CN102676541A - NAC transcription factor gene GmST2 of soybean holy bean No.9 and application of NAC transcription factor gene GmST2 - Google Patents
NAC transcription factor gene GmST2 of soybean holy bean No.9 and application of NAC transcription factor gene GmST2 Download PDFInfo
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Abstract
The invention discloses an NAC (N-acetyl-L-Cysteine) transcription factor gene GmST2 of soybean holy bean No.9 and a plant expression vector of the GmST2. The invention further discloses application of the gene GmST2 in improvement on the salt resistant/drought tolerance in arabidopsis thaliana and soybean plants. Provided by tests, the salt resistance/drought tolerance of a transgenic plant is greatly improved compared with that of a non transgenic plant and theoretical base and practice foundation for improving the salt resistance/drought tolerance of crops through genetic engineering means are provided. The NAC transcription factor gene GmST2 can be widely applied to cultivation of salt-resisting/drought tolerant plant varieties.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, relate in particular to No. 9 NAC transcription factor genes of the holy beans of soybean---GmST2 and application thereof.
Background technology
Soybean is important cash crop, therefore improves and optimizes soybean quality and become the focus that people pay close attention to.China is one of country of in the world lack of water, and arid is the main natural disaster of China's agriculture prodn, and the area that saltings and secondary salinization are ploughed increases year by year, and these hostile environment conditions have seriously restricted the crop yield of China.In order to adapt to these hostile environments, inside plants develops a series of defence measure, also needs the abiotic stress response gene to participate in.At present identified many salt and arid response gene and verified their function in abiotic stress, but remained unknown about the biological function of the relevant gene of a lot of abiotic stress.
Plant transcription factor research is an importance of functional genome research.Though transcription factor proportion in genome seldom plays a significant role in coercing in regulation and control growth and development of plant, response external environment.The NAC transcription factor is a newfound plant specific transcriptional regulator over past ten years.Aida in 1997 etc. have at first reported the NAC structural domain, and the N end of finding at petunia NAM gene, Arabidopis thaliana A TA F1/2 and CUC2 gene coded protein comprises one section conservative aminoacid sequence, gets three gene initial called after NAC.First NAC transcription factor is equaled from petunia, to clone in 1996 by Souer and obtains; In species such as Arabidopis thaliana, paddy rice, wheat, soybean, find in succession subsequently; In Arabidopis thaliana, find 105 NAC members at present altogether, then found 226 in the soybean.Research shows, the NAC transcription factor is built up at growth and development of plant, organ, hormone regulation and defence are resisted aspects such as multiple biology and abiotic stress and brought into play important effect.
The living environment of plant is complicated and changeable, often suffers environment stresses such as arid, high salt, low temperature and disease and pest, influences growth and development of plant, even can cause plant dead, has a strong impact on agriculture prodn and ecotope.The NAC transcription factor receives multiple biology to coerce the abduction delivering with abiotic stress, the stress response of involved in plant.Yet, do not see that also soybean NAC film combines the report of functional transcription factor.
Summary of the invention
The purpose of this invention is to provide holy No. 9 NAC transcription factor gene-GmST2 of beans of a kind of soybean and application thereof.
Technical scheme of the present invention is: from No. 9, holy beans, separate obtaining NAC transcription factor gene-GmST2; Forward this gene in the model plant Arabidopis thaliana its function of proof, this gene is forwarded to obtain the transfer-gen plant that anti-salt/drought tolerance improves in the former plant soybean then.
No. 9 NAC transcription factor genes of the holy beans of soybean of the present invention GmST2, it is characterized in that: the nucleotide sequence of said gene cDNA is shown in SEQ ID No.1.
The present invention also provides a kind of plant expression vector pK2GW7::GmST2 that contains No. 9 NAC transcription factor genes of the holy beans of above-mentioned soybean GmST2, it is characterized in that: said carrier cloning zone nucleotide sequence is shown in SEQ ID No.5.
The present invention also provides a kind of plant expression vector pB2GW7::GmST2 that contains No. 9 NAC transcription factor genes of the holy beans of above-mentioned soybean GmST2, it is characterized in that: said carrier cloning zone nucleotide sequence is shown in SEQ ID No.6.
No. 9 NAC transcription factor genes of holy beans according to the invention GmST2 expresses GmST2 gene and the application of putting forward high capability of anti-salt/drought tolerance in plant.。
Wherein: said plant is soybean or Arabidopis thaliana; Further, said Arabidopis thaliana is the Col-0 wild-type, and soybean varieties is Shandong beans 11.
Concrete, No. 9 NAC transcription factor genes of holy beans according to the invention GmST2 expresses the application of GmST2 in Arabidopis thaliana or soybean plant strain.
