CN1175115C

CN1175115C - Method for screening transgenic plant and its used bivalent plant expression vector

Info

Publication number: CN1175115C
Application number: CNB021520364A
Authority: CN
Inventors: 谢龙旭; 徐培林
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2002-11-25
Filing date: 2002-11-25
Publication date: 2004-11-10
Anticipated expiration: 2022-11-25
Also published as: CN1412323A

Abstract

The present invention provides a new method for sieving transductants of plants and a bivalent expression carrier of the plants, and the method utilizes glyphosate resistance genes to replace the existing antibiotic resistance genes as marking genes for sieving transgenic plants. The bivalent expression carrier of the plants in the method contains the glyphosate resistance genes and plant characteristic genes to be sieved. The present invention can sieve transductants of plants at high efficiency, and the glyphosate resistance genes as the sieving marking genes have double efficacy, wherein on one hand, the glyphosate resistance genes are used as herbicide resistance genes, on the other hand, the glyphosate resistance genes are used as the sieving marking genes of the plants. In addition, the glyphosate resistance genes and other important purpose characteristic genes are linked to convert the plants so as to be greatly convenient to convert the plants and pure line breeding varieties. The transgenic plants sieved by the method have good characteristics of purpose conversion genes and the characteristic of good glyphosate resistance.

Description

A kind of method of transgenic plant and used two valency plant expression vectors thereof of screening

Technical field

The invention belongs to the plant gene engineering technology field, be specifically related to a kind of with glyphosate resistance gene the serve as a mark methods of genescreen transgenic plant and used two valency plant expression vectors thereof.

Background technology

How screening vegetable transformant is a gordian technique that successfully obtains transgenic plant.Selective marker mainly contains several major types according to the character of its gene product: antibiotics, biochemical class, fluoresceins and antiweed class.At present, adopt the marker gene of antibiotics resistance gene mostly as the screening Plant Transformation, the most frequently used have neomycin phosphotransferase gene (NptII) and a hygromycin phosphotransferase gene (HPT). and the former has resistance to kantlex (km) and Xin Meisu (neo) by gene encoding enzyme, and latter's gene encoding enzyme produces resistance to Totomycin (hyg).Gene has the higher and convenient advantage of screening of transformation efficiency though these antibiotics resistance genes serve as a mark, and fears are entertained that in a single day these resistant genes are transferred in the pathogenic bacteria by transfer-gen plant, will make pathogenic bacteria that microbiotic is produced resistance.Only as reporter gene, there is not (screening) effect of selecting with the fluoresceins gene.Biochemical genoid mark as the dihydrofolate reductase gene mark, owing to cost an arm and a leg, should not be promoted.Above-mentioned three major types type selective marker can not be as the purpose character gene as just selection markers.Anti-herbicide gene commonly used at present has bar (the Basta acetyl-CoA transferring enzyme) gene of anti-careless fourth phosphine and bxn (nitrilase) gene of anti-bromobenzene cyanogen, but these two kinds of selective agents all are eliminated gradually, because careless fourth phosphine expensive and bromobenzene cyanogen can be influenced rodentine growth by skin absorption.

Glyphosate can also can be degraded by the physics and chemistry approach, so there is not the problem of environmental pollution basically by multiple microbiological degradation in environment to animal toxicity low (big white mouse LD 50, oral 11343mg/kg, subcutaneous injection 7500mg/kg).Utilizing serve as a mark gene pairs people's food chain of shikimic acid hydroxyl ethene transferase gene (aroA) safer, is a kind of ideal plant marker gene therefore.

