprises 35S promoter of cauliflower mosaic virus (CaMV) and the nopaline synthase promoter of agrobacterium tumefaciens T-DNA.These promotors all are effectively in most of vegetable cells, but its copy number that inserts site and insertion is different, will the activity of transcribing and translate of the protein coding sequence in its downstream be produced a very large impact.

Thereby, the present invention relates to strengthen the viral deutero-translation enhancement sequences of the translation ability of mRNA in vegetable cell that under DNA instructs, produces through transcription, make the translation process of mRNA at ribosome bind site, can correct terminated terminator codon sequence any read under yard situation all, the mRNA of being convenient to the mRNA that vegetable cell obtains through transcription is correctly cut and processes cuts and job sequence, and directly is added to the polyadenylic acid sequence of the acceleration mRNA stabilization on the mRNA 3 ' end and impels the polyadenylation signal sequence that adds this polyadenylic acid sequence.

According to a preferred embodiment of the invention, in of the present invention pair of valency combination of plant expression vector, said 5 ' end non-coding region is by two enhancer sequence, a promoter sequence, the individual translation enhancement sequences that is derived from plant virus capsid protein plasmagene and foreign gene in vegetable cell in the translation process favorable short nucleotide sequence form.

In integrative gene expression vector of the present invention, except the dna encoding sequence of the insect-killing protein of the invention described above, what other were arranged in said encoding sequence two flanks (i.e. 5 ' and 3 ' end borderline region) is used to that to regulate and control sequence that this encoding sequence transcribes and translate at vegetable cell all known in the art and obtain easily.For example, the said translation enhancement sequences that is derived from plant virus capsid protein plasmagene coding region of the present invention's use is the Ω sequence.According to known Ω consecutive nucleotides order, we have synthesized two sections nucleotide fragments SEQ IDNO:5 and SEQ ID NO:6, have synthesized the Ω sequence of being made up of 68bp by enzymatic reaction.This sequence enrichment TTAAC sequence constitutes rrna and rRNA binding site (Richards et a1., Eur.J.Biochem.84:513-519,1987) in the translation process of protein synthesis.Wherein promotion foreign gene short nucleotide sequence of translation process in vegetable cell of the present invention's use is Kozak sequence (Kozak et al., Nucleic Acids Research 12:857-872,1984 of describing as people such as Kozak; Cell, 44:283-292,1986).Our institute's synthetic Ω sequence and Kozak sequence are shown in SEQ ID NO:7.

Be suitable for being connected, and in vegetable cell, start this DNA sequences encoding and transcribe the promotor of beginning and comprise composing type, induction type, tissue or organ specificity or etap specificity promoter with insect-killing protein encoding sequence of the present invention.For example, they comprise but are not only limited to cauliflower mosaic virus (CaMV) 35S or 19S promotor, mannopine synthetic enzyme (MAS) promotor, nopaline synthetic enzyme and octopine synthase promoter, the maize alcohol dehydrogenase promotor, and diphosphoribulose carboxylase, oxydase small subunit promotor etc.Wherein, the present invention preferably has the CaMV 35S promoter that doubles enhancer element.This promotor complete sequence is shown in SEQ ID NO:8.

According to a preferred embodiment of the invention, in of the present invention pair of valency combination of plant expression vector, said 3 ' end non-coding region comprises a multi-joint termination codon subsequence, mRNA cutting sequence and a mRNA cutting post-treatment sequence, similar polyadenylation signal sequence and polyadenylic acid sequence.These are regulated and control sequence can be a natural inherent in the vector plasmid carrier, or after it is transformed in the suitable cloning host, under the existence of dNTP and suitable DNA synthetic enzyme and dna ligase and the effect, utilize various known technology addings.

According to a preferred embodiment of the invention, the said encoding sequence and 5 ' and 3 ' the distolateral wing sequence thereof are synthetic chemically.

Yet, in an embodiment preferred of the present invention, above-mentioned these 3 ' ending regulating sequences, all be basically according to we design in advance and artificial synthetic.It will be appreciated by those skilled in the art that, be based upon this embodiment of the present invention on a large amount of theoretical investigation working foundations, will more help improving expression efficiency and stability and the accuracy of our two kinds of prepared insect-killing protein encoding sequences in vegetable cell.

For example, in a particularly preferred embodiment of the present invention, we have designed and synthesized the multi-joint terminator sequence (SEQ ID NO:9) that is directly connected in aforementioned two kinds of insect-killing protein structure genes, 3 ' end, in the short sequence of this Nucleotide, include three terminator codons that lay respectively in the different reading frames.Even thereby occur mispronouncing at translation process to the structural gene sequence of its left wing, also will be able to correct termination.What for another example, this aspect was designed 3 ' rectifies and definitely to cut the correct termination (SEQID NO:10) that has further guaranteed protein translation with job sequence.In addition, in 3 ' end non-coding region of of the present invention pair of valency combination of plant expression vector bacillus thuringiensis insecticidal protein gene, our design also is connected with polyadenylic acid poly (A) sequence (SEQ ID NO:11) in 3 ' side of above-mentioned multi-joint terminator codon.Like this, will avoid both having made in transcribing the post-treatment process since unknown mechanism caused can not add this (poly (A)) sequence the time be unfavorable for the stable problem of translating of mRNA.

In addition, being applicable to that the two valency combination of plant expression vectors of the present invention can also comprise is derived from cauliflower mosaic virus, the terminator of Nopaline (Nos) gene and analogue thereof.In one embodiment of the invention, we have used the terminator of Nos gene.

In order to improve the expression efficiency of gene in the unifacial leaf Gramineae plant of the two valency insect-killing proteins of expression of the present invention, can between structure gene and promoter sequence, add the suitably intron sequences of size, for example, first intron of corn alcohol dehydrogenase gene (Gene andDevelopment 1:1183-1200,1987), be derived from first intron (the Tanaka et al. of the catalase gene of Viscotrol C plant, NucleicAcids Research18:6767-6770,1990) etc.

Can use method as known in the art with the two valency fusion gene constructs that are suitable in vegetable cell expressing two valency insect-killing proteins of the present invention be connected to any can the carrier of self-replacation in bacterial cell or vegetable cell on.Such carrier for example comprises and is derived from colibacillary plasmid vector pUC18, pUC19 (Gene 103:1985), and the plasmid vector (being produced by Promega company) that causes a series of pGEM of being referred to as of different polyclone restriction enzyme sites through different modifications.Especially plant expression vector pBI101, pBI121 and pBI131 system (Jefferson, et al., EMBOJ.16:3901,1987), or the like.In a series of preferred embodiments of the present invention, the carrier that carries the two valency fusion gene constructs of the invention described above is pUC19, pBI121.2 and pBI131, and both are particularly suitable for the back as the instrument plasmid vector for preparing plant expression vector.

Certainly, as previously mentioned, in order correctly to select and to identify by the plant transformed cell, but the fusion expression vector of above-mentioned reorganization of the present invention also further contains selective marker and reporter gene.The both sides of employed selective marker and reporter gene can have adjusting sequence separately, to impel their expression in plant.The selective marker and the reporter gene that are suitable for are known in the art.Foreign gene and other genes of coding selective marker can be contained in the same expression vector, perhaps are contained in when transforming in the simultaneously applied different carrier.The preferred selectable marker gene of the present invention is neomycin phosphotransferase (NPTII) gene and β-Pu Taotanggansuanmei (GUS) gene, and together is contained in the same recombinant expression vector, thereby has guaranteed the reliability selected by transformant or plant.

Therefore, according to another aspect of the present invention, the present invention further provides the two valency combination of plant expression vectors that are suitable in vegetable cell, expressing simultaneously bacillus thuringiensis insect-killing protein and Cowpea Trypsin Inhibitor, wherein comprise:

1). bacillus thuringiensis insecticidal protein gene expression cassette;

A) .5 ' end non-coding region;

B). N-terminal about 608 the amino acid whose nucleotide sequences of coding bacillus thuringiensis insect-killing protein;

C) .3 ' end non-coding region.