The present invention at first has been cloned into NAC transcription factor gene GmST2 in No. 9 plant of holy beans; Sharp gateway system excessively carries out the BP reaction with the GmST2 gene and is connected to the pDONR221 (see figure 1); Cloned in a large number in bacillus coli DH 5 alpha through the method that transforms then; Then carry out the LR reaction and be connected to this gene GmST2 on expression vector pK2GW7 (see figure 2) and the pB2GW7 (see figure 3), and in bacillus coli DH 5 alpha, express; Then screen the transgenic intestinal bacteria, and extract the conversion plasmid, will transform plasmid at last and change among the agrobacterium strains GV3101.To transform agrobacterium strains and change in Arabidopis thaliana and the soybean, thus the function of the GmST2 that checking is expressed.
Beneficial effect of the present invention: utilize existing plant gene engineering technology; The present invention clones first and has obtained holy beans No. 9 NAC transcription factor genes GmST2 and in Arabidopis thaliana, express, and the render transgenic Arabidopis thaliana has obtained the not available ability of putting forward high capability of anti-salt/drought tolerance of non-transgenic Arabidopis thaliana.Through the soybean auxiliary exogenous gene transforming method of embryo vacuum infiltration of sprouting this gene is changed in the soybean, prove through comparative analysis, transfer-gen plant improves than anti-salt/drought tolerance of non-transgenic plant.
Gene of the present invention can be widely used in cultivates anti-salt/drought-enduring plant variety.
Description of drawings
Fig. 1 is an entry vector pDONR221 collection of illustrative plates.
Fig. 2 is a plant expression vector pK2GW7 collection of illustrative plates.
Fig. 3 is a plant expression vector pB2GW7 collection of illustrative plates.
Fig. 4 is the cDNA electrophorogram of No. 9 NAC transcription factor genes of holy beans GmST2, and wherein: M is Marker, and swimming lane 1,2 is the cDNA of gene.
Fig. 5 is for changeing the Arabidopis thaliana pure lines screening of GmST2 gene.
Fig. 6 is No. 9 NAC transcription factor genes of holy beans GmST2 RealTime-PCR expression analysis in transgenic arabidopsis.
Fig. 7 is the analysis of salt resistance in the genetically modified Arabidopis thaliana.
Fig. 8 is the analysis of drought tolerance in the genetically modified Arabidopis thaliana.
Fig. 9 changes the soybean regeneration plant of GmST2 gene.
Figure 10 is that No. 9 NAC transcription factor genes of the holy beans of transgenic GmST2 Shandong beans 11PCR detects figure, and wherein: swimming lane M is Marker, the positive plasmid contrast of swimming lane positive control; The negative plant contrast of swimming lane negative control; Swimming lane 4,7,8 is changes GmST2 gene masculine plant.
Figure 11 is No. 9 NAC transcription factor genes of the holy beans of transgenic GmST2 Shandong beans 11 Southern-Blot analysiss, and wherein: swimming lane M is Marker, swimming lane+positive plasmid contrast; Swimming lane-negative plant contrast; Swimming lane 1,2,3 is changes GmST2 gene masculine plant.
Figure 12 is the analysis of salt resistance in the genetically modified soybean.
Embodiment
The clone of embodiment 1, GmST2
1.1 the extraction of No. 9 total RNA of holy beans
(1) No. 9 vegetable materials of holy beans is put into mortar, utilize liquid nitrogen to be ground into powder (directly applying to following experiment or freezing preserves subsequent use in-80 ℃ of Ultralow Temperature Freezers);
(2) etc. after the liquid nitrogen volatilization, immediately the 100-200mg plant powder is transferred in the 1.5ml centrifuge tube, adds 1ml Trizol extracting solution then rapidly, the vortex concussion is fully dissolved in the extracting solution sample, and room temperature is placed 5min;
(3) 4 ℃, 12,000rpm, centrifugal 10min transfers to the 0.9ml supernatant in the new 1.5ml centrifuge tube, adds 0.2ml chloroform thermal agitation mixing 15sec again, and room temperature is placed 2-5min;
(4) 4 ℃, 12,000rpm, centrifugal 10min transfers to the 0.4ml supernatant in the new 1.5ml centrifuge tube, adds the 0.4ml Virahol again, spins upside down mixing solution 15 times, and room temperature is placed 15mim;
(5) 4 ℃, 12,000rpm, centrifugal 10min abandons supernatant, with twice, 4 ℃ of the washing with alcohol of 1ml 75% deposition, 8,000rpm, centrifugal 5min;
(6) abandon supernatant, uncap and in Bechtop, go up the about 2-5min of dry RNA, add 40 μ l RNase-Free water, in 60 ℃, fully dissolve RNA 10min;
(7) survey the OD value and the concentration of RNA sample with ultraviolet spectrophotometer, A
260/ A
280It is good reaching 1.7-2.0; The quality that agarose gel electrophoresis detects.