Glyphosate is a kind of interior extinction spectrality weedicide, and it can not have the activity of the shikimic acid hydroxyl ethene transferring enzyme (EPSPS) that selectively suppresses all plants, blocks the synthetic of die aromatischen Aminosaeuren, thus the growth of blocking-up plant.EPSPS catalysis shikimic acid-3-phosphoric acid (S3P) in the die aromatischen Aminosaeuren route of synthesis is synthetic with phosphoenolpyruvic acid (PEP), forms 5-enol pyruvoyl shikimic acid-3-phosphoric acid (EPSP).The research that external using gene engineering is cultivated the herbicide-resistant crop had more than 15 years, had developed the genetically modified crops of multiple antiweed, and therefore, Antiglyphosate gene itself is again the purpose character gene.In the past, the main policies of Antiglyphosate gene engineering is to utilize rite-directed mutagenesis to improve the target protein EPSPS of glyphosate effect, but because the three-dimensional structure and the amino acid site of action of enzyme and imperfectly understand, so the effect of rite-directed mutagenesis is very limited.The problem that runs into is that though sudden change back enzyme has reduced the avidity to glyphosate, the avidity to the low thing of proenzyme also descends simultaneously, thereby has influenced the catalytic activity of enzyme.The CN1358858 patent application discloses a kind of resistance glyphosate resistant gene aroAM12 that adopts the gene optimization technology to be obtained, and it is characterized in that at the EPSPS amino acid position by proline 35 → L-Ala; L-Ala 40 → Xie Ansuan; Threonine 42 → methionine(Met); Arginine 83 → Histidine; Methionin 185 → L-glutamic acid; Isoleucine 201 → Serine; Arginine 330 → Threonine.Studies show that, because the change of a plurality of amino acid sites in the epsp synthase this enzyme is increased substrate PEP affinity, and descend with the glyphosate affinity, thereby raising is to the resistance of glyphosate.

Summary of the invention

For the single effect of selection markers or the deficiency of security in the method that overcomes existing screening transgenic plant, the invention provides a kind of novel screening method and used plant bivalent expression vector thereof.This method not only can efficiently be screened vegetable transformant, and the selection markers gene in the method itself is again good purpose character gene.

The method of screening transgenic plant of the present invention is: replace used in the past antibiotics resistance gene with the glyphosate resistance gene, as the marker gene of screening transgenic plant strain.

The concrete steps of the inventive method can be: at first insert the glyphosate resistance gene and (preferably pass through the glyphosate resistance gene that the gene optimization technology obtains in plant expression vector, aroAM12 for example) and the plant trait gene that will screen, constitute plant bivalent expression vector; Transform Agrobacterium with this plant bivalent expression vector, obtain to contain the engineering bacteria of this expression vector; Use this engineering bacteria infection plant explant again; Because glyphosate is toxic to plant, so metainfective explant breaks up a week in division culture medium after, the agent (glyphosate) for the treatment of to bring Selection In again after the glyphosate resistance gene gives full expression to is screened.

The plant bivalent expression vector that is used for the invention described above method, its constitutional features is: the plant trait gene that this carrier contains the glyphosate resistance gene and will screen.

It is underlying carrier that this plant bivalent expression vector can adopt plant expression vector pCAMBIA1300, inserts the glyphosate resistance gene at the multiple clone site MCS place of pCAMBIA1300 and constitutes with the plant trait gene that will screen.Contain the left and right sides border sequence (LB and RB) of T-DNA in the Ti-plasmids among the plant expression vector pCAMBIA1300, in this segment DNA, contain multiple clone site (MCS), outside two border sequences, contain kalamycin resistance gene km (r).

The used glyphosate resistance gene of above-mentioned plant bivalent expression vector preferably obtains by the gene optimization technology, for example aroAM12.

The present invention utilizes the double effects of glyphosate resistance gene, promptly on the one hand as anti-herbicide gene, on the other hand as a kind of plant screening mark gene, and transform plant with other free-revving engine character gene is chain, will conveniently greatly transform the screening of plant and the screening of pure line breeding kind.

Evidence, the transgenic plant with the inventive method screening not only have the proterties of good purpose transforming gene, and have good anti-glyphosate proterties.

Below the invention will be further described by drawings and Examples.

Description of drawings

Fig. 1 is the structure synoptic diagram that contains the pBtPCR2.1 plasmid of BtS1m gene.

Fig. 2 is the structure synoptic diagram of pGAT1300 plasmid.

Fig. 3 is the pCM12-BtS1m plant bivalent expression vector synoptic diagram that contains aroAM12 gene and BtS1m gene.

Fig. 4 is that the specific detection primer of aroAM12 and BtS1m gene carries out the result schematic diagram that PCR detects amplification.