2). the cowpea trypsin inhibitor gene expression cassette;

A) .5 ' end non-coding region;

B). about 87 the amino acid whose nucleotide sequences of coding Cowpea Trypsin Inhibitor aminoterminal;

C) .3 ' end non-coding region.

3). derive from the carrier part of pBI121:

A). neomycin phosphotransferase gene (NptII) expression cassette;

B). beta-glucosiduronatase gene (Gus) expression cassette; And

C). initial replicon and the functional structures such as left and right sides border sequence relevant with Plant Transformation.

According to a preferred embodiment of the invention, said 5 ' hold non-coding region and 3 ' end non-coding region to have foregoing composed component and function thereof respectively.

According to the present invention, said coding region has 64-1824 or the 11-271 as shown in 1-1824 nucleotide sequence and the SEQ ID NO:4 or 53-271 nucleotide sequence or its functional equivalent sequence or the mutant as shown in SEQ ID NO:2 respectively.

According to a preferred embodiment of the invention, the said coding gene sequence and 5 ' and 3 ' the distolateral wing sequence thereof are synthetic chemically.

Regulation according to budapest treaty, the applicant will be transformed into the fusion expression vector of naming to pGBI121S4ABC that the present invention in the bacillus coli DH 5 α host cell makes up on July 3rd, 1998 and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation registration number is CGMCC NO.0355.

Cotton is to import two valency killing genes, produces the exemplary target crop of insect resistance capacity effectively.Cotton is as a kind of cash crop, and we need not to consider its safety issue as food; Secondly, the financial loss that the cotton of worldwide extensively being planted is caused because of insect pest every year is very huge; In addition, made progress at present and the unit price Insect Resistant Cotton that begins to attempt using is faced with in the future insect to it tolerific potentially dangerous.Thereby according to an aspect of the present invention, our preferred cotton transforms the target crop of plant as two valency killing genes.

Thereby, the dna sequence dna of bacillus thuringiensis insect-killing protein and Cowpea Trypsin Inhibitor the present invention relates to encode, the two valency combination of plant expression vectors that comprise said dna sequence dna, with said expression vector transformed plant cells, the method of tissue and whole strain plant, and the consequent transgenic plant that plant insect is had control and poisoning ability, particularly to lepidopterous insects transgene cotton with remarkable toxic action such as bollworm for example.

As previously mentioned, in order in vegetable cell, to express two valency insect-killing proteins in the particularly whole strain plant, to give whole strain plant and seed thereof and offspring, must use in the recombinant expression vector conversion or transduce appropriate host cell or plant materials of the encoding sequence that carries of the present invention pair of valency insect-killing protein that appropriate means will prepare as stated above with killing ability.

With recombinant vectors importing host plant or its intracellular many methods of carrying foreign gene all is well known to those skilled in the art.These methods are including but not limited to 1) use Agrobacterium (Agrobacterium) as Agrobacterium-mediated Transformation method (Agrobacterium Mediated transformation), 2 that phytopathogen mediated) the physics method, as particle bombardment (Particle Bombardment﹠particle gun﹠amp; Gene gun), electric shock fusion method (Electroporation), microinjection (Microinjection), supersonic method (Ultrasonic waves), laser microbeam method (Laser microwave), silicon carbide fiber mediated method (Silicon carbide fiber), electrophoretic method (Electrophoretictransfection) etc.3) chemical method is as the conversion method of PEG mediation, liposome-mediated conversion method etc.4) germplasm system conversion method is as pollen-mediated method, pollen tube passage method, ovary injection, infusion method etc.5). with conversion methods that virus vector was mediated such as cauliflower mosaic virus (CaMV), geminivirus infection (Geminiviruses) or RNA viruses.

A kind of make us especially the interested method that in whole plant, imports foreign gene be in the so-called germplasm system conversion method that uses of the inventor improved ovary injection plant fertilization blastular method (referring to CN 95119563.8,1995; Zhou et al., Enzymol.Method.101:433-481,1983).

According to a preferred embodiment of the invention, said transgenic plant are transgene cottons.

According to another aspect of the present invention, toxic and insect is difficult to the method for tolerific plant to this toxicity to insect to the invention provides generation, and this method comprises:

1). make up said pair of valency combination of plant expression vector;

2). with any feasible method the two valency combination of plant expression that obtain in the step 1) are carried

Body imports in the vegetable cell, and obtains that thus insect is had resistance or kill capability

Transgenic plant and offspring thereof, comprise the plant tissue and the seed of any part.

What should particularly point out here is, though the generation with transgene cotton in the following embodiment of the present invention has described in further detail the present invention for example, this means that never the dna sequence dna of two valency insecticidal protein genes that the present invention prepares and the recombination, amalgamation and expression carrier that carries this dna sequence dna only are used to transform and produce the transgene cotton with insect resistance capacity.

Therefore, use has two valency killing gene expression vectors of feature described in the invention, import in any plant or its tissue or the cell with any method well known by persons skilled in the art, and the importing that obtains therefrom external source two valency insecticidal protein genes, have and kill and wound or the plant of the ability of the responsive insect of controlling plant, its offspring and seed thereof and plant part include within the present invention.

The purpose that provides following examples is to describe the present invention for example in detail, and does not constitute the await the reply restriction of interest field to the present invention.In the technology of the present invention feature and the claim scope that awaits the reply, those skilled in the art can make some change that is equal to and conspicuous improvement to the present invention.

The acquisition and the modification of embodiment 1 CpTI gene

1.1 PCR is to the preliminary modification of CpTI gene

CpTI gene with cowpea is a template, and (SEQ ID NO:13 and SEQ ID NO:14) carries out pcr amplification with designed synthetic primer, and the result obtains the dna fragmentation of about 300bp.PCR product behind the purifying precipitates behind Pst I, Sac I double digestion again.Carrier pUC19 is behind Pst I, Sac I double digestion, and electrophoresis reclaims the big fragment of 2.7kb.The 300bp fragment with after carrier is connected, is transformed DH5 α competent cell,, obtain recon and name and be pGC by the observation of α-Hu Bu phenomenon.Identify through Pst I, Sac I double digestion, confirmed the insertion of 300bp PCR product.

1.2 to CpTI Gene Sequence Analysis among the pGC

The preparation of (1) sex change template: with extracting plasmid DNA method (Sambrook J in a small amount, et al.Molecular Cloning:A Laboratory Manual.1989, ColdSpring Harbor Laborotory Press, New York), prepare pGC plasmid DNA to be measured.Get 1.5～2 μ g DNA (about 2～5 μ l) and add water to cumulative volume 32 μ l, add 8 μ l 2M NaOH then, mixing gently, room temperature sex change 10 minutes adds 4 μ lH then ₂O, 7 μ l 3M NaAc (pH 4.8), 120 μ l dehydrated alcohols, centrifugal 8 minutes of 12000rpm, with the precipitation that 70% ethanol is washed 2 gained, vacuum is drained, and is dissolved in the 10 μ l water, puts stand-by on ice.

(2) annealing: add 2 μ l annealing buffer in the template DNA after sex change, 2 μ l sequencing primers, mixing gently, instantaneous centrifugal back moves to 37 ℃ rapidly and continued incubations 10 minutes in 65 ℃ of incubations annealing 5 minutes, places more than 5 minutes in room temperature then.

(3) labeled reactant: inhale 2 μ l enzymes dilution buffer earlier, to wherein adding 0.5 μ lT7 Sequenase, place on ice then, the template after annealing adds labal Mix 3 μ l in the primer annealing mixture then, α- ³²P-dATP 1 μ l; T7 Sequenase 2 μ l after the dilution inhale gently in pipe and beat mixing, instantaneous centrifugal getting rid of, room temperature reaction 5 minutes.