1.2 the reverse transcription of RNA
(1) in centrifuge tube, add following material (40 μ l reaction system) successively:
(2) gently behind the mixing, 65 ℃ of sex change 5min are inserted on ice immediately, and ice bath is 1min at least;
(3) in centrifuge tube, add following material successively
(4) gently behind the mixing, 42 ℃ of water bath with thermostatic control 1h, 65 ℃ of sex change 10min ,-20 ℃ of preservations are subsequent use.
1.3 GmST2 gene clone
GmST2OX-F：5’-AAAAAGCAGGCTCGATGAAGGGAGAATTAGAGTTGCC-3’
GmST2OX-R：5’-AGAAAGCTGGGTTTCACATCTTCTGTAGGTACATGAACA-3’
(1) high-fidelity enzyme Primer Star carries out the reaction system (50 μ l system) as follows of the first step amplification of Gateway system:
Amplification condition is following:
After reaction finished, reaction solution detected in the 0.8%TAE agarose gel electrophoresis.
(2) the segmental purifying and recovering of clone gene (day root test kit)
1) cutting-out is had the segmental gel of purpose and put into the 1.5ml centrifuge tube and claim gel weight, add the sol solutions of 3 times of volumes, 60 ℃ of colloidal sol 10min, constantly upset during the colloidal sol;
2) after gel melts fully, all be drawn to and reclaim in the post, place a moment;
3) room temperature, 12000rpm, centrifugal 30Sec abandons solution;
4) rinsing liquid of adding 700 μ l in post, 12000rpm, centrifugal 1min abandons rinsing liquid;
5) rinsing liquid of adding 500 μ l in post, 12000rpm, centrifugal 1min abandons rinsing liquid;
6) void column, 12000rpm, centrifugal 2min;
7) the recovery post is uncapped and is dried 1-2min, puts into new clean 1.5ml centrifuge tube, adds the 40 μ l aqua sterilisas or the EB damping fluid of 60 ℃ of preheatings, places 2min;
8) the centrifugal 1min of 12000rpm, gained solution is the recovery fragment.
(3) high-fidelity enzyme Primer Star carries out second step amplification of Gateway system.
The attb primer sequence:
attb-F：5’-G?GGGACAAGT?TTG?TAC?AAAAAA?GCA?GGC?T-3’
attb-R：5’-GGG?GAC?CAC?TTT?GTA?CAA?GAA?AGC?TGG?GT-3’
Reaction system is (50 μ l system) as follows:
Amplification condition is following:
After reaction finished, reaction solution detected in the 0.8%TAE agarose gel electrophoresis, sees Fig. 4.
1.5 the segmental BP reaction of clone gene
BP reaction (Gateway system) system is following:
25 ℃ the reaction 8 hours-spend the night.
1.6 the competent preparation of intestinal bacteria (aseptic technique)
(1) gets DH5 α bacterial classification inoculation in 20ml LB liquid nutrient medium, 37 ℃ of shaking table overnight cultures;
(2) be inoculated in the 50ml LB liquid nutrient medium by 1: 100,37 ℃, 200rpm cultivated 1 hour, to OD
600Value is 0.4-0.6;
(3) bacterium liquid is placed on 30min on ice;
(4) 4 ℃, 4200rpm, centrifugal 10min abandons supernatant, adds the CaCl of the 0.1M of 10ml precooling
2The suspension thalline;
(5) bacterium liquid is placed on 10min on ice;
(6) 4 ℃, 4200rpm, centrifugal 10min abandons supernatant, adds the CaCl of the 0.1M of 2ml precooling
2The suspension thalline is sub-packed in the 1.5ml centrifuge tube, and existing usefulness or-80 ℃ of preservations behind liquid nitrogen flash freezer of adding final volume 30% glycerine are subsequent use.
1.7 colibacillary plasmid transforms (aseptic technique)
(1), flicks the centrifuge tube mixing, ice bath 30min with 1-5 μ l DNA or connect in the competent cell that product adds 50 μ l;
(2) 42 ℃, warm water bath heat shock 90sec, ice bath 2-3min immediately;
(3) add 1ml LB substratum, cultivate 40-50min for 37 ℃;
(4) room temperature, 4000rpm, centrifugal 3min collects thalline;
(5) bacterium is coated contained on the corresponding antibiotic culture plate, be inverted overnight cultures for 37 ℃.
