CN106350526A - Glycine max(L.)Merr Shengdou No.9 MYB transcription factor family gene GmMYB84 and application thereof - Google Patents
Glycine max(L.)Merr Shengdou No.9 MYB transcription factor family gene GmMYB84 and application thereof Download PDFInfo
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Abstract
The invention discloses a glycine max Shengdou No.9 MYB transcription factor family gene GmMYB84 and a plant expression vector containing the gene GmMYB84. The invention also discloses application of the gene GmMYB84 to arabidopsis and soybean plants for improving the drought stress tolerance. Experiments prove that the drought resistant capability of transgenic plants is greatly improved than that of non-transgenic plants, so that theoretical basis and practice basis are provided for the improvement on crop drought resistance; the condition indicates that the gene GmMYB84 can be widely applied to the culture of drought resistant plant varieties.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, more particularly, to a kind of Semen sojae atricolor (glycine max (l.) merr)
Myb transcription factor family gene gmmyb84 and its application in holy bean 9.
Background technology
Plant is exposed in the middle of natural environment, is often subject to the impact of various environment-stress, such as arid, extreme temperature
Degree, nutrient dificiency and pest and disease damage etc..These environment-stress not only constrain the growth promoter of plant, also result in grain simultaneously and make
The underproduction of thing.The growth of higher plant own growth and the response to environmental change are by regulating and controlling the expression of genes of interest Lai real
Existing.And the interaction of transcription factor and gene cis acting element, can be used as the molecular switch of gene expression regulation.Exactly
The interaction of transcription factor and degeneration-resistant functional gene promoter region cis acting element have activated the table of anti contravariance related gene
Reach, improve the comprehensive resistance of plant.
Myb transcription factor family is one of maximum transcription factor family, has and send out in almost all of eukaryote
Existing.Plant myb transcription factor family participates in regulating development of plants, metabolism, biology and abiotic stress response etc..At present,
Have been found that 206 genes belong to myb transcription factor family in Semen sojae atricolor.
Liao in 2009 etc. further determined that 156 genes from this 206 Semen sojae atricolor myb transcription factor family members,
Wherein at least has 43 genes to participate in abiotic stress response, author subsequently have extensively studied gmmyb76, gmmyb92 and
The function of this 3 genes of gmmyb177.Rt-pcr result shows, this 3 genes all show expression when drought stress is processed
The trend that amount substantially raises, transgenic Arabidopsis plants seed germination rate, n plant survival rate and plant height under drought stress conditions
Deng being all generally higher than col-0 plant.2014, the research such as rao showed overexpression gmcam4 gene (gmcam4-oe) in Semen sojae atricolor
The drought-enduring of plant can be significantly increased.Author passes through yeast two-hybrid assay (y2h) and bimolecular fluorescence complementary experiment (bifc),
Identify the interaction albumen gmmyb2 of gmcam4, gmmyb2 albumen is the r2r3-myb albumen containing 2 myb domains, enters
One step demonstrates important function in drought stress tolerance for the myb transcription factor.2013, mistake in the report arabidopsiss such as zhang
Expression gmgbp1 (glycine max gamyb-binding protein gene, genbank dq112540) gene can have
Effect improves the drought-resistance ability of plant.2015, the research such as wang showed that gmwrky27 albumen can be mutual with gmmyb174 albumen
Effect, in conjunction with the cis acting element of gmnac29 gene promoter upstream, the expression of suppression gmnac29 gene, thus increase
The strong drought-enduring ability of Semen sojae atricolor.
Since myb family transcription factor finds that people study to it and have been achieved for major progress so far first, it is related to
The many aspects such as growth and development of plants, hormone signal response.Of particular concern is, film combination transcription factor resists to plant
The adverse circumstances such as characteristic of disease, winter resistance, drought resistance and salt tolerance adapt to be respectively provided with highly important regulating and controlling effect.But at present its signal is passed
Lead and still lack breakthrough achievement in research with the research of regulatory mechanism, and focus mostly in model plant arabidopsiss.