(4) chain extension termination reaction: on 4 centrifuge tubes, put on A, C, G, T respectively, and add the corresponding chain termination liquid of 2.5 μ l (Mix short) respectively and be incubated 1 minute at least at 37 ℃, from (3), pipette 4.5 μ l reactants in the mark reaction solution respectively and join A, C, G, T, in four pipes, the rearmounted 37 ℃ of reactions of mixing 5 minutes, each added 5 μ l stop buffers, in 37 ℃ of insulations 2 minutes.

(5) preparation of sequencing gel: the order-checking instrument of using Bio-rad company.

Measure 50ml 6% order-checking acrylamide gel stock solution (6%Seqencing gel:1.5g methylene diacrylamide, 28.5g acrylamide, 240g urea, 50ml 10 * TBE adds water and is settled to 500ml), add 500 μ l, 10% Ammonium Persulfate 98.5,50 μ l TEMED, mixing slowly is injected in the clean order-checking panel assembly, insert comb, polymerized at room temperature is more than 45 minutes.

(6) installed electrophoresis apparatus, adding 1 * TBE is electrophoretic buffer (10 * TBE:108gTris, 9.3g Na ₂EDTA.2H ₂O, 55g boric acid is used H ₂O is dissolved in 1000ml (pH8.3) surely).With 80 ℃ of C sex change of sample 2 minutes, place on ice last sample electrophoresis.If necessary, when bromjophenol blue arrives the plate bottom, carry out sample on the secondary, continue electrophoresis to the secondary the sample bromjophenol blue to the plate bottom.

(7) stop electrophoresis, following glue, compressing tablet ,-70 ℃ of radioautograph are spent the night, and punching is read.

The result proves that having correct CpTI gene in this plasmid inserts fragment.This sequence and natural CpTI gene order are 5 ' ' and 3 ' end different, and do not contain the 5 ' leader sequence of holding about 50bp.

1.3 the removal in EcoRI site in the CpTI gene

Further by designing primer (SEQ ID NO:15 and SEQ ID NO:16), the EcoR I site among the sudden change pGC2 in the CpTI gene.Its roughly process be: obtaining earlier the dna fragmentation of about 140bp, 160bp respectively through two PCR reactions, is template with these two dna fragmentations then, about 300bp CpTI gene fragment of being suddenlyd change by PCR acquisition EcoRI site.As shown in table 1:

The removal in EcoRI site in the table 1.CpTI gene

Reaction 1 reaction 2 reaction 3 template CpTI gene (EcoRI+) CpTI gene (EcoRI+) Segment A and B5 ' primer SEQ ID NO:13 SEQ ID NO:15 SEQ ID NO:133 ' primer SEQ ID NO:16 SEQ ID NO:14 SEQ ID NO:14PCR product Segment A (160bp) fragment B (141bp) CpTI gene (EcoRI ^-, 282bp)

Response procedures is: 94 ℃ 2 minutes

93 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations

72 ℃ 5 minutes

Electrophoresis is identified and purified pcr product, is obtained the modified CpTI gene of removing the EcoRI site.CpTI gene clone after will suddenling change with Pst I, Sac I once more is in plasmid pUC19.Obtain recombinant plasmid pGCE.Sequential analysis (method therefor is with 1.2) to pGCE proves that the CpTI gene of being cloned meets preliminary design scheme fully.It is arranged in insertion nucleotide sequence between pUC19 PstI and SacI site shown in SEQ ID NO:4.

Embodiment 2: the structure of two valency killing gene plant expression vectors

2.1 modify the structure of back CpTI expression cassette

Comprise following gene expression regulation element in the constructed CpTI expression cassette of present embodiment: 35S promoter, the enhanser that doubles, Ω sequence and the kozak sequence of 5 ' end, the multi-joint terminator sequence of 3 ' end, cutting sequence, correct job sequence and NOS terminator.These expression regulation elements except that 35S promoter and enhanser all by chemical process synthetic (seeing Chinese patent CN 95119563.8,1995 for details).In preserving the plasmid pG4AB of the bacillus thuringiensis insecticidal protein gene expression cassette that has synthetic, this laboratory includes these elements.

Utilize plasmid pG4AB, behind Pst I and Sac I double digestion, reclaim the 3.5kb fragment, be connected to the carrier that is reclaimed after the CpTI gene among the pGCE is downcut with Pst I and Sac I and get on, obtain recombinant plasmid pG4ACE as carrier.In this plasmid, then have the CpTI expression casette.Phase shift mutation does not take place in the initiator codon ATG in initiator codon ATG in the carrier and the CpTI gene, as shown in Figure 1.

2.2 have the acquisition of two valency fusion expression vectors of two valency killing genes

Bacillus thuringiensis insecticidal protein gene among the pG4AB is downcut with HindIII, be cloned into the mode of connection of flush end on the Acc I site of pG4ACE.Fragment and cloning vector are all mended flat with the Semen Phaseoli radiati Germinatus nuclease.Obtained this killing gene with positive and negative both direction broadcast into recombinant plasmid: pGS4ABC and pGD4ABC.Adopt different restriction enzymes to cut evaluation, proved the correct of constructed plasmid through many group enzymes.

2.3 the structure of two valency killing gene plant expression vectors

With the 4.7kb killing gene among HindIII cutting-out pGS4ABC and the pGD4ABC, to be cloned on the HindIII site of plant expression vector pBI121, the acquisition recombinant plasmid is named and is pGBI121S4ABC.This with pBI121 be carrier the two valency combination of plant expression vector pGBI121S4ABC that carry two kinds of killing genes plasmid map as shown in Figure 2.

Importing and the evaluation of 3. pairs of valency combination of plant of embodiment expression vector in the model plant tobacco

3.1 the preparation of LBA4404 competent cell

The single colony inoculation of picking LBA4404 in 5ml YEB (containing Streptomycin sulphate 100 μ g/ml), 28 ℃, the 250rpm overnight incubation.Draw the 2ml bacterium colony and add in the 50ml YEB substratum, continue to be cultured to the OD value and be about 0.6.Bacterium liquid is gone in the aseptic centrifuge tube, ice bath 30 minutes, centrifugal 5 minutes of 5000rpm is with the CaCl of 2ml 20mM ₂Resuspended thalline is sub-packed in the aseptic little centrifuge tube by every pipe 200 μ l.

3.2 recombinant plasmid changes the LBA4404 cell over to

In 200 μ l LBA4404 competent cells, add 2ug recombinant plasmid pGBI121S4ABC, pGBI121D4ABC, pGBI131S4ABC and pGBI131D4ABC DNA respectively, put ice bath 5 minutes, went to then in the liquid nitrogen freezing 8 minutes, incubation after 5 minutes in 37 ℃ of water-baths rapidly, add 800 μ l YEB substratum, 28 ℃ of 250rpm express in advance and cultivated 4～5 hours, are coated with the YEB solid plate that kantlex is contained in the shop then, cultivate 24～48 hours for 28 ℃.The Agrobacterium bacterium colony that then grows has corresponding conversion plasmid.

3.3 the conversion of tobacco

(1) preparation of Agrobacterium: 28 ℃ of cultivations respectively of spending the night have the Agrobacterium 50ml of plant expression vector, and centrifugal 5 minutes of 5000rpm collects bacterial sediment, with the washing of liquid MS nutrient solution once, use MS resuspended to OD again ₆₀₀=0.2～0.4.

(2) contaminate: get tobacco aseptic seedling blade, be cut into the about 0.5 centimetre of dice of the length of side by knife, in the Agrobacterium suspension, soaked 5～10 minutes, blot with sterilization filter paper then.