Fig. 5 is the resistance experiment synoptic diagram of tobacco leaf to glyphosate.

Fig. 6 is the tobacco resistance experiment synoptic diagram of taking root.

Fig. 7 is the resistance synoptic diagram of tobacco plant to glyphosate.

Fig. 8 is the pest-resistant effect synoptic diagram that two valency transgene tobaccos show.

Embodiment

Embodiment is 1. pest-resistant, the structure of anti-glyphosate plants bivalent expression carrier

According to reaction of the HindIII+SalI double digestion shown in the table 1.1 and electrophoresis system, the plasmid pBtS1m (No. 01129519.8 patent application is open) that contains the BtS1m gene fragment with the HindIII+SalI cutting, behind agarose gel electrophoresis, utilize E.Z.N.A  GelExtraction kit (Omega, reclaim the band that size is located about 2.7kb down together), obtain the 2E-35sp+BtS1m fragment.With HindIII+XhoI cutting aroAM12PCR2.1 plasmid (the PCR2.1 plasmid that contains the aroAM12 gene, see Fig. 1), reclaim the band at 4100bp place, obtain containing the fragment of Nos terminator sequence, with the T4DNA ligase enzyme 2.7kb of recovery and two portions dna fragmentation of 4100bp are coupled together (the ligation system sees Table 1.2) then, obtain containing the pBtPCR2.1 plasmid (see figure 1) of BtS1m gene.Cut the pCAMBIA1300-M12 (insert the pCAMBIA1300 plasmid by the aroAM12 gene fragment and constitute, see Fig. 2) that contains the aroAM12 gene fragment with EcoRI+XhoI, reclaim the band at 2300bp place, obtain containing the fragment of CaMV+TSP+aroAM12; With EcoRI+XhoI cutting pCAMBIA1300 carrier, reclaim the band at 6800bp place, obtain removing the pCAMBIA1300 empty expression vector of the mould plain gene of moisture resistance; With the T4DNA ligase enzyme 2300bp and 6800bp two portions dna fragmentation are coupled together then, obtain containing the pGAT1300 plasmid (see figure 2) of the removal moisture resistance mycin antibiotic resistance gene of aroAM12.With HindIII+XbaI cutting pGAT1300 plasmid, obtain having the pGAT1300 plasmid of breach.With HindIII+XbaI cutting pBtPCR2.1 plasmid, reclaim the band at 3.0kb place, obtain the 2E-35sp+Bt+Nos fragment, use T4DNA Ligase connecting band pGAT1300 plasmid jaggy and 3.0kb two portions dna fragmentation then, promptly obtain two valency plasmid vector pCM12-s1m (see figure 3)s.Contain Antiglyphosate gene (aroAM12) and anti insect gene (BtS1m) on this plant bivalent expression vector pCM12-s1m, but do not contain the antibiotics resistance gene that in plant, to express.Wherein, the formation of Antiglyphosate gene (aroAM12) place expression cassette is: from 5 ' and to the promotor CaMV35s of 3 ' extreme direction (I) transcription regulatory region, (II) transduction peptide sequence (TSP), so that the synthetic product is transferred in its target site point chloroplast(id), (III) Antiglyphosate gene M12 encoding sequence and (IV) the non-translational region PolyA fragment of 3 ' end.The formation of anti insect gene BtS1m place expression cassette is: from 5 ' and to 3 ' extreme direction (I) transcription regulatory region, the 35s promotor that includes 2 enhansers, be 2E-35sp, (II) the Ω fragment (translational enhancer) of TMVRNA, (III) the chimeric Bt insecticidal proteins encoding sequence BtS1m of band secreting signal peptide, (IV) Nos, the transcription termination sequence of 3 ' end.

Reaction of table 1.1 HindIII+SalI double digestion and electrophoresis system

Plasmid vector DNA 20.0 μ l

HindIII(10U/μl) 3.0μl

SalI(10U/μl) 3.0μl

10x reaction buffer 8.0 μ l

ddH ₂0 46.0μl

Insulation is 3 hours under 37 ℃ of conditions, use 1 * TAE (0.04mol/L Tris-acetate then, 0.001mol/L the EDTA) sepharose of electrophoretic buffer preparation 1.0%, adding EB (ethidium bromide) in sepharose is 0.5 μ g/ml to final concentration, electrophoresis under the volts lost of 3-5V/cm.