Through retrieval, yet there are no Semen sojae atricolor sage bean No. 9 myb transcription factor family gene gmmyb84 and its correlation function
Report.
Content of the invention
For current technology situation, it is an object of the invention to provide a kind of Semen sojae atricolor (glycine max (l.) merr) is holy
Myb transcription factor family gene gmmyb84 and its application in bean 9.
Myb transcription factor family gene in Semen sojae atricolor of the present invention sage's bean 9 it is characterised in that: described unnamed gene
For Semen sojae atricolor myb transcription factor gene gmmyb84, the nucleotide sequence of this gene cdna is as shown in seq id no.1.
Present invention also offers a kind of plant expression vector containing above-mentioned Semen sojae atricolor myb transcription factor gene, its feature exists
In: described plant expression vector is named as expression vector pk2gw7::gmmyb84, this expression vector cloning region nucleotide sequence
As shown in seq id no.2.
Or, present invention also offers a kind of plant expression vector containing above-mentioned Semen sojae atricolor myb transcription factor gene, it is special
Levy and be: described plant expression vector is named as expression vector pb2gw7::gmmyb84, this expression vector cloning region nucleotide
Sequence is as shown in seq id no.3.
Myb transcription factor gene answering in improving plant drought stress tolerance in Semen sojae atricolor sage's bean 9 of the present invention
With.
Wherein: described plant is preferably Semen sojae atricolor or arabidopsiss.Further, described arabidopsiss are col-0 wild types, Semen sojae atricolor
Kind is Shandong bean 11.
In the present invention, applicant separates from Semen sojae atricolor sage's bean 9 and obtains myb transcription factor family gene gmmyb84, will
This gene goes to proves its function in model plant arabidopsiss, then return again to this gene in former plant Semen sojae atricolor, result obtains
The transfer-gen plant that drought tolerance improves.
Specifically, Semen sojae atricolor myb transcription factor family gene gmmyb84 expresses gmmyb84 in arabidopsiss or soybean plant strain
The illustration of application.
Applicant is cloned into myb transcription factor family gene gmmyb84 first in holy No. 9 plant of bean；Using
Gmmyb84 gene is passed through bp reaction forming to (see Fig. 1) on pdonr221 carrier by gateway system, then passes through conversion
Method is cloned in escherichia coli dh5 α in a large number, then carries out lr reaction and this gene gmmyb84 is connected to expression vector
On pk2gw7 (see Fig. 2) or pb2gw7 (see Fig. 3), and express in escherichia coli dh5 α；Then screening transgenic escherichia coli,
And extract conversion plasmid, finally conversion plasmid is proceeded in agrobacterium strains gv3101.Conversion agrobacterium strains are proceeded to plan south
In mustard and Semen sojae atricolor, obtain the transfer-gen plant of drought tolerance raising, checking have expressed the function of gene gmmyb84.
The invention has the beneficial effects as follows: using existing plant gene engineering technology, the present invention clones first and has obtained sage
No. 9 myb transcription factor family genes of bean are simultaneously expressed in arabidopsiss, and render transgenic arabidopsiss obtain non-transgenic and intend south
The ability of the drought stress tolerance not available for mustard.Pass through the gene transformation of Semen sojae atricolor Germinating Embryo vacuum infiltration auxiliary simultaneously
Method proceeds to this gene in Semen sojae atricolor, proves through comparative analysiss, and transfer-gen plant has very than the drought tolerance of nontransgenic plants
Big raising.Fully demonstrate myb transcription factor family gene gmmyb84 in Semen sojae atricolor sage's bean 9 of the present invention and improve plant
In strain drought stress tolerance, there is important effect, indicate that gene of the present invention can be widely used for cultivating drought-enduring plant
Kind.
Brief description
Fig. 1 is entry vector pdonr221 collection of illustrative plates.
Fig. 2 is plant expression vector pk2gw7 collection of illustrative plates.
Fig. 3 is plant expression vector pb2gw7 collection of illustrative plates.
