CN102010873B - Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof - Google Patents

Abstract

The invention provides an insect-resistant gene Cry1Ab-t and an encoding protein thereof. In the gene, by using a plant bias codon and reducing a sequence rich in AT, an inverted repeat sequence and a subsequence-containing undefined eucaryotic deoxyribose nucleic acid (DNA) sequence in the original DNA sequence on the premise of keeping part of amino acid sequences of a Bacillus thuringHansis (Bt) protein Cry1Ab unchanged, a new Cry1Ab-t gene sequence is synthesized by artificial modification, then a prokaryotic expression carrier and a plant expression carrier are constructed for the gene, and a corresponding host cell is converted. In vitro experiments prove that a synthesized Bt toxoprotein has an obvious insecticidal effect on corn borers. The insect-resistant gene Cry1Ab-t and a target protein expressed by the gene can be steadily and efficiently expressed in corns, so that the gene can be used for culturing Cry1Ab-t transgene insect-resistant corns. The artificially synthesized Bt insect-resistant gene Cry1Ab-t can be also used for improving the insect resistance of other crops, fruit trees, vegetables and the like.

Description

Synthetic Bt anti insect gene Cry1Ab-t and application thereof

Technical field

The present invention relates to genetically engineered and biological control field, be specifically related to a kind of synthetic Bt anti insect gene Cry1Ab-tAnd use.

Background technology

Corn is an important crops; Be again important feed and industrial raw material; Current corn insect pest (is main with Pyrausta nubilalis (Hubern).) is serious, causes a large amount of underproduction of corn, therefore adopt an effective measure its harm of control to improve corn yield, increasing farmers' income has great importance.Because lack suitable pest-resistant cultivar, the main method that solves insect pest at present is in process of growth, to spray chemical insecticide; But chemical insecticide kills off the insect pests and natural enemy simultaneously, causes ecological imbalance and environmental pollution.Can the Bt anti insect gene be imported in the corn variety through transgenic technology, and then improve the insect-resistance of transgenic corns, reduce the usage quantity of agricultural chemicals, save human and material resources and social resources.

Bt genes encoding insecticidal crystal protein, from bacillus thuringiensis ( Bacillus thuringHansis), be the Gram-positive edaphic bacillus.It produces the desinsection parasporal crystal albumen (the synthetic this proteinic gene of control is on plasmid) that is called delta-endotoxin in the gemma forming process, these albumen have very high insecticidal activity.Its action principle is that this pest-resistant albumen can be hydrolyzed to littler active toxin fragment by alkaline intestinal juice dissolving---core fragment ( Hofte and Whiteley, 1989).The further hydrolysis of active fragments ability protease inhibitor, the protein bound that is activated are on the brush shape vesicle on the insect gut, thereby causing to bore a hole influences osmotic equilibrium, cell expansion and dissolving, and it is also dead at last that target organisms stops to get food.Research show many Bt albumen, the intestinal epithelial cell of target pest all have the binding site of high-affinity ( Hofte and Whiteley, 1989) and in the past decades, confirmed the insecticidal crystal protein of their codings of tens of kinds of bacillus thuringiensis fungus strains and kind more than 130.The research proof; The Bt crystallin is to human body, Mammals, birds, fish and the equal toxicological harmless effect of a lot of useful insect; Free from environmental pollution, so the Bt preparation has been used nearly 50 years as a kind of non-harmful natural microbial sterilant at aspects such as agricultural, forestry and environmental healths.The Bt crystal proteins must be by insect's food-taking to the competence exertion insecticidal function; But Bt crystal proteins poor stability under the physical environment; Insecticidal effect receives weather effect big, then is prone to degraded after the sun shines, and can not be penetrated into plant tissue inside; Be prone to washed away by rainwater, dew, these factors have limited its development and application greatly.