1.8 colibacillus PCR checking
Reaction system is (20 μ l system) as follows:
Amplification condition is following:
After reaction finished, reaction solution detected in the 0.8%TAE agarose gel electrophoresis.
1.9DNA order-checking
The positive single bacterium colony that picking contains recombinant plasmid shakes with the liquid LB that contains Kan (25mg/L) and spends the night, and serves the order-checking of Hai Boya Bioisystech Co., Ltd then, and obtain sequencing result: gene cDNA sequence is shown in sequence table SEQ ID No.1.
Through sequential analysis; The nucleotide homology 98.02% of above-mentioned cDNA sequence and soybean Willms82; What three experiment proofs obtained is exactly No. 9 NAC transcription factor genes of holy beans, called after GmST2, and the nucleotide sequence of the cDNA of said gene GmST2 is shown in SEQ ID No.1.
1.10 the extraction of e. coli plasmid dna
(1) the picking mono-clonal is inoculated in 10ml and contains in the corresponding antibiotic LB liquid nutrient medium, 37 ℃ cultivate 8 hours-spend the night;
(2) room temperature, 12000rpm, centrifugal 1min collects thalline;
(3) abandon supernatant, add the solution I of 100 μ l low temperature precoolings, vibration suspension thalline;
(4) add the freshly prepared solution II of 200 μ l, fast fast upset mixing, ice bath 5min;
(5) solution clarification back adds the solution III of 150 μ l low temperature precoolings, ice bath 5min behind the mixing gently;
(6) 4 ℃, 12000rpm, centrifugal 10min draws in supernatant to the new 1.5ml centrifuge tube;
(7) add isopyknic phenol/chloroform/primary isoamyl alcohol (25/24/1), the vibration mixing;
(8) room temperature, 12000rpm, centrifugal 10min shifts the upper strata water in another new 1.5ml centrifuge tube, adds isopyknic chloroform: primary isoamyl alcohol (24: 1), again extracting once, the vibration mixing;
(9) room temperature, 12000rpm, centrifugal 10min shifts the upper strata water in another new 1.5ml centrifuge tube, adds isopyknic Virahol, and mixing is also placed 30min in-20 ℃;
(10) room temperature, 12000rpm, centrifugal 10min abandons supernatant and keeps deposition.
(11) with 75% washing with alcohol deposition twice, vacuum-drying 5min is dissolved in 40 μ l aqua sterilisas, puts to-20 ℃ of preservations subsequent use.
1.11 the segmental LR reaction of clone gene
LR reaction (Gateway system) system is following:
25 ℃ the reaction 8 hours-spend the night.
1.12 competent preparation of intestinal bacteria and plasmid transform (with 1.6,1.7)
1.13 colibacillus PCR checking (with 1.8)
1.4 the extraction of e. coli plasmid dna (with 1.10)
The clone zone nucleotide sequence of verifying the positive pK2GW7::GmST2 plasmid that changes the pK2GW7 carrier over to is shown in SEQID No.5.
The clone zone nucleotide sequence of verifying the positive pB2GW7::GmST2 plasmid that changes the pB2GW7 carrier over to is shown in SEQID No.6.
1.5 the competent preparation of Agrobacterium (aseptic technique)
(1) gets Agrobacterium GV3101 bacterial classification inoculation in 10ml YEP liquid nutrient medium, 28 ℃ of shaking table overnight cultures;
(2) be inoculated in the 50ml YEP liquid nutrient medium by 1: 50,28 ℃ shaking culture 3-4 hour, to OD
600Value is 0.4-0.6;
(3) 4 ℃, 4200rpm, centrifugal 10min collects thalline;
(4) abandon supernatant, add the NaCl suspension thalline of the 0.15M of 10ml precooling;
(5) repeating step 3;
(6) abandon supernatant, add the CaCl of the 20mM of 2ml precooling
2The suspension thalline is sub-packed in the 1.5ml centrifuge tube, and existing usefulness or adding final volume 7%DMSO-80 ℃ of preservations behind liquid nitrogen flash freezer are subsequent use.
1.6 the plasmid of Agrobacterium transforms (aseptic technique)
(1) 10 μ l DNAs is added in the competent cell of 50 μ l, flick the centrifuge tube mixing, ice bath 30min;
(2) liquid nitrogen flash freezer 1min; 37 ℃ of water-bath 5min, ice bath 2-3min immediately then;
(3) add 1ml YEP substratum, cultivate 2-4h for 28 ℃;
(4) room temperature, 4000rpm, centrifugal 3min collects thalline;
(5) bacterium is coated contained on the corresponding antibiotic YEP culture plate, be inverted for 28 ℃ and cultivate 48h.