Fig. 4 is the cds full-length clone electrophoretogram of myb transcription factor family gene gmmyb84 in holy bean 9
Wherein: m is marker, swimming lane 1,2,3,4 is the cds full-length clone of gene.
Fig. 5 is the arabidopsiss pure lines screening of 35s::gmmyb84 transgenic.
Fig. 6 is myb transcription factor family gene gmmyb84 rt-pcr expression point in transgenic arabidopsis in holy bean 9
Analysis.
Fig. 7 is 35s::gmmyb84 transgenic Arabidopsis plants under the conditions of 0,100,200,300mmol/l mannitol
Drought Resistance Analysis
Wherein: under the conditions of a:0mm mannitol；Under the conditions of b:100mm mannitol；C:200mm mannitol condition
Under；Under the conditions of d:300mm mannitol.
Fig. 8 be 35s::gmmyb84 genetically engineered soybean regeneration plant acquisition, be followed successively by the sprouting of a: seed, b: preculture,
C: co-culture, d: infect, co-culture, shoot regeneration of growing thickly, e: take root, f: strong sprout and transplanting.
Fig. 9 is the pcr detection figure that in holy bean 9, myb transcription factor family gene gmmyb84 turns Shandong bean 11
Wherein: upper figure is the result that 35s-f/r primer carries out pcr amplification, figure below is carried out for 35s-f+gmmyb84-r primer
The result of pcr amplification
Swimming lane m is marker, and swimming lane positive control compares for positive plasmid；Swimming lane negative control is that negative plant pair is shone；
Upper in figure: swimming lane 1,2,3,4,5,6,7,8,9 is and turns gmmyb84 transgenic positive plant.Lower in figure: swimming lane 1,2,3,4,5,
6th, 8 is to turn gmmyb84 transgenic positive plant.
Figure 10 is the southern blot analysis that in holy bean 9, myb transcription factor family gene gmmyb84 turns Shandong bean 11
Figure.
Wherein: swimming lane m be marker, swimming lane "+" for positive plasmid compare；Swimming lane "-" is that negative plant pair is shone；Swimming lane
" 1,2,3,4,5,6,7 " are gmmyb84 genetically engineered soybean positive plant.
Specific embodiment
Embodiment 1, the clone of gmmyb84
The extraction of the 1.1 holy total rna of bean 9
(1) holy No. 9 vegetable materials of bean are put in mortar, be ground into powder using liquid nitrogen and (directly apply to following
Experiment or freeze save backup in -80 DEG C of ultra cold storage freezers)；
Etc. (2), after liquid nitrogen volatilization, immediately 100-200mg plant powder is transferred in 1.5ml centrifuge tube, then rapidly
Add 1ml trizol extracting solution, vortex concussion makes sample be fully dissolved in extracting solution, room temperature places 5min；
(3) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.9ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.2ml chloroform and acutely vibrate mixing 15sec, room temperature places 2-5min；
(4) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.4ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.4ml isopropanol, spin upside down 15 mixing solution, room temperature places 15mim；
(5) 4 DEG C, 12,000rpm, it is centrifuged 10min, abandons supernatant, precipitate twice with the washing with alcohol of 1ml 75%, 4 DEG C, 8,
000rpm, is centrifuged 5min；
(6) abandon supernatant, uncap and rna about 2-5min is dried in the superclean bench, add 40 μ l rnase-free water,
Fully dissolving rna10min in 60 DEG C；
(7) od value and the concentration of rna sample, a is surveyed with ultraviolet spectrophotometer260/a280Reach 1.7-2.0 to be preferred；Agar
The quality of sugared detected through gel electrophoresis.
The reverse transcription of 1.2 rna
(1) sequentially add following material (40 μ l reaction system) in centrifuge tube:
(2) after gently mixing, 65 DEG C of degeneration 5min, it is inserted on ice immediately, ice bath at least 1min；
(3) sequentially add following material in centrifuge tube
(4) after gently mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of degeneration 10min, -20 DEG C save backup.
