CN104059135A - Bt protein as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses a Bt protein as well as a coding gene and application thereof. An amino acid sequence of the Bt protein is as shown in SEQ ID No.1, and a base sequence of the coding gene of the Bt protein is as shown in SEQ ID No.2. The application refers to application of the Bt protein in improving insect resistance of plants. Expression activity of an mBt gene of mBT transgenic rice obtained by transferring rice through the coding gene is 20%-50% stronger than that of a wBT gene; resistance on lepidopteron ice leaf folder and striped rice borer is also greatly improved as follows: the rice leaf folder can form a certain withered and yellow hazard symptoms on leaves of the wBt gene ruce, but the leaves of the mBt transgenic gene rice are not formed or only formed with withered and yellow hazard symptoms or only extremely small withered and yellow symptoms are formed near a shear mouth; on the wBt transgenic rice, a white head rate caused by striped rice borer is 1.1 +/-0.1%, and the white head rate on the mBt transgenic rice is 0.

Description

A kind of Bt albumen and encoding gene and application

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of Bt albumen and encoding gene and application.

Background technology

The lepidoptera pests such as the snout moth's larva of rice are one of important restriction factors of paddy rice high and stable yield.Up to now, not yet find any Rice Resources lepidoptera pests such as rice borer to resistance.Past, the control of rice borer mainly be take and sprayed chemical pesticide as main, but a large amount of residual toxicitys not only causes serious environmental pollution, and the health quality of rice is declined, affect the healthy of people.As Rice Production big country, lepidoptera pest has caused massive losses to every year China's Rice Production.

Bt albumen is that bacillus thuringiensis (Bacillus thuringiensis) produces at it a kind of crystal proteins producing in born of the same parents' process, has the narrow spectrum toxic action of lepidopterous insects.The eighties, the gene of first coding Bt albumen is cloned, and Bt albumen has efficient and single-minded insecticidal activity to target pest, be easy to degrade in environment, and without any residual, these advantages is all that general chemistry sterilant is incomparable.Exceed in century-old production application process reaching, never observe Bt albumen environment and non-target insect or Mammals and birds etc. are had to bad impact or toxic action.

The Bt gene that derives from bacterium, through artificial reconstructed, is imported to plant, can make plant self produce Bt albumen, thereby lepidopterous insects is had to resistance.In recent years, by trans Bt gene, cultivate insect-resistant transgenic crops, on the crops such as tobacco, tomato, cotton, potato, corn, paddy rice and rape, succeed.Transgenic Bt cotton and corn etc., successively in national large scale applications such as the U.S., Canada, Argentina, Brazil and India, have all been obtained the effect that increases income of producing of protecting.2009, the cultivated area of global transgenic Bt crop was over 3,800 ten thousand hectares; The cotton cultivated area of Bt of China also reaches 3,800,000 hectares, accounts for 66% of the total cultivated area in the whole nation, reduces approximately more than 80 ten thousand tons of chemical pesticide consumptions, and decrement reaches 70-80%, for cotton grower's increasing both production and income accumulative total is over 30,000,000,000 yuan.In November, 2009, the Ministry of Agriculture has ratified Transgenic Bt Rice China extensive No. 1 and with the extensive No. 1 Biosafety certificate for the cross combination Bt Shanyou 63 of restorer preparation of China (Nong Jian demonstrate,proves No. 072nd, word (2009): turn magnificent extensive No. 1 safety certificate in Hubei Province's production application of cry1Ab/cry1Ac gene pest-resistant paddy rice), thereby makes the commercial applications of China's transgenic paddy rice take the first step that the Liao Ling world is gazed at.

The encoding sequence of revising Bt gene is the important channel of further improving its using value in plant.Up to now, the engineered Bt protein gene that can be used for plant of many processes is disclosed.