1987, people such as Belgian Vaeck obtained to change the pest-resistant tobacco of Bt insecticidal proteins first, and the transgene tobacco of acquisition can only detect faint insect-resistance, its expressing protein almost detect less than, only account for 0.001% of soluble proteins.In China, 1992, people such as Guo's three heaps adopted plant optimizing codon method synthetic total length 1824bp at first at home GFMCry1AKilling gene has obtained Chinese first-generation unit price Insect Resistant Cotton; (1995) such as fourth groups of stars etc. (1993) and kingdom's English change the pB48.415 plasmid over to ovary injection and particle bombardment respectively and obtain transfer-gen plant in the maize calli, and insect-resistance is measured and shown that it has certain insect-resistance; China Agricultural University in 1994 at home and abroad first with the method for ovary injection anti insect gene BtChange corn over to and obtain transfer-gen plant; The Zhou Fengyong of the Chinese Academy of Agricultural Sciences etc. will in 1998 GFMKilling gene is building up on the pMG6 plasmid, imports in the corn, and the offspring detects and shows that the Bt gene arrives (LIU YJ etc., 2003) of future generation with mendelian inheritance pattern heredity.Current, the whole world has changed into Bt transgenic plant more than 26 kinds, comprises staple crops such as cotton, corn, paddy rice.Isolate although transgenic technology can be broken reproduction, realize the transgenosis between different plant species.But the gene that in a certain species, can efficiently express, be transformed into another species after, expression amount is not necessarily high, also may not necessarily have its original function, especially for the species that differ greatly.Wherein, the species codon preference is one of many factors that influence the exogenous gene expression amount.Different species often have different preference codons.Analyze this preference property, before transgenic, the goal gene that needs are transformed carries out the design again or the transformation of codon, has vital role for improving the expression amount of foreign gene in receptor biological.

Nineteen eighty-three, Washington, DC university announced successfully kalamycin resistance gene to be imported tobacco cell, and April in the same year, Univ Wisconsin-Madison USA announced successfully to change soybean gene over to birth that Sunflower Receptacle indicates plant transgenic technology jointly.1986, first batch of transgenic plant (pest-resistant, antiweed) went through to get into field test.Mammals heavy chain of antibody in 1989 and light chain gene successful expression and correctly be assembled into the antibody of function in tobacco.Nineteen ninety Gordon-Kamm reported first has obtained the transformant that can educate with particle gun maize transformation suspension cell line.Subsequently, corn gene technology beginning fast development, a large amount of transgenic plant are developed success successively.KozHal (1993) etc. has cultivated the insect-resistant transgenic corn, and transfer-gen plant can high level expression CryIA (b)Anti insect gene.Zhang Xiujun (1999) etc. contain the embryo callus that the lysine-rich protein plasmagene imports corn with particle bombardment with two.Liu Dawen (2000) etc. will Zm13-BarnaseGene transformation maize callus obtains positive plant through herbicide screening.Ishida (1996) etc. utilize ultra binary vector to set up the rataria of agrobacterium tumefaciens maize transformation self-mating system first, and Frame (2002) etc. has realized that successfully agrobacterium tumefaciens utilizes common binary vector to transform rataria.Zhang Yanzhen (2002) etc. import the superior corn self-mating system to agrobacterium tumefaciens-mediated transformation with the Bt killing gene and have carried out comparatively systematic research, and Huang (2005), Frame (2006) and Lee (2007) etc. utilize Agrobacterium successfully to transform the ordinary maize self-mating system respectively.

China had set up more sophisticated corn gene technical system already aspect transgenic technology research.As China Agricultural University at home early carried out the work that maize genetic transforms, in 1994 at home and abroad first with the method for ovary injection anti insect gene BtChange corn over to and obtain transfer-gen plant, with the independent intellectual property right gene BtFor the pest-resistant corn of representative has been showed the excellent development prospect.Simultaneously, had stronger innovation ability in marker-free, selectable marker gene deletion and core technology innovation aspect such as the target gene product is regularly degraded, plant tissue specificity predominant expression.Isolating at present anti insect gene is a lot, mainly comprises: the 1. anti insect gene of bacterial origin, mainly be the Bt toxoprotein gene ( Cry1Ab, Cry1Ac, Cry2, Cry3Deng); 2. the protease inhibitor gene of plant origin, characteristics are that pest-resistant spectrum is wide, derive from plant itself and are easy to accepted by the public; 3. the nutritive insecticidal albumen of bacterial origin (Vip1, Vip2, Vip3 etc.), the anti-lepidopteran of wide spectrum particularly has black cutworm, mythimna separata and beet leaves moth and pretends usefulness.Because the influence of gene pest-resistant ability and insect tolerance, transgenic pest-resistant product mainly obtain through obtaining approach such as more how more effective anti insect gene, the existing gene of transformation, two anti-plant materialss.

Cry1Ab albumen is one type of parasporal crystal that lepidoptera pest is had toxic action that is produced by bacillus thuringiensis, and it has important application prospects in the biological control field.Because the Cry1Ab gene source is in bacillus thuringiensis, it is applied directly to change base low with having expression amount after in the plant, expression product is unstable, problems such as insect-resistance weak effect.The codon service condition of research bacillus thuringiensis and corn; Can find that there is very big-difference in both on codon preference; Utilize the preference codon of plant, design, transform Cry1Ab again, improve the expression amount of insecticidal proteins; And then cultivate novel pest-resistant transgenic corns, for the corn safety in production provides strong guarantee.