(3) cultivate altogether: the leaf piece is placed in the common culture medium of added with antibiotic not (top pad two metafiltration paper) and cultivated altogether 3 days in 25% lucifuge.

(4) select to cultivate: with the blade subculture to selecting in the substratum in illumination box (illumination 12 hours, dark 12 hours) to be cultured to the appearance of resistant buds.

(5) acquisition of resistance seedling: callus is moved to the root culture gene, every two all succeeding transfer culture once, plant at last in the little polypots.

3.4 the molecular biology identification of transgene tobacco

3.4.1 PCR identifies:

Get 1～3 gram fresh tobacco blade, add equivalent Al ₂O ₃Use liquid nitrogen grinding, powder is placed the 50ml centrifuge tube, add 20ml DNA extraction damping fluid (100mM Tris.HCl pH8.0,500mMNaCl, 10mM beta-mercaptoethanol, 50mM EDTA pH8.0), thermal agitation 1 minute adds 4ml10%SDS, mixing gently, in 65 ℃ of heating 10～20 minutes, emerald green to color, add ice-cold 5M KAC 4ml, placed on ice 30 minutes, then at 4 ℃, centrifugal 15 minutes of 12000rpm sucts clearly, behind 0.6 times of isopropanol precipitating, handle with RNAase, purifying is standby.

Genomic dna with transgene tobacco is a template, and the Auele Specific Primer with two valency killing genes carries out following PCR reaction respectively:

In 0.5ml Eppendorf pipe, add:

H ₂O 30.5μl

10×PCR?buffer 5μl

MgCl2(25mM) 3μl

4 * dNTP (each 2.5mM), 5 μ l

Primer1(20pmol/μl) 2.5μl

Primer2(20pmol/μl) 2.5μl

Template plasmid pGUSO DNA (100ng/l) 1 μ l

Taq enzyme (3U/ μ l) 0.5 μ l

50μl

Paraffin oil 50 μ l

Response procedures is: 94 ℃ 3 minutes

93 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations

72 ℃ 5 minutes

Used bacillus thuringiensis insecticidal protein gene primer (20pmol/ul) is:

5’-GGGCCCGCTGAATCCAACTGGAGAGGC-3’(SEQ?ID?NO：17)

5’-CCATACAACTGCTTGAGTAACCCAGAAGTTG-3’(SEQ?ID?NO：18)

Used CpTI primer (20pmol/ul) is:

5’-CTACTGCAGCATGGATTTGAACCACCTCGGAAGT-3’(SEQ?ID?NO：13)

5’-GGGAGCTCTTACTCATCATCTTCATCCCTGGACT-3’(SEQ?ID?NO：14)

Found that the DNA cloning fragment that conforms to the expection size is arranged respectively.

3.4.2 the Southern of PCR product analyzes:

The Southern that further carries out PCR analyzes,

(1) the PCR product with DNA of plants carries out electrophoresis and photograph by 0.8% sepharose.

(2) glue is placed shallow bid add 200ml sex change liquid (0.5M NaOH, 1.5M NaCl) vibration 45 minutes.

(3) discard sex change liquid, with the distilled water rinsing for several times, add neutralizer (1M Tris, pH7.4,1.5M NaCl) 200ml, vibrate 30 minutes, the neutralizer that more renews then, in the continuation with 15 minutes.

(4) cut the nitrocellulose filter (NC film) of corresponding size, in distilled water evenly drenched after, the NC film is dipped among 10 * SSC (20 * SSC: every 1000ml contains NaCl 175.3g, Trisodium Citrate 88.2g pH 7.0), placed 20 minutes.

(5) use the commentaries on classics film instrument of Bio-rad company to change film 1 hour.

(6) remove blob of viscose, film was washed 5 minutes with 6 * SSC in the good well of mark position, and uv irradiating is 5 minutes then, with fixed dna.2 * SSC washes film, and room temperature is air-dry.

(7) the NC film is involved in the hybridization bottle, adds 20ml prehybridization solution (6 * SSC, 5 * Denhardt, 0.5%SDS, 200ug/ml milt DNA), make the film homogeneous immersion not have bubble, place 68 ℃ of prehybridizations of dna molecule hybridize instrument to spend the night then.

(8) mark of probe: adopt Promega company random primer test kit mark.Get an amount of dna fragmentation to be marked, 95-100 ℃ of sex change placed rapidly on ice in 2 minutes, in a new Eppendrof pipe, added successively:

5×labling?buffer 10μl

DCTP, dGTP, dTTP, mixture 2 μ l

Sex change template 25ng

BSA 2μl

α- ³²P-dATP 5μl

Enzyme 1 μ l

Add H ₂O is to cumulative volume 50 μ l

Mixing gently, room temperature reaction 60 minutes.95% sex change placed on ice in 2 minutes.Add 2 μ l0.5M EDTA.

(9) in the bag that prehybridization finishes, add sex change probe 30～50 μ l, seal again and continue at 68 ℃ of hybridization 8～10 hours.

(10) take out Hybond membrane with 2 * SSC, 0.5%SDS washed film 30 minutes, and with 2 * SSC, 0.1%SDS washed 15 minutes, used 0.1 * SSC again, and 0.5%SDS washes 30 minutes～a few hours of film, the film washing liquid that the centre more renews in 42 ℃.

(11) 0.1 * SSC washed film 2 minutes, inhaled the pearl of anhydrating with thieving paper, wrapped film with preservative film, compressing tablet in the darkroom.

(12)-70 ℃ autography was developed a film after 3～6 hours.

The result shows that the result of the PCR-Southern of two kinds of amplified productions is all positive.

3.5 the GUS of transgene tobacco analyzes

Get the blade of transgene tobacco, cut several fritter (0.5mm ²), be put in the 1.5mlEppendorf pipe and add 15ul GUS substrate, spend the night in 37 ℃ of processing.Use 95% ethanol decolorization 2 hours then, 70% ethanol continues decolouring for several times, and is each more than 1 hour.Observe and blue reaction whether occurs.The result proves that most of transfer-gen plant presents tangible GUS positive reaction.

3.6 the mensuration of the trypsin inhibitor activity of transgene tobacco is (with reference to Washington (Washington Biochemical Co., Decker, L.A.:Washington EnzymeManual, Freehold, New Jersey, 1977) configuration of (1) substrate solution: take by weighing 189mg right-tolylsulfonyl-L-arginine ethyl ester

(Na-p-TOSYL-L-ARGININE METHYL ESTER，TAME，

C ₁₂H ₂₂N ₄O ₄SHCl, FW=378.9, SIGMA company) be dissolved in the configuration of (2) enzyme solution in the 50ml deionized water: trypsin Trypsin, 1:250,2～4 μ

/ mg, Difco company) 25mg is dissolved in the blade that 100ml 0.001N HCl (3) chooses similar contrast of situation and transgene tobacco, and extracting with ordinary method can

The dissolubility total protein is with Xylene Brilliant Cyanine G standard measure (Braford, M.M., 1976, Anal.

Biochem 72:248-254), and is diluted to same concentrations.(4) operation steps: in 2.9ml Tris damping fluid reaction system, comprise the 0.30ml substrate

Solution and testing sample.Join in the quartz cuvette of 1cm 25 ℃ of guarantors after the mixing

Add the enzyme solution of 0.10ml behind the temperature 10min, survey absorbancy in the 247nm place, with

The Tris damping fluid compares.Read light absorption value every 30sec in the 3min.(5) enzymic activity is calculated: calculate with following formula:

E ₂₄₇×3/(0.54×Ew)＝U/mg

E ₂₄₇The extinction increase value of-----247nm place per minute

0.54-----1 μ mol is right-absorbancy that tolylsulfonyl-L-arginine/ml causes.