Table 1.2 ligation system

Dna fragmentation 1 5.0 μ l

Dna fragmentation 2 5.0 μ l

ddH ₂O 8.0μl

T4?Ligase(5weissU/ul) 0.5μl

T4 Ligase damping fluid (10x) 2.0 μ l

Ligation is spent the night under 16 ℃ of conditions.

The acquisition of embodiment 2. intestinal bacteria transformants

The preparation of E.coli XL-1-blue competent cell: 1. the single bacterium colony of XL1-blue is inserted 5mL and do not contain in the antibiotic LB liquid nutrient medium (seeing Table 2.1), 37 ℃, concussion spend the night; 2. the amount by 1% (v/v) changes in the fresh LB liquid nutrient medium, and 37 ℃ shake to 0D ₆₀₀=0.3; 3. the LB liquid nutrient medium (the LB preparation sees Table 2.1) with 50-100mL adds in two aseptic centrifuge tubes, places 30 minutes on ice; 4. 4 ℃, 4000rpm (rev/min) centrifugal 10 minutes, remove supernatant liquor, in each centrifuge tube, add the 0.1%CaCl of 10mL precooling ₂The resuspended thalline of solution, ice bath 30 minutes. 5., remove supernatant liquor, thalline is suspended in 0.1% CaCl of 2mL precooling centrifugal 10 minutes of 4 ℃, 4000rpm ₂In the solution, add the aseptic glycerine of 300 μ L again, mixing adds 100uL XL1-blue competent cell, in-70 ℃ of preservations in each aseptic centrifuge tube.

Get connection product (plant bivalent expression vector pCM12-s1m) the 10 μ l among the embodiment 1, just join in the intestinal bacteria XL-1-blue competent cell that melts, slight mixing, ice bath half an hour; 42 ℃ of 90 seconds of water-bath heat shock, ice bath 1-2 minute then.Add LB substratum 1ml in the Eppendorf pipe, 37 ℃ of water-baths were cultivated 1 hour, 13000rpm centrifugal 5 seconds removed supernatant, added 100 μ l LB substratum, and mixing is coated on LB+Km (kanamycine, the kantlex) flat board.37 ℃ when cultivating about 24 hours, picking list bacterium colony shakes bacterium and spends the night, and adopts E.Z.N.A  Plasmid miniprep Kit to reclaim plasmid, after PCR and enzyme are cut detection, picks out positive plasmid, preserves corresponding bacterium colony, i.e. transformant.This transformant can be used for amplification and extracts plant bivalent expression vector pCM12-s1m.

Table 2.1 LB liquid culture based component

Microbial culture tryptone 10g;

Microbial culture yeast extract 5g;

NaCl 10g；

Deionized water 950mL

(, the preparation solid medium adds agar if then pressing the amount of 18-20g/L again.)

PH7.2 is transferred with 5mol/LNaOH in the dissolving back fully, and adding deionized water again is 1L to cumulative volume, and autoclaving gets final product then.

The conversion of embodiment 3. Agrobacterium tumefaciens

The preparation of Agrobacterium tumefaciens LBA4404 competent cell: 1. picking LBA4404 bacterium colony from YEB solid medium (the seeing Table 3.1) flat board that contains Streptomycin sulphate (str) and Rifampin (Rif), being inoculated into 5ml contains corresponding antibiotic YEB liquid nutrient medium and (presses table 3.1, do not add agar) in, 28 ℃, the 200rpm concussion is spent the night.2. be diluted to 50ml with the YEB liquid nutrient medium, 28 ℃ then, 200rpm were cultivated 6-12 hour, to 0D ₆₀₀Value is about 0.6.3. placed 5 minutes on ice, then in centrifugal 7 minutes of 4 ℃, 2000rpm.4. getting 1ml concentration is the ice-cold CaCl of 20mmol/L ₂The resuspended precipitation of solution, mixing.5. be distributed into 10 parts, every part 100 μ l preserves standby in-70 ℃ of refrigerators.