Summary of the invention

The technical problem that the present invention will solve provide a kind of can be in crop stably express and expression amount is high, pest-resistant effective synthetic anti insect gene Cry1Ab-tAnd proteins encoded, and be applied to the aspects such as conversion structure of carrier, host cell.

Another object of the present invention provides anti insect gene Cry1Ab-tApplication in improving the transgenic plant insect-resistance.

For solving the problems of the technologies described above, the present invention realizes through following technical scheme:

Cry1Ab-tGene is in wild-type Cry1AbTransform on the basis; Only stay one section base sequence SEQ ID No.2 of the N end 1845bp of excalation; And form under the overall constant situation at the amino acid that keeps this section sequence; With the corresponding codon of codon constant series SEQ ID No.2 of plant-preference, get rid of exist in the dna sequence dna cause the unsettled AT of being rich in sequence of plant transcription and restriction enzyme site commonly used (SacI), the method through permutation cipher corrects elimination then; And add terminator codon TAG at 3 ends, obtain the dna sequence dna of a transformation; Confirm the encoding sequence of Bt gene shown in sequence table SEQ .ID NO:1 that also chemosynthesis is transformed, its corresponding amino acid sequence is shown in SEQ ID NO:3; According to the needs of gene function analysis, further right Cry1Ab-tGene is cloned, and is adding that at the sequence two ends restriction enzyme enzyme recognition site sequence is so that be building up on the corresponding expression vector.

Gene of the present invention is operably connected with prokaryotic expression carrier, fast Preliminary detection synthetic Bt of the present invention anti insect gene Cry1Ab-tExpression product is to the toxicity of Pyrausta nubilalis (Hubern)..Gene of the present invention is operably connected with plant expression vector, expression vector is imported (LBA4404) in the corresponding Agrobacterium, and carry out the genetic transformation of maize calli and rataria, obtain changeing synthetic through agrobacterium-mediated transformation Cry1Ab-tThe corn transformant of gene is cultivated the insect-resistant transgenic corn.Also can carry out genetic transformation, make it possess anti-insect activity other farm crop or fruit tree etc.

Those skilled in the art can with farm crop such as its maize transformation, cotton, paddy rice, vegetables, make it possess corresponding anti-insect activity according to gene disclosed by the invention.

Those skilled in the art can also produce Bt albumen through large scale fermentation with gene transformation bacterium of the present invention or fungi, and it is prepared into sterilant, are used for the control of crop pests.

The present invention has actively useful effect:

Anti insect gene of the present invention Cry1Ab-tSequence and original Cry1AbSequence relatively, improved gene design disappearance and dna sequence dna change, strengthened its expression in plant greatly.Use plant-preference property codon, reduced the inverted repeats that is rich in AT sequence and existence and indefinite eukaryotic DNA sequence intron sequences in the original DNA sequence.For improved Cry1Ab-tGene in 1845 bases, has changed 1221 bases, thereby has made G+C content for to bring up to 62.7% by 37.3%. Cry1Ab-tThe homology of gene and original DNA sequence is 66.2%.Anti insect gene of the present invention Cry1Ab-tCan be in vegetable cell the expression of efficient stable.

Synthetic is made in this person's wage reform Cry1Ab-tAfter gene imports corn through means such as agriculture bacillus mediated, ultrasonication and particle gun conversions, can obtain the pest-resistant Cry1Ab-t transformant of genetic stability.Simultaneously also can be this Bt anti insect gene Cry1Ab-tBe transformed in the farm crop such as cotton, paddy rice, vegetables, make it possess corresponding anti-insect activity, thereby reduce the usage quantity of agricultural chemicals,, have important economic value and wide application prospect to reduce environmental pollution.

Description of drawings

Fig. 1 is new modifying gene Cry1Ab-tSynthetic, identify collection of illustrative plates, wherein A, B do Cry1Ab-tThe salvage collection of illustrative plates of gene, C are final synthetic Cry1Ab-tGene mapping, D are prokaryotic vector pET28b- Cry1Ab-t(NdeI and HindIII) enzyme is cut and is identified collection of illustrative plates).

Fig. 2 is plant expression vector figure: pCAMBIA1300-35S-MCS- Bar

Fig. 3 is pCAMBIA1300- Cry1Ab-t- BarMake up collection of illustrative plates.

Fig. 4 is for transforming anti insect gene through agrobacterium-mediated transformation Cry1Ab-tThe screening of back callus (left side), regeneration (in) and transfer-gen plant be transplanted to flowerpot (right side); In the screening of corn resistant calli and the regenerative process of resistant plant; Substratum contains high-caliber selective reagents-bialaphos; Have only transformant to grow with this understanding; The growth of non-transformed cell has received complete inhibition, and is all dead with this understanding as the non-transformed calli and the plant of control group.