Publication number is that the Chinese patent literature of CN1083884C discloses two kinds of encoding insecticidal protein genes and bivalent fusion expression vector and application thereof, by the method for double chain synthesising DNA, synthetic the GFM killing gene that merges of the Gry1Ab of total length 1824bp and Gry1Ac, the expression amount of result Bt albumen in plant increases substantially, and complete synthesis gene has improved nearly a hundred times than the expression of protogene.

Fan Yunliu etc. are succeeding in developing Bacillus thuringiensis (Bt) delta-endotoxin cry1Ab/Ac hybrid protein sequence (hereinafter to be referred as wBt), and in 1996, offer the applicant and use in paddy rice, obtained the pest-resistant transformant (Tu J., et al1998) of expressing wBt hybrid albumen.

But the insecticidal activity of existing Bt albumen still needs further to be improved.

Summary of the invention

The invention provides a kind of Bt albumen, this Bt albumen is stronger than existing Bt albumen to the resistance of lepidopterous insects.

A Bt albumen, aminoacid sequence is as shown in SEQ ID No.1.

The present invention also provides the encoding gene of described Bt albumen, and base sequence is as shown in SEQ ID No.2.

Bt albumen of the present invention (mBt albumen) and encoding gene thereof are transformed and are obtained on the basis of wBt hybrid albumen (aminoacid sequence is as shown in SEQ ID No.3) and encoding gene (base sequence is as shown in SEQ ID No.4) thereof.Its transformation program is:

(1) between wBt hybrid albumen the 3rd, 4 amino acids residues, insert one section of polypeptide, the aminoacid sequence of this polypeptide is NPNINECI, and the space structure of mBt albumen is had to stabilization;

Between wBt hybrid albumen the 3rd, 4 amino acids residues, insert this polypeptide, can cause the change of wBt hybrid albumen sterie configuration, thereby make the space structure of the mBt albumen that obtains more stable;

(2) (D of the 97th replaces with Q wBt hybrid albumen the 97th, 480,521 amino acids residues to be carried out to the modification of amino acid substitution type, the R of the 480th replaces with K, the N of the 521st replaces with H), these amino acid play a crucial role to insecticidal activity and the specificity of wBt hybrid albumen, carry out after the modification of amino acid substitution type, insecticidal activity and the specificity of mBt albumen are further enhanced;

(3) according to the codon preference of gene expression in plants, do not changing under the prerequisite of aminoacid sequence, the encoding sequence that step (2) is obtained carries out codon optimized;

(4) after codon optimized completing, at 5 ' end interpolation plant ribosome binding sequence AACA of mBt albumen coded sequence, to strengthen its expression activity.

The base sequence consistence of thus obtained mBt gene and wBt gene is that the consensus amino acid sequence of 96.7%, mBt albumen and wBt albumen is 98.2%.

The present invention also provides expression unit, recombinant vectors, the transformant that contains described encoding gene, and the initial carrier of described recombinant vectors is pSB130-Actin::Tnos.

The present invention also provides the application of described Bt albumen in improving plant anti-insect performance.As preferably, described plant is paddy rice.With turn wBt trans-genetic hybrid rice, what the encoding gene of Bt albumen of the present invention (mBt gene) rice transformation was obtained turns mBt trans-genetic hybrid rice, the expression activity of its mBt gene is stronger by approximately 20～50% than wBt gene; The resistance of lepidopterous insects Cnaphalocrocis medinali(rice leaf roller) and striped rice borer is also improved greatly, be mainly manifested in: Cnaphalocrocis medinali(rice leaf roller) can form certain withered and yellow hazard symptoms on the blade that turns wBt trans-genetic hybrid rice, on the blade that turns mBt trans-genetic hybrid rice, do not form or only near clip, form minimum withered and yellow hazard symptoms; Turning on wBt trans-genetic hybrid rice, the dead ears rate that striped rice borer harm causes is 1.1 ± 0.1%, and is turning on mBt trans-genetic hybrid rice, and dead ears rate is 0.

Compared with prior art, beneficial effect of the present invention is:

The expression activity of mBt gene of the present invention in paddy rice obviously strengthens, and turns mBt trans-genetic hybrid rice the resistance of lepidopterous insects is also improved greatly, can effectively prevent and treat lepidopterous insects, guarantees paddy rice high and stable yield.