1.3 gmmyb84 gene clonings
Gmmyb84 ox-f:5 '-aaaaagcaggctcaatgtctacttcaaagagcgtcag-3 '
Gmmyb84 ox-r:5 '-agaaagctgggttttatttttgtaacttgctaaattgc-3 '
(1) high-fidelity enzyme primer star carries out following (the 50 μ l of reaction system of the first step amplification of gateway system
System):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
(2) purification of clone gene fragment reclaims (Tiangeng test kit)
1) gel cutting with purpose fragment is put into 1.5ml centrifuge tube and claim gel weight, add the molten of 3 times of volumes
Glue, 60 DEG C of colloidal sol 10min, constantly overturn during colloidal sol；
2) after gel melts completely, all it is drawn in recovery column, place a moment；
3) room temperature, 12000rpm, it is centrifuged 30sec, abandon solution；
4) add the rinsing liquid of 700 μ l, 12000rpm in post, be centrifuged 1min, abandon rinsing liquid；
5) add the rinsing liquid of 500 μ l, 12000rpm in post, be centrifuged 1min, abandon rinsing liquid；
6) void column, 12000rpm, it is centrifuged 2min；
7) recovery column is uncapped and is dried 1-2min, puts into new clean 1.5ml centrifuge tube, adds 40 μ l of 60 DEG C of preheatings to go out
Bacterium water or eb buffer, place 2min；
8) 12000rpm centrifugation 1min, resulting solution as reclaims fragment.
(3) high-fidelity enzyme primer star carries out the second step amplification of gateway system.
Attb primer sequence:
Attb-f:5 '-g ggg aca agt ttg tac aaa aaa gca ggc t-3 '
Attb-r:5 '-ggg gac cac ttt gta caa gaa agc tgg gt-3 '
Reaction system is following (50 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies, sees Fig. 4.
The bp reaction of 1.4 clone gene fragments
Bp reaction (gateway system) system is as follows:
Note: pdonr221 purchases in invitrogen (Shanghai) commerce and trade company limited.
25 DEG C react 8 hours-overnight.
The preparation (sterile working) of 1.5 E. coli competent
(1) dh5 α strain is taken to be inoculated in 20ml lb fluid medium, 37 DEG C of shaking table cultures are overnight；
(2) it is inoculated in 50ml lb fluid medium by 1:100,37 DEG C, 200rpm cultivates 1 hour, to od600It is worth and be
0.4-0.6；
(3) bacterium solution is put on ice for 30min；
(4) 4 DEG C, 4200rpm, it is centrifuged 10min, abandons supernatant, add the cacl of the 0.1m of 10ml pre-cooling2Suspension thalline；
(5) bacterium solution is put on ice for 10min；
(6) 4 DEG C, 4200rpm, it is centrifuged 10min, abandons supernatant, add the cacl of the 0.1m of 2ml pre-cooling2Suspension thalline, subpackage
In 1.5ml centrifuge tube, now with or add final volume 30% glycerol save backup for -80 DEG C after liquid nitrogen flash freezer.
1.6 colibacillary plasmids convert (sterile working)
(1) 1-5 μ l plasmid dna or connection product are added in the competent cell of 50 μ l, flick centrifuge tube and mix, ice
Bath 30min；
(2) 42 DEG C, tepidarium heat shock 90sec, ice bath 2-3min immediately；
(3) 1ml lb culture medium, 37 DEG C of culture 40-50min are added；
(4) room temperature, 4000rpm, it is centrifuged 3min, collects thalline；
(5) bacterium is coated on the culture plate containing corresponding antibiotic, 37 DEG C of inversion overnight incubation.
1.7 escherichia coli pcr checkings
Reaction system is following (20 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
1.8 dna sequencings
The positive single bacterium colony that picking contains recombiant plasmid is shaken overnight with the liquid lb containing kan (25mg/l), then send Ji
Nan Boshang Bioisystech Co., Ltd is sequenced, and obtains sequencing result: gene cdna sequence is as shown in sequence table seq id no.1.