Fig. 5 is T ₀For the transformant goal gene Cry1Ab-tPCR detect, M:DL2000 plus is CK1 wherein: the positive plasmid contrast, CK2: non-transgenic seedling negative control, blank: the distilled water contrast, 1-6 is Cry1Ab-t1Arrive Cry1Ab-t6.

Fig. 6 is T ₀For the detection collection of illustrative plates (arrow is designated as target protein among the figure) of transformant target protein Cry1Ab-t, CK1: transgenic seedling negative control not; 1-6 is Cry1Ab-t1Arrive Cry1Ab-t6.

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.TP among the following embodiment if no special instructions, is ordinary method.Used test materials and reagent among the following embodiment, if no special instructions, all available from routine biochemistry reagent company.

Embodiment 1 Cry1Ab-tThe design of gene and synthetic

Cry1Ab-tGene is at the Bt gene Cry1AbThe basis of parent nucleotide sequence on; Stay one section base sequence SEQ ID No.2 of the N end 1845bp of excalation; And keeping under the overall constant situation of reservation queue amino acid composition; The preferences codon of employing plant gene is replaced the corresponding codon of original Bt; ATTTA, AATGAA etc. are rich in AT sequence and indefinite intron sequences in the elimination Bt gene, and reject big inverted repeats and the restriction enzyme enzyme recognition site sequence commonly used that exists in the gene, and the encoding sequence of designing target synthetic Bt gene is shown in sequence table SEQ ID No.1.

Original Bt anti insect gene Cry1Ab and the new Bt gene C of synthetic ry1Ab-t nucleotide coding sequence are carried out the discovery of Blast2 compare of analysis: 5 ' the end 1845bp nucleotide sequence of reservation and the new Bt gene nucleotide series homology of transformation are 66.2%.The statistics of based composition is: for improved Cry1Ab-tGene in 1845 bases, has changed 1221 bases, thereby has made G+C content for to bring up to 62.7% by 37.3%.

Transform synthetic new Bt anti insect gene Cry1Ab-tEntrust Shanghai biotechnology ltd (in June, 2010) synthetic.

Agents useful for same: Shanghai biotechnology ltd.

The concrete grammar step is following;

(1) synthetic as required with DNA work software Cry1Ab-tGene order design primer (oligonucleotide strand) 58, every 35-45bp, every treaty 33ug is about 1OD.

(2) PCR connection primer is a double-stranded DNA, two-wheeled PCR.

First round PCR: Cry1Ab-tDividing three sections increases

1-10 primer 9-23 primer 33-58 primer

The first round: reaction system

Primer 1 1ul (1 OD/400ul H ₂O) about 6 nmol

Primer 2 1ul (1 OD/400ul H ₂O) about 6 nmol

dNTP 1ul（10mM）

Pfu 10×Buffer 5ul (200mM TrisHCl,pH8.8；100mM KCl；20mM MgSO ₄；160mM (NH ₄) ₂HSO ₄；1％Triton and 1mg/mlBSA)

Template (primer 1-10 or 95-23 or 33-28) 26 * 0.2ul; 28 * 0.2ul; 24 * 0.2ul

Pfu enzyme 0.25 (5U/ul)

Add water to 50ul

Reaction parameter: 95 ℃ of 3min; 94 ℃ of 1min, 55 ℃ of 45S, 72 ℃ of 1min, 20 circulations; 72 ℃ are extended 2min

Second takes turns: reaction system

Primer 1 1ul (1 OD/400ul H ₂O) about 6 nmol

Primer 2 1ul (1 OD/400ul H ₂O) about 6 nmol

dNTP 1ul（10mM）

Pfu 10×Buffer5ul (200mM Tris-HCl，pH8.8；100mM KCl；20mM MgSO4；160mM (NH ₄) ₂HSO ₄；1％Triton and 1mg/mlBSA)

Template first round reaction solution 1-26,25-52, each 2ul of 51-48

Pfu enzyme 0.25 (5U/ul)

Add water to 50ul

(above agents useful for same is Shanghai biotechnology ltd and produces)

Reaction parameter: 95 ℃ of 3min; 94 ℃ of 1min, 59 ℃ of 45S, 72 ℃ of 1min, 22 circulations; 2 ℃ are extended 3min

(3) 1% agarose gel electrophoresis, visible three sections specific bands clearly, size fulfills the expectation and closes.

(4) reclaim test kit with gel and reclaim purified pcr product.