Accompanying drawing explanation

Fig. 1 is the plasmid map of recombinant vectors pSBmBt; Wherein, goal gene mBt ceneme is in Yi T-DNA district, marker gene hph ceneme is in Er T-DNA district, for the border sequence of its transfer, is respectively LB1 and RB1 and LB2 and RB2, for the marker gene of plasmid self screening, is anti-kanamycin gene kan+;

Fig. 2 is that the PCR that carries 5 fine independent transformant of Japan of mBt gene detects analytical results; Wherein, M is DNA molecular amount mark; P is mBt plasmid positive control; N is the fine negative control of not genetically modified Japan; 1-5 is respectively the fine independent transformant of 5 Japan that turns mBt gene;

Fig. 3 is the silver label immunity test strip analytical results that carries 5 fine independent transformant of Japan of mBt gene; Wherein, CK+ is the fine positive control of Japan that turns wBt gene; CK-is the fine negative control of not genetically modified Japan; 1-5 is respectively the fine independent transformant of 5 Japan that turns mBt gene;

Fig. 4 be the fine blade of not genetically modified Japan (on), turn mBt gene rice leaf (in) and turn wBt gene rice leaf (under) insect-resistance qualification result comparison to Cnaphalocrocis medinali(rice leaf roller).

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.

The acquisition of embodiment 1mBt gene

The modification transformation of mBt gene is to utilize technology well known to those skilled in the art, and PCR primer fixed point introducing mutating alkali yl technology PCR side-directed mutagenesis is realized, and its concrete steps are:

(1) between wBt hybrid albumen the 3rd, 4 amino acids residues, insert one section of polypeptide that this protein is played to stabilization, the aminoacid sequence of this polypeptide is NPNINECI;

(2) on the 97th, 480 and No. 521 residues that insecticidal activity and specificity are played a crucial role, carry out the modification of amino acid substitution type, wherein, the D of the 97th replaces with Q, and the R of the 480th replaces with K, and the N of the 521st replaces with H;

(3) according to the codon preference of paddy gene, do not changing under the prerequisite of aminoacid sequence, the encoding sequence that step (2) is obtained carries out codon optimized;

(4) after codon optimized completing, at its 5 ' end, add plant ribosome binding sequence AACA, to strengthen its expression activity, finally obtain mBt gene, its base sequence is as shown in SEQ ID No.2.

The consensus dna sequence of thus obtained mBt gene and wBt gene is 96.7%, and consensus amino acid sequence is 98.2%; And with the consensus amino acid sequence of the publication number disclosed GFM killing gene of Chinese patent literature that is CN1083884C be 97.9%.

Embodiment 2 prepares transgenic paddy rice

1 builds recombinant vectors

(1) preparation of mBt gene Insert Fragment: by design of primers and pcr amplification, 5 ends and 3 ends at mBt gene coded sequence are introduced respectively restriction enzyme site Nco I and Kpn I, use the AxyPrep PCR of Axygen company cleaning agents box purified pcr product, operation steps is as follows:

1). in PCR reaction solution, add the Buffer PCR-A (if Buffer PCR-A less than 100 μ l add to 100 μ l) of 3 volumes; After mixing, transfer to and prepare in pipe, by preparing pipe, be placed in 2ml centrifuge tube (providing in test kit), the centrifugal 1min of 12000rpm, abandons filtrate.

2). by preparing pipe, put back in 2ml centrifuge tube, add 700 μ l Buffer W2, the centrifugal 1min of 12000rpm, abandons filtrate; Note: confirm to add dehydrated alcohol by the designated volume on reagent bottle in Buffer W2 stoste.

3). (optional step) puts back in 2ml centrifuge tube preparing pipe, adds 400 μ l Buffer W2, the centrifugal 1min of 12000rpm; Note: while taking out 2ml centrifuge tube from whizzer, note not allowing the Buffer W2 at the pipe end touch preparation pipe.