Add the plasmid 5 μ l that extracted among the embodiment 2 among the bacillus LBA4404 competent cell 100 μ l that preserve in-70 ℃ of refrigerators, 37 ℃ of water-baths 5 minutes add 1ml YEB liquid nutrient medium afterwards, place 3 hours under the room temperature.Centrifugal 30 seconds of 6000rpm outwells supernatant, adds 100 μ l YEB, and mixing is coated on the YEB+Str+Rif+Km flat board.In the time of about 48 hours, picking list bacterium colony (cut the detection with PCR through enzyme, confirm as and contain the bivalent gene plasmid vector) shakes bacterium and spends the night, and is cultured to OD ₆₀₀Value is used for during for 0.4-0.6 infecting.

Table 3.1 YEB solid culture based component

Microbial culture tryptone 5g/L

Microbial culture yeast extract 5g/L

Sucrose 5g/L

Sal epsom 2mmol/L

Adjust pH to 7.2, the amount of pressing 18g/L adds agar.

Embodiment 4. contains the engineering bacteria of two valency carriers to the conversion of tobacco and the acquisition of resistant plant

The engineering bacteria that embodiment 3 is obtained infects tobacco explant (blade, be cut into 0.5 * 0.5cm size) 5 minutes, with blotting through autoclaved filter paper, be moved into MS+6-BA (6-benzyladenine, the 6-benzyl purine) cultivated 48 hours altogether the dark place in the substratum of 1mg/L+NAA (naphthalene acetic acid, naphthylacetic acid) 0.1mg/L+3% sucrose+0.7% agar.With aseptic water washing 3 times, aseptic filter paper blots, and is moved into then in MS+6-BA (1mg/L)+NAA (0.1mg/L) division culture medium that contains cephamycin (400mg/L), and 28 ± 2 ℃, diffuse light conditions is cultivated, and induces differentiation.Cultivated for 1 week, transfer to again [MS+6-BA (1mg/L)+NAA (0.1mg/L)+glyphosate (30 μ mol/L)] on the division culture medium that contains the selective agent glyphosate.Continue to cultivate for some time (3 week), when bud grows to 2-3cm, the resistant buds cutting-out that produces is moved in MS+NAA (0.1mg/L) root media that does not contain glyphosate, induce the formation of root.The back adventive root appearance of one week.After spending for two weeks, opened the bottle cap hardening 2-3 days, seedling is taken out, wash away the root substratum, move on in the basin, can survive after 1 week of preserving moisture with tap water.

The molecular biology identification of embodiment 5. transfer-gen plants

Get tobacco plant to be measured (resistant plant that embodiment 4 screenings obtain) young leaflet tablet, adopt CTAB (cetyldimethylethylambromide bromide ammonium) method trace to extract DNA: to get about 1-2cm ²Fresh young leaflet tablet, 1 ice-cold CTAB (-) damping fluid (the 50mmol/LTris-HCl pH8.0 of 400 μ l is housed, 0.7mmol/L NaCl, 10mmol/LEDTA pH8.0) Guan Zhongyong glass stick grinds, add 500 μ l then in the CTAB of 65 ℃ of preheatings (+) damping fluid { CTAB (-), the 2 mercapto ethanol of adding 1% before 2%CTAB. uses }, mixing, 65 ℃ are incubated 90 minutes, put upside down mixing during this time frequently, take out the back, adds 450ul chloroform/primary isoamyl alcohol (volume ratio is 24: 1) after waiting to be chilled to room temperature, put upside down mixing to solution and be the milkiness shape, centrifugal 10 minutes.Upper phase is transferred in the clean Eppendorf pipe, adds the equal-volume Virahol, room temperature was placed 20 minutes, and centrifugal then 5 minutes, collecting precipitation.With 70% washing with alcohol DNA precipitation, behind air drying, add the dissolving of 20 μ l distilled waters.

Use the specific detection primer (according to table 5.1 and table 5.2 system) of aroAM12 and BtS1m gene to carry out the PCR detection respectively.