(5) be the clone to three sections PCR products respectively, propose plasmid, enzyme is cut and is detected and order-checking.Do stencil design total length PCR scheme obtaining three correct plasmids then, carry out that pcr amplification just newly transformed Cry1Ab-tGene, full length gene 1848bp, gene order is shown in sequence table SEQ ID No.1.

(6) and then right Cry1Ab-tFull length gene PCR product is done cloning and sequencing work, obtains a flush end then and is cloned into the plasmid on the pUC57, and this plasmid contains the Cry1Ab-t gene of synthetic, called after pUC1Ab-t carrier.

The expression of embodiment 2 Cry1Ab-t genes in Bacillus coli cells and the toxicity detection of expression product

Be the toxicity situation of the new pest-resistant PROTEIN C ry1Ab-t of Bt that transforms of rapid detection to Pyrausta nubilalis (Hubern)., we have made up Cry1Ab-t Prokaryotic Expression carrier, and pest-resistant PROTEIN C ry1Ab-t toxicity is carried out vitro detection.

Plasmid pUC1Ab-t is provided by Shanghai bio-engineering corporation, contains Cry1Ab-tGene.According to structure Cry1Ab-tThe needs of gene prokaryotic carrier are in 5 ' end interpolation of primer sequence NdeIRestriction endonuclease recognition site sequence C ATATG, 3 ends add HindIIIRestriction endonuclease recognition site sequence A AGCTT.The design primer sequence is (like SEQ ID NO:4, shown in 5):

Upstream primer F1:5-CATATGGACAACAACCCAAACATC-3

Downstream primer R1:5-AAGCTTCTAGTACTCAGCCTCGAATG-3

Prokaryotic expression carrier is the pET-28b of Novgen company, and its promotor is T7 lac; Used expression strain is BL21 (DE3), and this bacterial strain also is applicable to the expression vector of other band T7 lac promotor.The carrier construction method flow process is following:

The first step: pcr amplification Cry1Ab-tGene is with pUC 1Ab-yPlasmid is a template, is primer with F1 and R1, and 5 ' end adds NdeIThe restriction endonuclease recognition site, 3 ' end adds HindIIIThe restriction endonuclease recognition site.Reclaim test kit with gel and reclaim purifying Cry1Ab-tGene fragment;

Second step: use restriction enzyme NdeIWith HindIIIEnzyme is cut pET28b, and gel reclaims test kit and reclaims purifying 5.3kb fragment;

The 3rd step: the fragment behind two purifying is carried out ligation, make up gained prokaryotic expression plasmid called after pET28b 1Ab-t, carry out enzyme with restriction enzyme and cut evaluation, show the correct (see figure 1) of vector construction;

The 4th step: with the pET28b that builds 1Ab-tPlasmid transforms BL21 (DE3) competent cell, and does not have to change over to that to insert the BL21 that pulsating expression vector plasmid pET28b transforms be control strain.

With containing pET 1Ab-tE. coli bl21 bacterium liquid after IPEG induces, extract Bt albumen, be contrast with clear water with containing empty carrier pET28b e. coli bl21 bacterium liquid, carry out the worm examination with Pyrausta nubilalis (Hubern)., concrete steps are following:

1. choose mono-clonal from flat board and be seeded in the LB liquid nutrient medium that 5ml contains 50mg/ml Kan 37 ^oC shaking table overnight cultures;

2. the bacterium that will spend the night is inoculated in the 5ml LB liquid nutrient medium 37 with the ratio of 1:100 ^oThe C shaking table is cultured to OD ₆₀₀Reach 0.4-1;

3. add IPTG to final concentration be 0.5mM, cultivated 4 hours;

4. the centrifugal 10min of 4000rpm collects thalline;

5. add 36ml lysis buffer (2mM Tris-HCl; 0.2mM CaCl ₂PH=8.0), the thalline that suspends again adds N,O-Diacetylmuramidase to final concentration 1mg/ml, places 30min on ice;

6. ultrasonic disruption thalline (broken parameter: ultrasonic 1 second, 2 seconds at interval), the centrifugal 10min of 4000rpm collects supernatant;

7. the supernatant of collecting is joined in the feed that oneself disposes.To contain empty carrier pET28b e. coli bl21 bacterium liquid is contrast, carries out the worm examination with Pyrausta nubilalis (Hubern).: in each test tube, put into a feed, and incubate the Pyrausta nubilalis (Hubern). of not taking food at the beginning of inserting 10, respectively connect 10 test tubes.Put into 26～28 ℃ of temperature, cultivate in the environment of relative humidity about 70%, carry out toxicity and identify.Test-results shows: artificial reconstructed synthetic Cry1Ab-tAlbumen has insecticidal effect preferably, and the mortality ratio of Pyrausta nubilalis (Hubern). reaches 86.63%, and the growth of Pyrausta nubilalis (Hubern). is had the obvious suppression effect, and single the worm of worm that lives is heavy only to have 0.1610 (seeing table 1).