The weight (mg) that contains enzyme in the used enzyme liquid of Ew-------0.1ml

The cumulative volume of 3--------reaction solution finds that by calculating the trypsinase vigor of transgene tobacco is than the transgenosis contrast is not low.Significant difference.

3.6 the insect-resistance of transgene tobacco test

Get the group training seedling leaves of transgene tobacco, place plate respectively, petiole base wraps up with moistening rayon balls.The bollworm that each plate connects one 1～2 age carries out insecticidal test, has proved that the seedling of transgene tobacco has had tangible insect-resistance.Bollworm is got food in a large number in transgene tobacco not, grows normally, and growth rapidly; Bollworm the food refusal reaction promptly occurs after getting food on a small quantity in transgene tobacco, and growth is suppressed, and is last dead.

Get the transgene tobacco blade of intermediate house, place plate respectively, petiole base wraps up with moistening rayon balls.Each plate connects 12 bollworm newly hatched larvaes and carries out insecticidal test, in the 62 strain transgene tobaccos of being tested, has 43.5% (27/62) to have tangible insect-resistance, and 21.0% (13/62) has the moderate insect-resistance, and 35.5% (22/62) is not pest-resistant.The killing ability of the transgene tobacco that different carriers obtained does not have marked difference.Result's statistics sees Table 2.Continue to carry out insecticidal test with an age or two instar bollworm grubs, the high tobacco leaf of discovery part insect-resistance can be killed an age or two instars.

Table 2: the high resistant strain number of the pest-resistant experimental result test material/strain number/% moderate resistant strain number/% of the transgene tobacco blade that is obtained hangs down resistant strain number/% non-resistant strain number/%pGBI131S4ABC/33 16/48% 2/6% 4/12% 11/,33%,pGB,I13,1D4,ABC,/29 11/38% 5/17% 2/7% 11/38% and annotates: " high resistance " insect-resistance is remarkable, it is all dead that worm is got the food back on a small quantity, and blade is impaired slight:

" moderate resistance " moderate insect-resistance, worm are got the whole death in food back on a small quantity or are grown and obviously be suppressed, and blade is impaired heavier:

It is more that " low resistance " more weak insect-resistance, worm are got food, but it grows and be suppressed, and blade is impaired serious.

" non-resistant " is not pest-resistant substantially, and worm is only often got food, and it is normal to grow, and blade is impaired serious unusually.

Embodiment 4 preparations transform with two valency combination of plant expression vector pGBI121S4ABC

4.1 large quantity extracting plasmid pGBI 121S4ABC

(1) the order bacterium colony has in the corresponding antibiotic liquid LB substratum in 20ml, and 37 ℃,

Shaking culture is spent the night under the 250rpm condition.

(2) bacterium liquid is changed in the 4000ml liquid LB substratum, continue to cultivate 8-12 hour;

(3) use centrifugal 5 minutes of 5000rpm, collect thalline;

(4) with STE 100ml suspension washing thalline, merge each pipe in 2 500ml centrifuge tubes,

Again the centrifugal supernatant that goes;

(5) respectively to every pipe add 80ml Solution I (50mM sucrose, 10mM EDTA,

25mM Tris-HCl pH8.0), thalline suspension back adding 160ml is newly prepared

Solution II (0.2M NaOH, 1%SDS), place on ice for several times gently by mixing

10 minutes, (every 100ml contained 5M KAC to add the SolutionIII of 120ml precooling again

60ml, glacial acetic acid 11.5ml, distilled water 28.5ml), mixing is put on ice gently

Put 30 minutes;

(6) 8000rpm is centrifugal 15 minutes, collects supernatant, adds different third of 0.6 times of volume

Alcohol, room temperature was placed 10 minutes;

(7) 8000rpm is centrifugal 15 minutes, and 70% washing with alcohol once is dissolved in 4ml

Among the TE (10mM Tris, 1mM EDTA pH8.0), add RNAase 8 μ l

(10mg/ml) handled 1 hour for 37 ℃;

(8) with phenol, chloroform extracting and purifying once, be dissolved among the 3mlTE behind the ethanol sedimentation.

4.2 ultracentrifugation is further purified the plasmid DNA of extracting (with Beckman company ultracentrifuge)

(1) add 2.9mlDNA solution in a 50ml centrifuge tube, the saturated cesium chloride of 6.6ml is molten

Liquid, (EB, 10mg/ml), mixing is stand-by for the 1ml ethidium bromide solution.

(2) if any many precipitations, then 8000rpm is centrifugal 5 minutes in going on foot on.

(3) above-mentioned mixed solution is changed in two 5.2ml ultracentrifugation pipes, exceed the speed limit with Beckman

The whizzer specific equipment heat-sealing mouth of pipe.

(4) use vTi80 rotary head room temperature traditional vacuum 20 hours, 6,5000rpm.

(5) under the medium ultraviolet light of darkroom, bore a hole with syringe needle on centrifuge tube top earlier, use 5ml then

Disposable syringe sucking-off plasmid bright band.

(6) remove EB for several times with the water-saturated n-butanol extracting.

(7) with after three times of the TE dilutions with 4 ℃ of precipitations of two times of dehydrated alcohols, centrifugal, dilute the dissolving back

Stand-by.

Embodiment 5 plant fertilization blastular injection converting cottons

Whether the bivalent gene of coded insect-killing protein efficiently expresses for producing transgenic plant to be the most key, can to succeed but foreign gene transforms plant, also be to cause to close important link in plant.Before the present invention,, all there is adverse factors though the methods conversion plants such as agrobacterium-mediated transformation, particle bombardment, PEG method, electrization that utilize that this area scientific and technical personnel know have many successful examples.Not only be subjected to high etc. the restriction of laboratory condition and expensive instrument and expense as above-mentioned method, the more important thing is that aforesaid method all has a disadvantageous general character, that is exactly the restriction that is subjected to the plant gene type.On producing, bringing into play the good plant kind of important effect at present, because genotypic difference can not be passed through callus approach regeneration plant; Or regeneration plant is very difficult.Under these circumstances, the present inventor is by plant fertilization blastular injection transformed technology or claim plant fertilization blastular injection conversion method (CN 95119563.8), has successfully obtained to have the insect-resistant transgenic cotton of high insect resistance capacity.Though the preferred cotton of the present invention is not limited to cotton as the initial plant of ovary injection foreign gene conversion plant fertilization blastular method.The advantage that ovary injection foreign gene transforms plant fertilization blastular technology is: 1). be suitable for the genotype of all plants; 2). method is simple, effectively; 3). be not subjected to the restriction of laboratory condition, instrument, and expense is low; 4). speed is fast, can obtain transgenic plant seed and offspring in 1 year.

In this example, ovary injection foreign gene transforms the method for plant fertilization blastular, is between about 10-24 hour after cotton blooms pollination.As before the sealing of megarchidium duct, usefulness micro-injection system in ovary, wants material to be diffused into gradually or to be extruded in the blastular of firm after fertilization in the process of megarchidium duct sealing by the hole of bead and megarchidium duct outward the foreign gene injection of solution.In zygote splitted process, foreign gene is absorbed in the genome that is incorporated into the fertilized egg cell, imports and integration process thereby finish foreign gene.