4). by preparing pipe, be placed in clean 1.5ml centrifuge tube (providing in test kit), add 25-30 μ l deionized water, the standing l min of room temperature preparing periosteum central authorities; The centrifugal 1min eluted dna of 12000rpm, obtains mBt gene Insert Fragment.

(2) double digestion of expression vector and Insert Fragment: expression vector pSB130-Actin::Tnos and mBt gene Insert Fragment all use Noc I and the Kpn I enzyme that spends the night at 37 ℃ to cut, and endonuclease reaction system is in Table 1.

Table 1 endonuclease reaction system

Enzyme cuts purifying, uses the AxyPrep DNA Gel Extraction Kit of Axygen company purifying enzyme to cut product, and operation steps is as follows:

1). under ultraviolet lamp, cut the sepharose (noting as far as possible shortening the time exposing under ultraviolet lamp) that contains target DNA, take gel weight.

2). add the Buffer DE-A (100mg gel, meter 100 μ l volumes) of 3 times of gel volumes, after mixing, in 65 ℃ of heating 6-8min, until gel melts completely.

3). add the Buffer DE-B of 0.5 times of Buffer DE-A volume, mix, when separated DNA fragmentation is less than 400bp, add the Virahol of 1 times of gel volume.

4). draw the mixed solution in step 3, transfer to DNA preparation pipe (being placed in 2ml centrifuge tube), the centrifugal 1min of 12000 * g.Abandon filtrate.

5). by preparing pipe, put back in 2ml centrifuge tube, add 700 μ l Buffer W1, the centrifugal 30sec of 12000 * g, abandons filtrate.

6). by preparing pipe, put back in 2ml centrifuge tube, add 700 μ l Buffer W2, the centrifugal 30sec of 12000 * g, abandons filtrate.Repeat once.

7). by preparing pipe, put back in 2ml centrifuge tube the centrifugal 1min of 12000 * g.Thoroughly remove remaining liquid.

8). by preparing pipe, be placed in 1.5ml centrifuge tube, preparing film central authorities, add elutriant or deionized water that 25～30 μ l are preheated to 65 ℃, the standing 1min of room temperature.The centrifugal 1min eluted dna of 12000 * g.

(3) connection of expression vector and Insert Fragment: the carrier segments and the goal gene that reclaim are carried out to concentration determination, then spend the night in 16 ℃ of connections, ligation system is in Table 2.

Table 2 ligation system

2 transform intestinal bacteria

To connect product and transform bacillus coli DH 5 ɑ competent cell, method is as follows: draw that 10 μ l connect in the intestinal bacteria competence that products join 100 μ l and with liquid-transfering gun piping and druming evenly, in on ice, place 30min, after 42 ℃ of thermal shock 90s, be placed in rapidly 2min on ice; To pipe, add again the LB liquid nutrient medium of 1ml, in 37 degree shaker vibration 1h; After bacterium liquid recovery, centrifugal and absorb 800 μ l supernatant liquors, will be left 200 μ l supernatant liquors and bacterium piece and mix, coat uniformly LB solid and screen (kantlex of 50mg/l) media surface, 37 ℃ of incubated overnight.

3 positive bacterium colonies are identified

Select transparent bacterium colony and pick as template with toothpick, carrying out colony PCR amplification.PCR reaction system is in Table 3.

The PCR reaction system of table 3.mBt gene

PCR response procedures is: 94 ℃ of sex change 5min; 96 ℃ of sex change 15s,, 55 ℃ of annealing 30s, 72 ℃ extend 2min, 35 circulations; 72 ℃ are extended 10min,, proceed to 10 ℃ of preservations.

PCR product is identified through 0.8% agarose gel electrophoresis, after taking pictures, preserves.Then, select PCR and be accredited as positive bacterium colony and shake bacterium, by the plasmid purification test kit of Axygen company, extract plasmid, and plasmid is carried out to enzyme (the handsome company in the Shanghai) checking of cutting and check order.