Table 1 Cry1Ab-tThe toxicity of gene prokaryotic product is identified

Handle (three repetitions)	Examination borer population (only)	Average mortality (%)	Single worm of worm heavy (mg) lives
				Clear water	100	0	2.5646
Bare pET28b	100	23.26	2.5419
				Cry1Ab-t	100	86.63	0.1610

Embodiment 3 plant transgene vector construction and Agrobacterium-mediated Transformation

The first step: use XbaI, SacIEnzyme is cut T vector plasmid pUC Cry1Ab-t, reclaim purification kit with gel and reclaim Cry1Ab-tFragment.

Second step: use XbaI, SacIThe enzyme expression vector CPB that cuts plant reclaims purification kit with gel and reclaims the CPB fragment after enzyme is cut.

The 3rd step: two fragments behind the purifying are carried out ligation, make up plant expression vector pCAMBIA1300- Cry1Ab-t- Bar(referring to Fig. 2), enzyme carry out enzyme and cut evaluation, show the correct (see figure 3) of vector construction.

Adopt agriculture bacillus mediated genetic transforming method (bacterial classification LBA4404) to carry out the conversion of maize calli and rataria, will Cry1Ab-tIn the gene corn genome, after the screening of weedicide bialaphos, obtain transfer-gen plant,, when transformed plant grows the 7-8 leaf, get blade and extract DNA, goal gene through carrying out the transplanting (see figure 4) of transgenic corns behind screening, regeneration, the refining seedling Cry1Ab-tEmploying round pcr Preliminary detection foreign gene ( Cry1Ab-t) insertion situation (see figure 5).

Primer sequence (like SEQ ID NO:6, shown in 7):

Cry1Ab-t: 5'-CAACGAGTGCATCCCTTACAAC-3'，

Cry1Ab-t: 5'-CCGACGGGGCTGGCCATGATC-3'，

Purpose clip size: 960bp annealing temperature 58 degree, 35 circulations.

Detect with Bt-Cry1Ab/1Ac immunodiagnosis test strip and (to purchase true Nice, Beijing Bioisystech Co., Ltd; Article No.: STX06200/0050), the result shows that target protein obtains efficiently expressing (see figure 6) in transgenic corns.

This carrier contains Cry1Ab-tGene is two valency carriers, and directly maize transformation utilizes codon optimized principle reforming composite Cry1Ab-tGene adopts the CaMV35S promotor, is responsible for startup Cry1Ab-tGenetic expression.Plasmid pCAMBIA1300-35S-MCS-Bar (CPB) is for Shandong University provides, and is that the plasmid that on pCAMBIA1300 plasmid basis, makes up contains a goal gene and inserts box (35S-polyclone restriction enzyme site-Tnos) and the anti-herbicide gene from streptomyces hygroscopicus BarSelect marker gene BarGene (bialaphos resistance gene), long 552bp, 217 amino acid of encoding.Can make the plant opposing is weedicide (De-Block etc., 1987 of activeconstituents with L-Phosphioth ricin (PPT, γ-hydroxymethyl phosphinidene-α-tyrosine, phosphine Wheat flavone); Wohlleben etc., 1988), like weedicide Bialaphos (bialaphos) and Glufosinate (careless fourth phosphine). BarGene is the (Wohlleben etc. that from the streptomyces hygroscopicus (Streptomyces hygroscopicus) of synthetic Bialaphos, separate; 1988); Be the protection gene that S.Hygroscopicus avoids self product B ialaphos to poison, coding PPT Transacetylase (PAT).The ethanoyl of PAT catalysis acetyl-CoA is transferred on the amino of PPT, forms acetyl PPT, and makes the PPT inactivation.

At present, the report that also people, animal and plant is not had disadvantageous effect about said gene and coded product thereof.Therefore, the Bt anti insect gene of this transformation Cry1Ab-MaIn improving the transgenic corns insect-resistance, can well be used.Also can use and other farm crop, fruit tree or vegetables, like corn, paddy rice, yam, cotton etc.