Cotton is normal cross pollinated plant, and for keeping the relative purity of kind (being), in the noon before that day of blooming, the flower bud that will bloom at second day is tight with cableties, flower is distinguished be difficult for opening, so that its self-pollination.Bloomed back about about 10 hours, and after pollen tube enters blastular about 6 o'clock of evening of promptly blooming the same day, can carry out the injection of foreign gene.Before the injection flower distinguished to peel off together with stamen and expose young bell, erase the vertical style of young bell, our the micro-injection system of design draws the foreign gene solution of precooling, top from the young bell of erasing, vertically insert 2/3 place (as shown in the figure) of young bell size along axis, again microsyringe is upwards mentioned 1/3 place, stayed the insertion space of young approximately bell 1/3, slowly the external source injection of solution in the injection device is arrived and insert in the space.After this, exogenous DNA solution will be along the hole of bead and megarchidium duct to fertilization blastular internal diffusion, or owing to its integration is realized in the fertilization blastular sealing gradually to be extruded in the megarchidium duct.The foreign gene solution of general injection is 10 μ l, and total amount is about 0.25-0.5 μ g.Certainly, can suitably increase or reduce the injection volume of foreign gene according to what of ovule number in its ovary for different crops.After the foreign gene injection, in order to prevent and to reduce coming off of young bell, improve into the bell rate, smear the Plant hormones regulators,gibberellins of 40ppm with writing brush or be clipped between young bell handle base portion and the stem (branch) at young bell handle base portion with the cotton balls that is soaked with 40ppm Plant hormones regulators,gibberellins, branch Xiang Duan with the young bell place of said injection foreign gene removes simultaneously, keeping the abundant nutrition of young bell, thereby will help into the growth of seed in bell and the bell.

The molecular Biological Detection of embodiment 6 divalent insect-resistant cottons

The method of Application Example 3.4 has been carried out PCR and PCR-Southern analysis to transgenic cotton and offspring thereof respectively, has confirmed that two valency combination of plant expression vectors have been transferred in the cotton gene group.

The biology insecticidal test of embodiment 7 divalent insect-resistant cottons

Carrying out pest-resistant qualification test with the pest-resistant method of biology, is the most sensitive and effective method.Divalent insect-resistant cotton R1 generation, R2 generation and R3 that harvesting screens are for (contemporary seed is R0 generation, the plant that seed germination grows is R1 generation) two of plant top or branch top unfolded tender leafs, be placed on respectively in two culture dish, each culture dish is put 12 three day old larva, put in 28 ℃ of thermostatic chambers, after connecing worm, calculated dead borer population in 72 hours, the borer population of living, and the observation blade extent of damage, triplicate.The formula that calculates mortality ratio or corrected mortality is as follows:

Also carried out simultaneously a large amount of field insecticidal tests.The result proves, transgenic cotton against pests is compared with contrasting not transgenic cotton, has high insect-resistance, and this resistance can be stable entails the offspring.Insect Resistant Cotton strain after isozygotying can keep the insect-resistance more than 80%.Its insect resistance capacity and unit price Insect Resistant Cotton (CN 95119563.8,1995) are not significantly improved, but it is in pest-resistant scope and effectively slow down or prevent that insect from producing transgenic cotton and be better than the former aspect the resistance.

In a word, the synthetic gene of the coding bacillus thuringiensis insecticidal proteins of the present invention's preparation and the cowpea trypsin inhibitor gene after the modification all have the base sequence that adapts with plant.Recombinate with the highly expression regulating gene element sequences, the efficient combination of plant expression vector of the bivalent gene that is built into, no matter be to import tobacco by agrobacterium-mediated transformation, still transform fertilization blastular method converting cotton by the ovary injection, prove that all this pair valency combination of plant expression vector can efficiently express in plant, and killing gene can be entailed offspring and seed, make it to have high insect-resistance.Thereby, the invention provides a kind of acquisition and have high insect-resistance, and insect is difficult to the method to they tolerific divalent insect-resistant transgenic plant.

When brief description of drawings: figure one explanation made up the CpTI expression casette, this gene was arranged in correct reading frame.Thereby no matter translate and betide first, second reading frame, all producing has active Cowpea Trypsin Inhibitor.Among the figure, pGCE is the middle plasmid vector that has the CpTI gene, and pG4ACE and pG4AB are respectively the middle plasmid vector that has expression regulation element that carries CpTI gene and synthetic B.t. insecticidal protein gene.Figure two has illustrated that the present invention is the plasmid structural representation of two valency combination of plant expression vector pGBI121S4ABC of vector construction with pBI121

Sequence table (1) general information: (i) applicant:

(A) title: Biological Technology Research Center, Chinese Academy of Agricultural Sciences

(B) address: No. 30, Baishiqiao Road, Haidian District, Beijing

(C) country: China

(D) postcode: 100081

(F) phone: (010) 62184096

(G) fax: (010) 62184096 (ii) denomination of invention: two kinds of encoding insecticidal protein genes and two valency combination of plant expression vectors and use (iii) sequence number: the information of 18 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 608 amino acid

(B) type: amino acid

(C) chain: strand

( D ) ： ( ii ) ： ( iii ) ：B.t.Kurstaki HD-1/HD-73 ( iv ) ：SEQ ID NO：1Met Asp Cys Arg Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu1 10 20Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu21 30 40Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile41 50 60Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg61 70 80Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr81 90 100Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg101 110 120Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu121 130 140Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu141 150 160Asn Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala161 170 180Thr Ile Asn Ser Arg Tyr Asn Asp Leu Tyr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala181 190 200Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Ile201 210 220Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe221 230 240Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile241 250 260Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile261 270 280Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr281 290 300Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly301 310 320Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln321 330 340Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr341 350 360Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu361 370 380Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val381 390 400Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser401 410 420His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile421 430 440Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala441 450 460Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn Phe Leu Phe Asn Gly Ser Val461 470 480Ile Ser Gly Pro Gly Phe Thr Gly Gly Asp Leu Val Arg Leu Asn Ser Ser Gly Gly Asn481 490 500Ile Gln Asn Arg Arg Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr501 510 520Arg Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn Trp Gly Asn521 530 540Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr Ser Leu Asp Asn Leu Gln Ser541 550 560Ser Asp Phe Gly Tyr Phe Glu Ser Ala Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val561 570 580Gly Val Arg Asn Phe Ser Gly Thr Thr Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro581 590 600Val Thr Ala Thr Leu Glu Ala Glu601 608 ( 3 ) SEQ ID NO：2： ( i ) ：