The aroAM12 primer sequence is: primer 1:5 '-CATGCCATGGAATCCCTGACGTTACAA-3 '

Primer 2: 5 '-CGCGGATCCTTAGCAGGCTACTCATTC-3 '

The Bts1m primer sequence is: primer 1 ': 5 ' TATCCCATTGTTCGCAGTCC 3 '

Primer 2 ': 5 ' CATCACGACTCAAGTTGTTA 3 '

Table 5.1 PCR reaction solution

2 * TaqDNA polymerase buffer, 4 kinds of each 200 μ M of dNTP, according to each 3pM of special primer (BIOASIA) of each stencil design, each From Template DNA about 10 ²-10 ⁴Copy, the about .0.5-5U of TaqDNA polysaccharase (worker is given birth in Shanghai).Be specially:

dNTPs 2μl

Template DNA 1 μ l

TaqDNA polysaccharase 0.5 μ l

Primer 1 0.5 μ l

Primer 2 0.5 μ l

ddH ₂O 16.5μl

Table 5.2 PCR response procedures

Pre-sex change: 94 ℃, 5 minutes

The circulation: 94 ℃ 60 seconds, 58 ℃ 60 seconds, 72 ℃ 60 seconds

Carry out altogether 30 times.

72 ℃ were extended 10 minutes.

After reaction finishes, electrophoresis detection pcr amplification result.Electrode buffer is 1 * TAB, 1.0% sepharose, and electrophoresis is 1 hour under the volts lost of 3-5V/cm.

Detected result as shown in Figure 4.Wherein M is DNA ladder, and ck is a blank, and last figure a, b, c, d are the detected result behind the adding BtS1m special primer, and figure below a, b, c, d are the detected result behind the adding aroAM12 special primer.Therefrom as can be seen, two groups of primers locate to have occurred amplified band at 1.3kb (aroAM12 fragment) and 0.98kb (BtS1m part fragment) respectively.As can be seen from Figure 4 show that with the glyphosate resistance gene DNA cloning result that 4 strains that GeneScreen selects transform seedling that serves as a mark this bivalent gene is incorporated in the plant genome DNA.

Embodiment 6. plant detect the resistance of glyphosate

Get the embodiment 4 transformation of tobacco seedling spire that obtains, be cut into 0.5 * 0.5cm size, be placed in the division culture medium of MS+6-BA (1mg/L)+NAA (0.1mg/L)+3% sucrose+0.7% agar that contains 10 to 100 μ mol/L glyphosates, at 28 ℃ of constant temperature, 16 hours every days, illumination condition was cultivated down.With the tobacco leaf of unconverted in contrast, the tobacco that the check conversion is obtained is to the resistance of glyphosate, experimental result is as shown in Figure 5: the top A of delegation is the transgene tobacco blade, from left to right in the substratum concentration of glyphosate be followed successively by 0,0.05,0.4,0.8,1.2mmol/L; The following B of delegation is wild contrast tobacco leaf, from left to right in the substratum concentration of glyphosate be followed successively by 0,0.05,0.4,0.8,1.2mmol/L.As can be seen from the figure the blade of transfer-gen plant still can break up on the substratum of 1.2mmol/L glyphosate and sprouts, and just do not have bud to break up out to time on the substratum that impinges upon the 0.05mmol/L glyphosate, show that the transformed plant that is obtained has good glyphosate resistance.

The bud that the resistant buds that induces and blank differentiate is transferred in the root media of MS+NAA (0.1mg/L)+3% sucrose+0.7% agar+30 μ mol/L glyphosates, at 28 ℃ of constant temperature, 16 hours every days, illumination condition was cultivated 20 days down, observed the situation of taking root.The result is as shown in Figure 6: wherein a is two valency transgene tobaccos, and CK is the blank tobacco; Transfer-gen plant can normally be taken root in the root media of 30 μ mol/L glyphosates, and contrast is not then taken root or seldom taken root.

To be transplanted to the glyphosate resistance tobacco seedling of booth, directly use the Roundup (activeconstituents is for being diluted to the 1.55g/L glyphosate) of Monsanto Company to spray.The result is as shown in Figure 7: wherein a1 is that blank tobacco seedling sprays the preceding upgrowth situation of glyphosate (1.55g/L), and a2 is for spraying back 15 days upgrowth situation; B1 is that two valency transgenic seedlings spray the preceding upgrowth situation of glyphosate (1.55g/L), and b2 is for spraying back 15 days upgrowth situation.After spraying a week, the tobacco that transgenosis obtains does not have impaired phenomenon; After for the second time spraying medicine, some xanchromatic spots occurred on the minority blade, but do not influenced the growth of whole tobacco and grow young leaves, contrast is then dead after 10 days.