Sequence table

SEQUENCE LISTING

< 110>Henan Academy of Agricultural Sciences

< 120>synthetic Bt anti insect gene Cry1Ab-t and application thereof

< 130>research of transgenic Bt corn

<160> 7

<170> PatentIn version 3.2

<210> 1

<211> 1848

<212> DNA

< 213>synthetic

<400> 1

atggacaaca acccaaacat caacgagtgc atcccttaca actgtctgag caaccctgag 60

gtggaggtgc tgggcggtga gcgtatcgag actggctaca ccccgatcga tatcagcctc 120

agcctgaccc agttcctcct gtccgaattc gtcccgggcg ccggcttcgt gctgggcctg 180

gtcgacatca tctggggcat cttcggcccc agccagtggg acgcgttcct cgtccagatc 240

gagcagctga tcaaccagcg catcgaggag ttcgctcgca accaggcgat cagccgcctg 300

gagggcctgt ccaacctgta ccagatctac gcggaatcct tccgcgagtg ggaggcggac 360

cccaccaacc ccgccctgag ggaagagatg cgcatccagt tcaacgacat gaactccgcg 420

ctgacgaccg ccatccccct gttcgccgtg cagaattacc aggtgcccct gctgagcgtc 480

tatgtgcagg ccgccaacct gcatctgtcg gtcctccgcg acgtcagcgt cttcggccag 540

cgctggggct tcgacgccgc cacgatcaac tctcgctaca atgatctgac ccgcctgatc 600

ggcaactaca ctgaccacgc cgtgcgctgg tacaacaccg gtctggaacg cgtgtggggt 660

cccgacagcc gcgactggat caggtacaac caattccgcc gcgagctgac gctgactgtg 720

ctcgatatcg tcagcctgtt ccccaactac gacagccgca cataccccat ccgcaccgtg 780

agccagctga cgcgcgagat ttacaccaac cccgtgctgg agaacttcga cggctccttc 840

cgcggcagcg cgcagggcat cgaggggagc atccgctccc cccacctgat ggacatcctc 900

aactcgatca ccatctacac ggacgcccac cgcggcgagt actattggag cggccaccag 960

atcatggcca gccccgtcgg cttctcgggc cctgagttca cgttccccct gtacggcacc 1020

atgggcaacg ctgcacctca gcagcgcatc gtggcacagc tgggccaggg agtgtaccgc 1080

acgctcagca gcacgctgta ccgtcgtcct ttcaacatag gcatcaacaa ccaacagctg 1140

agcgtgctgg atggcaccga gttcgcgtat ggcacgagca gcaacctgcc cagcgccgtg 1200

taccgcaagt cgggcacggt ggacagcctg gatgagatcc cccctcagaa caacaacgtc 1260

ccgcctcgac agggcttcag ccatcgtctg tcgcatgtca gcatgttccg cagtggcttc 1320

agcaactcca gcgtctcgat catccgtgca ccgatgttca gctggatcca ccgctcggcc 1380

gaattcaaca acatcatccc cagcagccag atcacgcaga tccccctgac gaagagcacg 1440

aacctgggca gcggcaccag cgtggtcaag ggccccgggt tcaccggcgg cgacatcctg 1500

cgccgcacca gccccgggca gatctcgaca ttgcgcgtga acatcaccgc ccccctgagc 1560

cagcgctacc gcgtccgcat ccgctacgcc agcacgacca acctgcagtt ccacaccagc 1620

atcgacggcc gccccatcaa ccagggcaac ttcagcgcca cgatgagttc ggggtcgaac 1680

ctgcagagcg gttcgttccg cacggtaggc ttcaccaccc ccttcaactt cagcaatggc 1740

agctcggtgt tcacgctgag cgcccacgtc ttcaacagcg gcaacgaggt gtacatcgat 1800

cgcatcgagt tcgtccccgc ggaggttaca ttcgaggctg agtactag 1848

<210> 2

<211> 1845

<212> DNA

<213> Bacillus thuringHansis

<400> 2

atggataaca atccgaacat caatgaatgc attccttata attgtttaag taaccctgaa 60

gtagaagtat taggtggaga aagaatagaa actggttaca ccccaatcga tatttccttg 120

tcgctaacgc aatttctttt gagtgaattt gttcccggtg ctggatttgt gttaggacta 180

gttgatataa tatggggaat ttttggtccc tctcaatggg acgcatttct tgtacaaatt 240

gaacagttaa ttaaccaaag aatagaagaa ttcgctagga accaagccat ttctagatta 300

gaaggactaa gcaatcttta tcaaatttac gcagaatctt ttagagagtg ggaagcagat 360

cctactaatc cagcattaag agaagagatg cgtattcaat tcaatgacat gaacagtgcc 420

cttacaaccg ctattcctct ttttgcagtt caaaattatc aagttcctct tttatcagta 480

tatgttcaag ctgcaaattt acatttatca gttttgagag atgtttcagt gtttggacaa 540

aggtggggat ttgatgccgc gactatcaat agtcgttata atgatttaac taggcttatt 600

ggcaactata cagatcatgc tgtacgctgg tacaatacgg gattagagcg tgtatgggga 660

ccggattcta gagattggat aagatataat caatttagaa gagaattaac actaactgta 720

ttagatactg tttctctatt tccgaactat gatagtagaa cgtatccaat tcgaacagtt 780

tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt 840

cgaggctcgg ctcagggcat agaaggaagt attaggagtc cacatttgat ggatatactt 900

aacagtataa ccatctatac ggatgctcat agaggagaat attattggtc agggcatcaa 960

ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct atatggaact 1020

atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cgtgtataga 1080

acattatcgt ccactttata tagaagacct tttaatatag ggataaataa tcaacaacta 1140

tctgttcttg acgagacaga atttgcttat ggaacctcct caaatttgcc atccgctgta 1200

tacagaaaaa gcggaacggt agattcgctg gatgaaatac cgccacagaa taacaacgtg 1260

ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg ttcaggcttt 1320

agtaatagta gtgtaagtat aataagagct cctatgttct cttggataca tcgtagcgct 1380

gaatttaata atataattcc ttcatcacaa attacacaaa tacctttaac aaaatctact 1440

aatcttggct ctggaacttc tgtcgttaaa ggaccaggat ttacaggagg agatattctt 1500