(E) length: 1824 base pairs

(F) type: Nucleotide

(G) chain: two strands

( H ) ： ( ii ) ：DNA ( iii ) ： ( iv ) ：SEQ ID NO：2ATGGACTGCA GGCCATACAA CTGCTTGAGT AACCCAGAAG TTGAAGTACT TGGTGGAGAA 60CGCATTGAAA CCGGTTACAC TCCCATCGAC ATCTCCTTGT CCTTGACACA GTTTCTGCTC 120AGCGAGTTCG TGCCAGGTGC TGGGTTCGTT CTCGGACTAG TTGACATCAT CTGGGGTATC 180TTTGGTCCAT CTCAATGGGA TGCATTCCTG GTGCAAATTG AGCAGTTGAT CAACCAGAGG 240ATCGAAGAGT TCGCCAGGAA CCAGGCCATC TCTAGGTTGG AAGGATTGAG CAATCTCTAC 300CAAATCTATG CAGAGAGCTT CAGAGAGTGG GAAGCCGATC CTACTAACCC AGCTCTCCGC 360GAGGAAATGC GTATTCAATT CAACGACATG AACAGCGCCT TGACCACAGC TATCCCATTG 420TTCGCAGTCC AGAACTACCA AGTTCCTCTC TTGTCCGTGT ACGTTCAAGC AGCTAATCTT 480CACCTCAGCG TGCTTCGAGA CGTTAGCGTG TTTGGGCAAA GGTGGGGATT CGATGCTGCA 540ACCATCAATA GCCGTTACAA CGACCTTACT AGGCTGATTG GAAACTACAC CGACCACGCT 600GTTCGTTGGT ACAACACTGG CTTGGAGCGT GTCTGGGGTC CTGATTCTAG AGATTGGATT 660AGATACAACC AGTTCAGGAG AGAATTGACC CTCACAGTTT TGGACATTGT GTCTCTCTTC 720CCGAACTATG ACTCCAGAAC CTACCCTATC CGTACAGTGT CCCAACTTAC CAGAGAAATC 780TATACTAACC CAGTTCTTGA GAACTTCGAC GGTAGCTTCC GTGGTTCTGC CCAAGGTATC 840GAAGGCTCCA TCAGGAGCCC ACACTTGATG GACATCTTGA ACAGCATAAC TATCTACACC 900GATGCTCACA GAGGAGAGTA TTACTGGTCT GGACACCAGA TCATGGCCTC TCCAGTTGGA 960TTCAGCGGGC CCGAGTTTAC CTTTCCTCTC TATGGAACTA TGGGAAACGC CGCTCCACAA 1020CAACGTATCG TTGCTCAACT AGGTCAGGGT GTCTACAGAA CCTTGTCTTC CACCTTGTAC 1080AGAAGACCCT TCAATATCGG TATCAACAAC CAGCAACTTT CCGTTCTTGA CGGAACAGAG 1140TTCGCCTATG GAACCTCTTC TAACTTGCCC TCCGCTGTTT ACAGAAAGAG CGGAACCGTT 1200GATTCCTTGG ACGAAATCCC ACCACAGAAC AACAATGTGC CACCCAGGCA AGGATTCTCC 1260CACAGGTTGA GCCACGTGTC CATGTTCCGT TCCGGATTCA GCAACAGTTC CGTGAGCATC 1320ATCAGGGCTC CTATGTTCTC TTGGATACAC CGTAGTGCTG AGTTCAACAA CATCATCGCA 1380TCCGATAGTA TTACTCAAAT CCCTGCAGTG AAGGGAAACT TTCTCTTCAA CGGTTCTGTC 1440ATTTCAGGAC CAGGATTCAC TGGTGGAGAC CTCGTTAGAC TCAACAGCAG TGGAAACAAC 1500ATTCAGAATA GGAGGTATAT TGAAGTTCCA ATTCACTTCC CATCCACATC TACCAGATAT 1560AGAGTTCGTG TGAGGTATGC TTCTGTGACC CCTATTCACC TCAACGTTAA TTGGGGTAAT 1620TCATCCATCT TCTCCAATAC AGTTCCAGCT ACAGCTACCT CCTTGGATAA TCTCCAATCC 1680AGCGATTTCG GTTACTTTGA AAGTGCCAAT GCTTTTACAT CTTCACTCGG TAACATCGTG 1740GGTGTTAGAA ACTTTAGTGG GACTGCTGGA GTGATTATCG ACAGATTCGA GTTCATTCCA 1800GTTACTGCAA CACTCGAGGC TGAA 1824 ( 4 ) SEQ ID NO：3：

(i) sequence signature:

(A) length: 87

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(iii) out of Memory: CpTI

(iv) sequence description: the information of SEQ ID NO:3Met Asp Leu Lys His Leu Gly Ser Asp His His Asp Asp Ser Ser Asp Glu Pro Ser Glu 1 10 20Ser Ser Glu Pro Cys Cys Asp Ser Cys Ile Cys Thr Lys Ser Ile Pro Pro Gln Cys His 21 30 40Cys Thr Asp Ile Arg Trp Asn Ser Cys His Ser Ala Cys Lys Ser Cys Met Cys Thr Arg 41 50 60Ser Met Pro Gly Lys Cys Arg Cys Leu Asp Ile Ala Asp Phe Cys Tyr Lys Pro Cys Lys 61 70 80Ser Arg Asp Glu Asp Asp Glu81 87 (5) SEQ ID NO:4: (i) sequence signature:

(A) length: 282 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological structure: linear (ii) molecule type: DNA (iii) out of Memory: CpTI gene order (iv) sequence description that process is modified: the information of SEQ ID NO:4CTACTGCAGG ATGGATTTGA AGCACCTCGG AAGTAATCAT CATGATGACT CAAGCGATGA 60ACCTTCTGAG TCTTCAGAAC CATGCTGCGA TTCATGCATC TGCACTAAAT CAATACCTCC 120TCAATGCCAT TGTACAGATA TCAGGTGGAA CTCGTGTCAC TCGGCTTGCA AATCCTGCAT 180GTGTACACGA TCAATGCCAG GCAAGTGTCG TTGCCTTGAC ATTGCTGATT TCTGTTACAA 240ACCTTGCAAG TCCAGGGATG AAGATGATGA GTAAGAGCTC CC 282 (6) SEQ ID NO:5: (i) sequence signature:

(A) length: 57 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): Ω sequence fragment primer-1 is sequence description (iv): the information of SEQ ID NO:5CCTAGGATCC TATTTTTACA ACAATTACCA ACAACAACAA ACAACAAACA ACATTAC 57 (7) SEQ ID NO:6: (i) sequence signature:

(A) length: 50 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): Ω sequence fragment primer-2 is sequence description (iv): the information of SEQ ID NO:6 GTTGTTTGTT GTAATGTTAA TGATAAATGT TATTGTTACC TGACGTCGGG 50b (8) SEQ ID NO:7: (i) sequence signature:

(A) length: 91 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): synthetic Ω sequence and Kozack fragment be sequence description (iv): the information of SEQ ID NO:7 CCTAGGATCC TATTTTTACA ACAATTACCA ACAACAACAA ACAACAAACA ACATTACAAT TACTATTTAC AATAACAATG GACTGCAGCC C 91bp (9) SEQ ID NO:8: (i) sequence signature:

(A) length: 802 base pairs

(B) type: Nucleotide

(C) chain: two strands

( D ) ： ( ii ) ：DNA ( iii ) ：5’35S ( iv ) ：SEQ ID NO：8CAAGCTTCAT ACAGAGCTCA ATGACAAGAA GAAAATCTTC GTCAACATGG TGGAGCTCTC 60bTTACGAACGA CACGATTGTC TACTCCAAAA ATATCAAAGA TACAGTCTCA GAAGACCAAA 120bGGGCAATTGA GACTTTTCAA CAAAGGGTAA TATCCGGAAA CCTCCTCGGA TTCCATTGCC 180bCAGCTATCTG TCACTTTATT GTGAAGATAG TGGAAAAGGA AGGTGGCTCC TTACAATGCC 240bATCATTGCGA TAAAGGAAAG GCCATCGTTG AAGATGCCTC TGCCGACAGT GGTCCCAAAG 300bATGGACCCCC ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA 360bAGCAAGTGGA TTGATGTGAT ACTCCAAAAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG 420bGGCAATTGAG ACTTTTCAAC AAAGGGTAAT ATCCGGAAAC CTCCTCGGAT TCCATTGCCC 480bAGCTATCTGT CACTTTATTG TGAAGATAGT GGAAAAGGAA GGTGGCTCCT TACAATGCCA 540bTCATTGCGAT AAAGGAAAGG CCATCGTTGA AGATGCCTCT GCCGACAGTG GTCCCAAAGA 600bTGGACCCCCA CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA 660bGCAAGTGGAT TGATGTGATA TCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC 720bTTCGCAAGAC CCTTCCTCTA TATAAGGAAG TTCATTTCAT TTGGAGAGGA CACGCTGAAA 780bTCACCTCTAG AGGATCCCCG GG 802b ( 10 ) SEQ ID NO：9： ( i ) ：

(A) length: 35 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): the different terminator encoding sequence (UT) of reading frame is sequence description (iv): the information of SEQ ID NO:9 CACTCGAGGC TGAATGAGTA AGTGAGTAGG TTAAC 35bp (11) SEQ ID NO:10: (i) sequence signature:

(A) length: 95 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): correctly cut and (iv) sequence description of modification and plant polyadenous glycosides signal peptide sequence: the information of SEQ ID NO:10 GGTTAACTTT GAGTATTATG GCATTGGAAA AGCCATTGTT CTGCTTGTAA TTTACTGTGT TTCTTTCAGT TTTTGTTTTC GGAAATAAAG TTAAC 95bp (12) SEQ ID NO:11: (i) sequence signature:

(A) length: 137 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological structure: linearity, (ii) molecule type: DNA, (iii) out of Memory: polyadenylic acid sequence, (iv) sequence description: SEQ ID NO:11GTTAACAAAA AAAAAAAAAA AAAAAAAAAA ATTTAACAAA AAAAAAAAAA AAAAAAAAAA 60bpAATTTAACAA AAAAAAAAAA AAAAAAAAAA AAATTTAACA AAAAAAAAAA AAAAAAAAAA 120bpAAAATTTAAA AGAGCTC 137bp, (13) information of SEQ ID NO:12:, (i) sequence signature:

(A) length: 261 base pairs

(B) type: Nucleotide

(C) chain: two strands

(D) topological structure: linear (ii) molecule type: DNA (iii) out of Memory: 3 ' non-coding sequence (iv) sequence description: the information of SEQ ID NO:12CACTCGAGGC TGAATGAGTA AGTGAGTAGG TAACTTTGAG TATTATGGCA TTGGAAAAGC 60bpCATTGTTCTG CTTGTAATTT ACTGTGTTCT TTCAGTTTTT GTTTTCGGAA ATAAAGTTAA 120bpCAAAAAAAAA AAAAAAAAAA AAAAAATTTA ACAAAAAAAA AAAAAAAAAA AAAAAAATTT 180bpAACAAAAAAA AAAAAAAAAA AAAAAAATTT AACAAAAAAA AAAAAAAAAA AAAAAAAATT 240bpTAAAAGAGCT C 261bp (14) SEQ ID NO:13:

(i) sequence signature:

(A) length: 34 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): the PCR primer-1 of CpTI gene is sequence description (iv): the information of SEQ ID NO:13 CTACTGCAGC ATGGATTTGA ACCACCTCGG AAGT 34b (15) SEQ ID NO:14:

(i) sequence signature:

(A) length: 34 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): the PCR primer-2 of CpTI gene is sequence description (iv): the information of SEQ ID NO:14 GGGAGCTCTT ACTCATCATC TTCATCCCTG GACT 34b (16) SEQ ID NO:15: (i) sequence signature:

(A) length: 19 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): CpTI sudden change EcoRI primer-1 is sequence description (iv): SEQ ID NO:15

The information of CAGGTGGAAC TCGTGTCAC 19b (17) SEQ ID NO:16: (i) sequence signature:

(A) length: 19 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA is out of Memory (iii): CpTI sudden change EcoRI primer-2 is sequence description (iv): SEQ ID NO:16

The information of GTGACACGAG TTCCACCTG 19b (18) SEQ ID NO:17:

(i) sequence signature:

(A) length: 27 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(iii) out of Memory: the PCR of bacillus thuringiensis insecticidal protein gene detects primer-1

(iv) sequence description: SEQ ID NO:17

The information of GGGCCCGCTG AATCCAACTG GAGAGGC 27b (19) SEQID NO:18:

(i) sequence signature:

(A) length: 31 bases

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(iii) out of Memory: the PCR of bacillus thuringiensis insecticidal protein gene detects primer-2

(iv) sequence description: SEQ ID NO:18

CCATACAACT?GCTTGAGTAA?CCCAGAAGTT?G?31b

Claims (16)

1. be suitable for two valency fusion gene plant expression vectors, wherein comprise at bacillus thuringiensis insect-killing protein of expressing aminoacid sequence shown in SEQ ID NO:1 in the vegetable cell simultaneously and the coding Cowpea Trypsin Inhibitor that the process of aminoacid sequence is modified shown in SEQ ID NO:3:

1). bacillus thuringiensis insecticidal protein gene expression cassette;

2). the cowpea trypsin inhibitor gene expression cassette:

2. bacillus thuringiensis insecticidal protein gene expression cassette according to two valency fusion gene plant expression vectors of claim 1, wherein 1) comprises:

A) .5 ' end non-coding region

B) nucleotide sequence shown in the .SEQ ID NO:2

C) .3 ' end non-coding region

3. cowpea trypsin inhibitor gene expression cassette according to two valency fusion gene plant expression vectors of claim 1, wherein 2) comprises:

A) .5 ' end non-coding region

B) nucleotide sequence shown in the .SEQ ID NO:4

C) .3 ' end non-coding region

4. according to two valency fusion gene plant expression vectors of claim 2 or 3, wherein a) said 5 ' hold non-coding region by two enhancer sequence, a promoter sequence, one is derived from the translation enhancement sequences of plant virus capsid protein plasmagene and the sequence composition of a coding ribophorin.

5. according to two valency fusion gene plant expression vectors of claim 4, the wherein said enhancer element that doubles that has is that CaMV 35S promoter sequence has nucleotide sequence or its functional equivalent sequence as SEQ IDNO:8.

6. according to two valency fusion gene plant expression vectors of claim 4, wherein said translation enhancement sequences is Ω sequence and Kozak sequence, has nucleotide sequence or its functional equivalent sequence as SEQ ID NO:7.

7. said nucleotide sequence has nucleotide sequence or its functional equivalent sequence of the 1-1824 shown in SEQ ID NO:2 according to the bivalent gene combination of plant expression vector of claim 2, b wherein).

8. said nucleotide sequence has 64 to 1824 nucleotide sequence or its functional equivalent sequence as shown in SEQ ID NO:2 according to the bivalent gene combination of plant expression vector of claim 2, b wherein).

9. said nucleotide sequence has nucleotide sequence or its functional equivalent sequence of the 11-271 shown in SEQ ID NO:4 according to the bivalent gene combination of plant expression vector of claim 3, b wherein).

10. said nucleotide sequence has 53 to 271 nucleotide sequence or its functional equivalent sequence as shown in SEQ ID NO:4 according to the bivalent gene combination of plant expression vector of claim 3, b wherein).

11. bivalent gene combination of plant expression vector according to claim 2, c wherein) said 3 ' end non-coding region has nucleotide sequence or its functional equivalent sequence shown in SEQ ID NO:12 in, this sequence comprises a multi-joint terminator sequence, the cutting sequence of a fusion gene transcription product and the job sequence of a fusion gene transcription product, a similar polyadenylation signal sequence and a polyadenylic acid sequence are formed.

12. according to the bivalent gene combination of plant expression vector of claim 3, wherein c) in said 3 ' end non-coding region comprise a multi-joint terminator sequence, have nucleotide sequence or its functional equivalent sequence shown in SEQ IDNO:9; With the cutting sequence of a fusion gene transcription product and the job sequence of a fusion gene transcription product, have nucleotides sequence or its functional equivalent sequence shown in SEQ IDNO:10.

13. be suitable in vegetable cell or whole strain plant, expressing the bivalent gene combination of plant expression vector of bacillus thuringiensis insect-killing protein and Cowpea Trypsin Inhibitor, it has the China Committee for Culture Collection of Microorganisms of being preserved in common micro-organisms center, and preservation registration number is the feature that is carried on the recombinant expression vector in the bacillus coli DH 5 alpha of CGMCC NO.0355.

14. produce to insect toxic and insect this toxicity is difficult to the method for tolerific plant, this method comprises:

1). make up said pair of valency combination of plant expression vector, have as claim 1 to

13 described features,

2). with any feasible method the two valency combination of plant that obtain in the step 1) are expressed

Carrier imports in the vegetable cell, and obtains thus insect is had resistance or kills and wounds

The transgenic plant of ability and offspring thereof comprise the plant of seed and any part

Tissue.

15. according to the method for claim 14, wherein said plant is any plant that can express two valency combination of plant expression vectors of claim 1.

16. according to the method for claim 14, wherein said plant is a cotton.

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