Synthesizing map 5, Fig. 6, Fig. 7 result show: transfer-gen plant has shown good resistance.

Embodiment 7. transgene tobaccos are to the insecticidal activity assay of bollworm

Test design transfer-gen plant aroAM12, aroAM12+BtS1m and three processing of conventional tobacco (CK).The tobacco leaf that is used to handle 1,2 instar larvae is put into and is covered with the culture dish that soaks filter paper, and petiole base is with moistening rayon balls parcel, in case here blade withers rapidly.Test group and control group respectively insert 1 age 8 of bollworms and 2 age 3 of bollworms, weigh before connecing worm.Connect worm and be placed on 28 ± 2 ℃, illumination: dark=16: in the greenhouse of 8hrs, observe the existence of larva and the situation of growth after 3 days.The result is shown in Fig. 8 and table 7.1.As can be seen, two valency transgene tobaccos show good pest-resistant effect from figure and table.

Table 7.1 transgene tobacco is to the influence of cotton bollworm larvae existence and growth

PCR result is to glyphosate for material mortality ratio (%) survival larva body weight

The resistance of code name 3d 6d (mg) 3d BtS1m aroAM12

o 12.5 12.5 5.0±0.3 - + +

a 88.5 100 0.6±0.3 + + +

b 80.0 90 0.7±0.2 + + +

c 85.0 100 0.6±0.35 + + +

d 80.0 100 0.5±0.28 + + +

CK 10.0 10.0 5.5±0.4 - - -

In the table, o is for containing aroAM12 unit price gene plant, and a, b, c, d are for containing the two valency transfer-gen plants of aroAM12+BtS1m, and CK is a blank.

Claims

1. the method with marker gene screening transgenic plant replaces antibiotics resistance gene with the glyphosate resistance gene, as the marker gene of screening transgenic plant strain; It is characterized in that described glyphosate resistance gene is aroAM12.

2. in accordance with the method for claim 1, it is characterized in that: the plant trait gene that in plant expression vector, inserts glyphosate resistance gene aroAM12 and will screen at first, formation plant bivalent expression vector; Transform Agrobacterium with this expression vector, obtain to contain the engineering bacteria of this expression vector; Use this engineering bacteria infection plant explant again; Metainfective explant breaks up the agent glyphosate that brings Selection In again after a thoughtful glyphosate resistance gene gives full expression to and screens in division culture medium.

3.. it is characterized in that in accordance with the method for claim 2: described plant bivalent expression vector inserts glyphosate resistance gene aroAM12 at the multiple clone site MCS place of underlying carrier pCAMBIA1300 and constitutes with the plant trait gene that will screen.

4. according to claim 2 or 3 described methods, it is characterized in that described glyphosate resistance gene aroAM12 obtains by the gene optimization technology.

5. according to claim 2 or 3 described methods, it is characterized in that the contained plant trait gene that will screen of described plant bivalent expression vector is anti insect gene BtS1m.

6. in accordance with the method for claim 5, it is characterized in that described plant bivalent expression vector is pCM12-s1m, wherein, the formation of Antiglyphosate gene aroAM12 place expression cassette is: from 5 ' and to the promotor CaMV35s of 3 ' extreme direction (I) transcription regulatory region, (II) transduction peptide sequence TSP, (III) Antiglyphosate gene aroAM12 encoding sequence and (IV) the non-translational region PolyA fragment of 3 ' end; The formation of anti insect gene BtS1m place expression cassette is: from 5 ' and to 3 ' extreme direction (I) transcription regulatory region, the 35s promotor that includes 2 enhansers, be 2E-35sp, (II) the Ω fragment translational enhancer of TMVRNA, (III) the chimeric Bt insecticidal proteins encoding sequence BtS1m of band secreting signal peptide, (IV) Nos, the transcription termination sequence of 3 ' end.