cgaagaactt cacctggcca gatttcaacc ttaagagtaa atattactgc accattatca 1560

caaagatatc gggtaagaat tcgctacgct tctaccacaa atttacaatt ccatacatca 1620

attgacggaa gacctattaa tcaggggaat ttttcagcaa ctatgagtag tgggagtaat 1680

ctacagtccg gaagctttag gactgtaggt tttactactc cgtttaactt ttcaaatgga 1740

tcaagtgtat ttacgttaag tgctcatgtc ttcaattcag gcaatgaagt ttatatagat 1800

cgaattgaat ttgttccggc agaagtaacc tttgaggcag aatat 1845

<210> 3

<211> 615

<212> PRT

< 213>synthetic

<400> 3

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly

20 25 30

Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser

35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile

50 55 60

Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile

65 70 75 80

Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala

85 90 95

Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala

130 135 140

Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val

145 150 155 160

Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser

165 170 175

Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg

180 185 190

Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val

195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

210 215 220

Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val

225 230 235 240

Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro

245 250 255

Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val

260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu

275 280 285

Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr

290 295 300

Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln

305 310 315 320

Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro

325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala

340 345 350

Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg

355 360 365

Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp

370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val

385 390 395 400

Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln

405 410 415

Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His

420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile

435 440 445

Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn

450 455 460

Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr

465 470 475 480

Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly

485 490 495

Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg

500 505 510

Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg

515 520 525

Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg

530 535 540

Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn

545 550 555 560

Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn

565 570 575

Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn

580 585 590

Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu

595 600 605

Val Thr Phe Glu Ala Glu Tyr

610 615

<210> 4

<211> 24

<212> DNA

< 213>artificial sequence

<400> 4

catatggaca acaacccaaa catc 24

<210> 5

<211> 26

<212> DNA

< 213>artificial sequence

<400> 5

aagcttctag tactcagcct cgaatg 26

<210> 6

<211> 22

<212> DNA

< 213>artificial sequence

<400> 6

caacgagtgc atcccttaca ac 22

<210> 7

<211> 21

<212> DNA

< 213>artificial sequence

<400> 7

ccgacggggc tggccatgat c 21

Claims (9)

1. synthetic Bt anti insect gene Cry1Ab-t, its nucleotide sequence is shown in SEQ ID NO:1.

2. according to the said synthetic Bt of claim 1 anti insect gene Cry1Ab-t, its aminoacid sequence is shown in SEQ ID NO:3.

3. one kind contains claim 1 or 2 said synthetic Bt anti insect genes Cry1Ab-tProkaryotic expression carrier.

4. one kind by the said prokaryotic expression carrier transformed host cells of claim 3 BL21 (DE3).

5. one kind by the said synthetic Bt of claim 1 anti insect gene Cry1Ab-tThe insecticidal proteins of coding.

6. sterilant that contains the said insecticidal proteins of claim 5.

7. one kind contains claim 1 or 2 said synthetic Bt anti insect genes Cry1Ab-tPlant expression vector.

8. one kind by the said plant expression vector transformed host cells of claim 7 LBA4404.

9. claim 1 or 2 said synthetic Bt anti insect genes Cry1Ab-tOr the said prokaryotic expression carrier of claim 3 carries out the method that maize genetic transforms through agriculture bacillus mediated method.

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