1.7 transform the PCR checking of Agrobacterium
Reaction system is (20 μ l system) as follows:
Amplification condition is following:
After reaction finished, reaction solution detected in the 0.8%TAE agarose gel electrophoresis.
2.1 flower infestation method arabidopsis thaliana transformation
(1) Arabidopis thaliana (Col-0 wild-type) when growing to bolting 1cm cuts the top to induce the generation of adnation inflorescence;
(2) transforming previous day, the Agrobacterium GV3101 that contains the expression vector plasmid that gets the 1ml activation is added in the 40ml YEP substratum that contains corresponding microbiotic and 50 μ g/ml Rifampins, and 28 ℃ of concussions are cultured to OD
600Be about 1.0-1.2;
(3) room temperature, 4200rpm, centrifugal 10min collects thalline, with the resuspended thalline of dip-dyeing solution (5% sucrose, 0.05% Silwet L-77), makes OD
600Be about 0.8;
(4) with pipettor Agrobacterium is dripped on the inflorescence and contaminates, treat that all inflorescences are all infected after, Arabidopis thaliana is put into vacuum drier vacuumizes 1min;
(5) cover inflorescence with freshness protection package, cut off the top in one day as for 20-22 ℃ of lucifuge cultivation and expose inflorescence, cultivate again after one day and throw off freshness protection package, be cultured to seed maturity.
2.2 the surface sterilization of Arabidopis thaliana seed
Arabidopis thaliana neutron to be sterilized is in right amount put into the 1.5ml centrifuge tube; Ethanol (TritonX-100 that contains 0.03% volume ratio) the concussion sterilization 1min that adds 1ml 75%; Again with 70% ethanol concussion sterilization 1min (twice); With suction nozzle seed is drawn onto on the aseptic filter paper at last and dries up, with aseptic toothpick it is clicked and entered in the substratum then.
2.3 the screening of transfer-gen plant
T0 to results carries out surface sterilization for seed, evenly coats on the 1/2MS flat board after then (to contain corresponding microbiotic Baste).Vernalization treatment moves into the phytotron growth after 3 days.Sprouted about 10 days, the bottle-green plant of cotyledon is a transfer-gen plant, and cotyledon is sent out light green even the plant of yellow is the non-transgenic plant.Change in the soil transfer-gen plant over to growth and obtain T1 for transgenic seed until results; T1 receives seed for the plant individual plant; The seed continuation screening that every strain is collected separates the offspring than being 3: 1 (positives: grow to results T2 after positive plant feminine gender) is transplanted for transgenic seed, after individual plant is received and planted; The seed that every strain is collected can obtain being sheerly T2 for transgenic seed through screening, sees Fig. 5.
2.4 the extraction of plant RNA
(1) vegetable material is put into mortar, utilize liquid nitrogen to be ground into powder (directly applying to following experiment or freezing preserves subsequent use in-80 ℃ of Ultralow Temperature Freezers);
(2) etc. after the liquid nitrogen volatilization, immediately the 100-200mg plant powder is transferred in the 1.5ml centrifuge tube, adds 1ml Trizol extracting solution then rapidly, the vortex concussion is fully dissolved in the extracting solution sample, and room temperature is placed 5min;
(3) 4 ℃, 12,000rpm, centrifugal 10min transfers to the 0.9ml supernatant in the new 1.5ml centrifuge tube, adds 0.2ml chloroform thermal agitation mixing 15sec again, and room temperature is placed 2-5min;
(4) 4 ℃, 12,000rpm, centrifugal 10min transfers to the 0.4ml supernatant in the new 1.5ml centrifuge tube, adds the 0.4ml Virahol again, spins upside down mixing solution 15 times, and room temperature is placed 15mim;
(5) 4 ℃, 12,000rpm, centrifugal 10min abandons supernatant, with twice, 4 ℃ of the washing with alcohol of 1ml 75% deposition, 8,000rpm, centrifugal 5min;
(6) abandon supernatant, uncap and in Bechtop, go up the about 2-5min of dry RNA, add 40 μ l RNase-Free water, in 60 ℃, fully dissolve RNA 10min;
(7) survey the OD value and the concentration of RNA sample with ultraviolet spectrophotometer, A
260/ A
280It is good reaching 1.7-2.0; The quality that agarose gel electrophoresis detects.
2.6 the reverse transcription of RNA
(1) in centrifuge tube, add following material (40 μ l reaction system) successively:
(2) gently behind the mixing, 65 ℃ of sex change 5min are inserted on ice immediately, and ice bath is 1min at least;
(3) in centrifuge tube, add following material successively
(4) gently behind the mixing, 42 ℃ of water bath with thermostatic control 1h, 65 ℃ of sex change 10min ,-20 ℃ of preservations are subsequent use.