4 Agrobacterium-mediated Transformation

Get 0.5 μ l and cut the plasmid with sequence verification through enzyme, join in the centrifuge tube (1.5ml) that contains 60 μ l Agrobacteriums electric shock competence EHA105, until rifle head, inhale to beat after mixing and move in pole cup; After electric shock, add rapidly 1ml LB liquid nutrient medium, suction is beaten and is mixed in the previous 1.5ml centrifuge tube of rear immigration, in 28 ℃ of shakers, turns up 1h; After the recovery of bacterium liquid, draw 100 μ l bacterium liquid, evenly coat the LB solid screening and culturing primary surface containing the kantlex of 50mg/l and the Rifampin of 25mg/l, cultivate 2 days for 28 ℃; After bacterium colony PCR checking positive colony, choose positive colony and shake under the glycerol concentration of bacterium in 50% and-80 ℃ of conditions and save backup.

5 turn the cultivation of mBt trans-genetic hybrid rice

The fine paddy rice of Japan of take is acceptor, by agrobacterium-mediated transformation, carries out gene transformation, and concrete steps are as follows:

(1) induction of embryo callus

The mature seed shelling is first used 70% alcohol immersion 1-2 minute, then with 3% chlorine bleach liquor, soak 10-15 minute, carry out surface sterilization (be preferably on shaking table and carry out), aseptic water washing 3-4 time, again seed is placed on aseptic filter paper after suck dry moisture, be inoculated on MS calli induction media 28 ℃ of dark cultivations.After about 10-15 days, peel the callus that mature embryo scultellum grows, proceed on MS subculture medium, under the same conditions succeeding transfer culture.Later every two weeks succeeding transfer culture once, until form color and luster cadmium yellow, quality is fine and close and become granular embryo callus subculture.Afterwards, before facing conversion, select embryo callus subculture succeeding transfer culture 4-5 days, for Agrobacterium, infect immediately and be total to cultivation.

(2) cultivation of Agrobacterium

The Agrobacterium EHA105 that contains recombinant vectors is inoculated on the LB substratum that contains 50mg/l kantlex, in 28 ℃ of dark culturing 1 day, centrifugal, be resuspended in AA liquid nutrient medium, adjust cell concentration to OD600 be 0.2-0.6, add Syringylethanone, making Syringylethanone final concentration is 100m Μ, is the agrobacterium suspension that common cultivation rice transformation is used.

(3) Agrobacterium is infected and is total to cultivation

The pre-incubated Rice Callus of step (1) is put in appropriate agrobacterium suspension, and room temperature is placed 20min, and frequently rocks.With the spoon with filter screen, take out callus and be placed in the batch cultur ware that is covered with aseptic filter paper, on Bechtop, dry 5-10min, on aseptic filter paper, suck unnecessary bacterium liquid, transfer to immediately the solid 28 ℃ of dark culturing 2 days on substratum altogether that are covered with one deck aseptic filter paper.

(4) turn the screening of mBt gene resistant calli

Callus after common cultivation is inoculated in the N6 screening culture medium that contains 500mg/l cephamycin and 50mg/l or appropriate Totomycin, 28 ℃ of dark 12-14 days that cultivate, the every 12-14 of the kanamycin-resistant callus tissue days subculture newly growing once, have the situation that accelerating growth appears in kanamycin-resistant callus tissue to second～four-wheel, again subculture can take out for 1-2 time wherein half carry out the differentiation of green seedling, second half subculture is standby.

(5) turn the differentiation of the green seedling of mBt gene

The resistant calli that screening obtains through screening culture medium is gone on the pre-division culture medium of N6 that contains 50mg/l or appropriate Totomycin, first secretly cultivate 7-9 days for 28 ℃, then go on N6 division culture medium differentiation culture under 25 ℃ and 16h/d illumination condition, until green seedling forms.