2.6 the RealTime-PCR of transfer-gen plant detects
The PCR method detects positive transfer-gen plant: resistant plant cDNA carries out PCR with GmST2 gene RealTime aligning primer and detects.
GmST2 gene RealTime primer sequence:
GmST2RealTime-F：5’-TTGAGCACGAGAACGAGACG-3’
GmST2RealTime-R：5’-CACATCGGGCGAAACCAC-3’
The reaction that general T aq enzyme carries out RealTime-PCR amplification is (15 μ l system) as follows:
Amplification condition is following:
Annotate: each sample is established 3 repetitions, and internal standard gene is selected TUB2 or ACTIN for use.
The result sees Fig. 6.
2.7 N.F,USP MANNITOL and Nacl handle Arabidopis thaliana
(1) surface sterilization of Arabidopis thaliana seed
With 2.2.
(2) N.F,USP MANNITOL and Nacl handle
Colo-0, GmST2-7, the GmST2-9 seed of bacterium of will having gone out evenly coated respectively on the 1/2MS flat board and (contained corresponding microbiotic Baste).Vernalization treatment moves into the phytotron growth after 3 days.Sprouted about 4 days, root grows to about 1cm, the Arabidopis thaliana seedling is moved on the 1/2MS culture medium flat plate of the N.F,USP MANNITOL that adds different concns (250,300,350,400mM) or Nacl (50,100,150,200mM).Treat that seedling grows to 11 days, observe the seedling upgrowth situation.
The result sees Fig. 7,8.
The auxiliary foreign gene conversion method of the embryo vacuum infiltration soybean transformation 3.1 soybean sprouts
(1) seed disinfection and cultivation processing in advance
Soybean seeds with 70% ethanol disinfection 5 minutes, is gone to use 0.1% HgCl behind the clean ethanol
2Sterilized 10 minutes, aseptic water washing 5-6 time, 25 ℃-28 ℃ following sterilized water seed soaking 12 hours.
Seed soaking back embryo is expanded the soybean that sprouts put into preparatory culture medium, 28 ℃, light were cultivated 1 day, and wherein said preparatory culture medium prescription is: MS+3.0mg/L 6-BA (6-benzyl aminoadenine)+20g/L sucrose+7g/L agar, pH5.8.
(2) the seed embryo expands and sprouts after cut, and exposes or the damage embryo position of sprouting
When pre-incubated soybean embryo integral body is expanded, radicle grows to 0.5 ± 0.1mm, during prominent breaking in the seed coat, peel off kind of a skin, cut radicle, keep the cotyledon of plumule, plumular axis and 1/2, locate pricker with dissecting needle at the plumule point simultaneously, make pinprick be covered with the plumule point.
(3) the vacuum infiltration auxiliary law Agrobacterium that will contain goal gene is infected the embryo of sprouting
The Agrobacterium that has plant expression vector of 4 ℃ of preservations is rule on the YEP solid medium that has added the 50mg/L Rifampin; 28 ℃ of dark culturing picking list bacterium colony after 3 days; Access contains the YEP liquid nutrient medium of 50mg/L Rifampin; Dark following 28 ℃, the switching once more after 24 hours of 200r/min shaking culture, the same terms was cultivated 16 hours down.Bacterium liquid is placed the centrifuge tube of sterilization, and 5000rpm collected thalline in centrifugal 5 minutes, with infecting the resuspended thalline of liquid, was adjusted to OD
600Value is 0.6, and adding 30mg/L Syringylethanone is subsequent use.The wherein said liquid formula that infects is: 0.1MS is a large amount of+and 0.1MS trace+B5 VITAMINs+0.5MES+1% glucose+2% sucrose, pH5.4.
The soybean of step (2) is immersed in the Agrobacterium bacterium liquid, vacuumized and keep 0.05MPa pressure 5-8min, dark following 28 ℃, 200r/min shaking culture infect 15-20min.
(4) cultivate altogether
The soybean of step (3) is taken out, inhale with aseptic filter paper and remove unnecessary bacterium liquid, tangent plane places on the common culture medium down, secretly cultivates 4 days down for 25 ℃-28 ℃.Wherein said culture medium prescription altogether is: MS+30mg/L Syringylethanone+20g/L sucrose+7g/L agar, pH5.8.
(5) grow thickly shoot regeneration, screening and little seedling rooting, screening
Soybean after cultivating altogether with sterile water wash 4 times, is blotted with aseptic filter paper again, be forwarded on the inducing clumping bud substratum, under 14 hours conditions of 25 ℃ and illumination every day, per two weeks change to new substratum, and cultured continuously 20 ± 2 days differentiates the bud of growing thickly.Wherein said inducing clumping bud screening and culturing based formulas is: MS+3.0mg/L 6-BA (6-benzyl aminoadenine)+0.2mg/L IAA (indolylacetic acid)+0.125 μ L/mL Baste+200mg/L CEFOTAXIME SODIUM STERILE+20g/L sucrose+7g/L agar, pH5.8.
Grow to 1-2 centimetre the bud of growing thickly after jigging choosing, budlet is cut separately, transfer on the screening culture medium of taking root, under 14 ± 1 hours conditions of 25 ℃ ℃ and illumination every day, cultured continuously 14 days.The wherein said screening and culturing based formulas of taking root is: MS+0.4mg/L IBA (second diindyl butyric acid)+0.125 μ L/mL Baste+200mg/L CEFOTAXIME SODIUM STERILE+20g/L sucrose+7g/L agar, pH5.8.
(6) seedling strong sprout and transplanting
Select the good seedling of root growth to transfer on the strong seedling culture base, under 14 hours conditions of 25 ℃ ℃ and illumination every day, cultivated 7-10 days.When seedling after screening survival and root growth are good, it is not injured root takes out, remove remaining substratum and move into soil, cover flowerpot to improve humidity with film.Wherein said strong seedling culture based formulas is: MS+2.0mg/LKT (kinetin)+0.4mg/LNAA (NAA)+20g/L sucrose+7g/L agar, pH5.8 sees Fig. 9.
3.2CTAB method is extracted the transfer-gen plant genomic dna
Take by weighing the regeneration plant blade of 0.5g; Shred the back and use liquid nitrogen grinding; Rapidly with powder transfer to the 1.5ml centrifuge tube, add 700 μ l then and be preheated to the thin basic ethanol that 65 ℃ 2xCTAB extracts damping fluid and 0.1%, put upside down the centrifuge tube mixing gently; Place 65 ℃ of water bath heat preservation 2h then, put upside down mixing frequently.Mixture adds isopyknic phenol after being cooled to room temperature: atmosphere is imitative: primary isoamyl alcohol (25: 24: 1), 4 ℃ of centrifugal 10min of following 10000g.Water is transferred in the clean centrifuge tube, adds isopyknic chloroform: primary isoamyl alcohol (24: 1), 4 ℃ of centrifugal 10min of following 10000g.Water is transferred in the clean centrifuge tube, adds the two volumes absolute ethyl alcohol, gentle mixing is placed 30min deposit D NA for-20 ℃.4 ℃ of centrifugal 10min of following 10000g discard liquid, and with 70% washing with alcohol twice, behind the drying at room temperature DNA, add the TE liquid dissolving that 100 sudden strains of a muscle contain the RNA enzyme of no DNA enzyme, 37 ℃ of water-bath 30min.Add 200 μ l absolute ethyl alcohols, gentle mixing is placed 30min deposit D NA for-20 ℃.4 ℃ of centrifugal 10min of following 10000g discard liquid, and with 70% washing with alcohol twice, behind the drying at room temperature DNA, add 40 μ l sterilized water dissolving DNAs, and 4 ℃ of preservations are for use.
3.3PCR detect transfer-gen plant with Sothern blot
3.3.1PCR method detects positive transfer-gen plant
The total DNA of resistant plant is respectively with 35S promoter primer, GmST2 gene primer be connected with Gm ST2 gene fragment and plant expression vector fragments sequence primer carries out PCR and detects.
35S fragment promoter primer sequence:
35S-F：5’-GCAGAGGCATCTTCAACG-3’
35S-R：5’-GACGATCTACCCGAGCAA-3’
GmST2 gene primer sequence:
GmST2-F：5’-ATGAAGGGAGAATTAGAGTTGCC-3’
GmST2-R：5’-TCACATCTTCTGTAGGTACATGAACA-3’
35S promoter F+GmST2 gene R primer sequence:
35S-F：5’-GCAGAGGCATCTTCAACG-3’
GmST2-R：5’-TCACATCTTCTGTAGGTACATGAACA-3’
Reaction system is (20 μ l system) as follows:
Amplification condition is following:
After reaction finished, reaction solution detected in the 0.8%TAE agarose gel electrophoresis, sees Figure 10.