(6) turn the cultivation of the green seedling of mBt gene

When green seedling grows to 8-12cm when high, open the bottle cap of regeneration bottle, and add 20-40ml tap water for hardening in bottle, afterwards, from regeneration bottle, extract green seedling and wash away substratum with tap water, being transplanted in the potted plant alms bowl in greenhouse or the cement pit of isolation, cultivation is to ripe.

(7) turn the Molecular of the green seedling of mBt gene

The Molecular that turns the green seedling of mBt gene is undertaken by conventional pcr amplification and Bt albumen silver label immunity test strip (silver soil, Beijing Bioisystech Co., Ltd) method.

Test-results shows: the transformant of PCR tests positive (Fig. 2) all manifests object tape (Fig. 3) on the detection line of silver label immunity test strip, its detection signal is mostly strong than the detection signal that turns the contrast of wBt gene masculine, illustrates that mBt gene is stronger than the expression activity of wBt gene.

Embodiment 3 turns the insect-resistance of mBt trans-genetic hybrid rice to be identified

The fine paddy rice of Japan that turns mBt gene of take is object, identifies the resistance of mBt gene pairs paddy rice Cnaphalocrocis medinali(rice leaf roller) and striped rice borer, and to turn the fine plant of wBt gene and not genetically modified Japan, does positive and negative contrast respectively.

Resistance to rice leaffold identify to adopt Leaf method to carry out: in rice tillering mid-term, from each, supply in the strain of examination rice respectively, at random from 6,3 long blades of clip 2～4cm of tillering (fall a leaf), and at blade two ends, press with 0.1g/L benzene a pair of horses going side by side and narrow the impregnated little filter paper of azoles fresh-keeping liquid.Then, in the little flat glass-tube of blade dislocation (9.5cm * 1.5cm), every pipe accesses respectively 5 ant snout moth's larvas, mouth of pipe jam-pack degreasing cotton plug, repetitive identified 3 strains/transformant.After starting to connect worm, within 4-7 days, check day by day the victimization state of blade, and the record of taking pictures.

Striped rice borer Resistance Identification adopts individual plant guard identification method to carry out: be about to rice strain to be measured and plant respectively in adding in the potted plant bucket of plastic lousing (diameter 12cm, high 70cm), at the beginning of the heading every strain in left and right in first 7 days is inoculated 15, incubate striped rice borer ant snout moth's larva, inoculation 3 strains/transformant.Connect worm and remove plastic lousing after 2 weeks, the dead ears rate that the harm of 3 weeks " Invest, Then Investigate " striped rice borer causes, investigation result is in Table 4 and Fig. 4.

Each insect-resistance qualification result for examination paddy rice of table 4

Experimental result shows: the wBt gene of modifying the transformation of improved mBt gene and unmodified is high resistance Cnaphalocrocis medinali(rice leaf roller) and striped rice borer all, but the former is better than the latter to the resistance of two target pests.Be mainly manifested in: Cnaphalocrocis medinali(rice leaf roller) can form certain withered and yellow hazard symptoms on the blade that turns wBt trans-genetic hybrid rice, on the blade that turns mBt trans-genetic hybrid rice, do not form or only near clip, form minimum withered and yellow hazard symptoms (Fig. 4); On dead ears rate that striped rice borer harm causes is fine in Japan, being 56.8 ± 1.2%, is 1.1 ± 0.1% turning on wBt trans-genetic hybrid rice, is all 0 (table 4) on 5 that turn mBt gene independent transformant plant.

Claims (8)

1. a Bt albumen, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.

2. the encoding gene of Bt albumen as claimed in claim 1, is characterized in that, base sequence is as shown in SEQ ID No.2.

3. contain the expression unit of encoding gene as claimed in claim 2.

4. contain the recombinant vectors of encoding gene as claimed in claim 2.

5. recombinant vectors as claimed in claim 4, is characterized in that, its initial carrier is pSB130-Actin::Tnos.

6. contain the transformant of encoding gene as claimed in claim 2.

7. the application of Bt albumen in improving plant anti-insect performance as claimed in claim 1.

8. application as claimed in claim 7, is characterized in that, described plant is paddy rice.