3.3.2 Southern is hybridized checking
Choose 3 strain positive plants (GmST2OX-1, GmST2OX-2, GmST2OX-3) and carry out Southern hybridization checking.Step is following:
(1) the CTAB method is extracted positive plant, wild-type plant DNA
(2) the SpeI enzyme is cut positive plant and wild-type plant DNA, electrophoresis
(3) change film
1. alkaline denaturation: under the room temperature gel is immersed 30min in the sex change liquid of several times volume.Sex change liquid: 0.5M NaOH; 1.5MNaCl.
2. neutralization: gel is transferred to neutralizer 15min.Neutralizer: 1M Tris-HCl (pH 7.4); 1.5M NaCl;
3. shift: cut out NC film or nylon membrane and cut off one jiao by the size of gel and serve as a mark, after the water-soaked, immerse and shift 5min in the liquid.Cut one than the wide slightly rectangular Whatman 3mm filter paper of film as salt bridge, cut by the size of gel again that 3-5 opens filter paper and a large amount of paper handkerchief is subsequent use.Shift.(transfer process generally needs 8-24hr, and every separated number hr changes the paper handkerchief that has wet.Shift liquid with 20 * SSC.Attention can not have bubble between film and glue.To prevent to be infected with on the film other dirts in the entire operation process.) transfer liquid (20 * SSC): NaCl 175.3g; Trisodium citrate 82.2g, NaOH transfers pH to 7.0, adds ddH2O to 1000ml.
4. shift and finish back taking-up NC film, immerse 6 * SSC solution and count min, the gel particle of being infected with on the flush away film places between two filter paper, and 80 ℃ of baking 2h are clipped in the NC film between two layers of filter paper then, are stored in dry place.
(4), and hybridize and detect according to Roche DIG High Prime DNA Labeling and Detection Starter Kit II specification sheets mark GmST2.
Detect the genome of finding selected GmST2 transfer-gen plant (GmST2OX-1, GmST2OX-2, GmST2OX-3) and all integrated goal gene, see Figure 11.
3.4 the drought resisting of genetically engineered soybean/salt tolerance analysis
Tl is for the genetically engineered soybean seed and contrast seed behind presoaking and germinating, is transplanted to respectively in the polypots (1/2Hogland liquid nutrient medium), under 25 ℃ of conditions of room temperature; After making its 2 weeks of growing; Grow to one heart stage of two leaves with the 1/2Hogland liquid nutrient medium pouring that contains NaCl 50rnmol/L, for avoiding the salt shock effect, concentration with the mode that increases progressively 25rnrnol/L every day with salt; Until handling predetermined concentration 250rnmol/L, transfer-gen plant and wild-type plant phenotype are observed.See Figure 12.
1/2Hogland liquid culture based formulas:
Claims (5)
1. No. 9 NAC transcription factor genes of the holy beans of soybean GmST2, it is characterized in that: the nucleotide sequence of said gene cDNA is shown in SEQ ID No.1.
2. plant expression vector pK2GW7::GmST2 who contains No. 9 NAC transcription factor genes of the holy beans of the said soybean of claim 1 GmST2, it is characterized in that: said carrier cloning zone nucleotide sequence is shown in SEQ ID No.5.
3. plant expression vector pB2GW7::GmST2 who contains No. 9 NAC transcription factor genes of the holy beans of the said soybean of claim 1 GmST2, it is characterized in that: said carrier cloning zone nucleotide sequence is shown in SEQ ID No.6.
4. No. 9 NAC transcription factor genes of the holy beans of the said soybean of claim 1 GmST2 expresses GmST2 gene and the application of putting forward high capability of anti-salt/drought tolerance in plant.
5. application as claimed in claim 4 is characterized in that: said plant is soybean or Arabidopis thaliana.
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| CN102660554A (en) * | 2012-04-27 | 2012-09-12 | 山东大学 | Soybean holy bean 9# NAC transcription factor gene GmST1 and application thereof |
| CN102660554B (en) * | 2012-04-27 | 2013-10-16 | 山东大学 | Soybean holy bean 9# NAC transcription factor gene GmST1 and application thereof |
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| CN106367423A (en) * | 2016-09-30 | 2017-02-01 | 山东大学 | Membrane-bound NAC transcription factor gene GmNTL7 in soybean Williams 82 and application thereof |
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| CN106399333A (en) * | 2016-11-30 | 2017-02-15 | 山东大学 | Soybean holy bean 9# LAMMER protein kinase gene GmPK6 and application thereof |
| CN111763251A (en) * | 2020-07-21 | 2020-10-13 | 四川农业大学 | Trifolium repens transcription factor TrNAC and coding sequence and application thereof |
| CN111763251B (en) * | 2020-07-21 | 2022-08-19 | 四川农业大学 | Trifolium repens transcription factor TrNAC and coding sequence and application thereof |
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