Through sequence analysis, above-mentioned cdna sequence and the nucleotide homology 100% of Semen sojae atricolor willms82, test proof three times
Obtain is exactly willms82myb transcription factor family gene, is named as gmmyb84, the core of the cdna of described gene gmmyb84
Nucleotide sequence is as shown in seq id no.1.
The extraction of 1.9 escherichia coli plasmid dna
(1) picking monoclonal is inoculated in the lb fluid medium that 10ml contains corresponding antibiotic, 37 DEG C of culture 8h；
(2) room temperature, 12,000rpm, it is centrifuged 1min, collects thalline；
(3) abandon supernatant, add the solutions i of 100 μ l low temperature pre-coolings, vibrate suspension thalline；
(4) add freshly prepared solutions i i of 200 μ l, fast upset soon mixes, ice bath 5min；
(5) solutions i ii of 150 μ l low temperature pre-coolings, ice bath 5min after gently mixing are added after solution clarification；
(6) 4 DEG C, 12000rpm, it is centrifuged 10min, draw supernatant to new 1.5ml centrifuge tube；
(7) add isopyknic phenol/chloroform/isoamyl alcohol (25/24/1), vibration mixes；
(8) room temperature, 12,000rpm, it is centrifuged 10min, transfer upper strata aqueous phase, in another new 1.5ml centrifuge tube, adds
Isopyknic chloroform: isoamyl alcohol (24: 1), then extract once, vibration mixes；
(9) room temperature, 12,000rpm, it is centrifuged 10min, transfer upper strata aqueous phase, in another new 1.5ml centrifuge tube, adds
Isopyknic isopropanol, mixes and places 30min in -20 DEG C；
(10) room temperature, 12,000rpm, it is centrifuged 10min, abandon supernatant and retain precipitation.
(11) precipitated twice with 75% washing with alcohol, be vacuum dried 5min, be dissolved in 40 μ l aquesterilisa, put and preserve to -20 DEG C
Standby.
The lr reaction of 1.10 clone gene fragments
Lr reaction (gateway system) system is as follows:
Note: pk2gw7, pb2gw7 purchase in invitrogen (Shanghai) commerce and trade company limited.
25 DEG C are reacted 8 hours.
The preparation of 1.11 E. coli competent and plasmid conversion (with 1.5,1.6)
1.12 escherichia coli pcr checkings (with 1.7)
The extraction (with 1.9) of 1.13 escherichia coli plasmid dna
Checking proceeds to the cloning region nucleotide sequence such as seq of the positive pk2gw7::gmmyb84 plasmid of pk2gw7 carrier
Shown in id no.2.
Checking proceeds to the cloning region nucleotide sequence such as seq of the positive pb2gw7::gmmyb84 plasmid of pb2gw7 carrier
Shown in id no.3.
The competent preparation of 1.14 Agrobacteriums (sterile working)
(1) Agrobacterium gv3101 strain is taken to be inoculated in 10ml yep fluid medium, 28 DEG C of shaking table cultures are overnight；
(2) it is inoculated in 50ml yep fluid medium by 1:50,28 DEG C of shaken cultivation 3-4 hours, to od600It is worth and be
0.4-0.6；
(3) 4 DEG C, 4,200rpm, it is centrifuged 10min, collects thalline；
(4) abandon supernatant, add the nacl suspension thalline of the 0.15mol/l of 10ml pre-cooling；
(5) repeat step 3；
(6) abandon supernatant, add the cacl of the 20mmol/l of 2ml pre-cooling2Suspension thalline, is sub-packed in 1.5ml centrifuge tube, existing
With or add final volume 7%dmso save backup for -80 DEG C after liquid nitrogen flash freezer.
Plasmid conversion (sterile working) of 1.15 Agrobacteriums
(1) 10 μ l plasmid dnas are added in the competent cell of 50 μ l, flick centrifuge tube and mix, ice bath 30min；
(2) liquid nitrogen flash freezer 1min；Then 37 DEG C of water-bath 5min, ice bath 2-3min immediately；
(3) 1ml yep culture medium, 28 DEG C of culture 2-4h are added；
(4) room temperature, 4,000rpm, it is centrifuged 3min, collects thalline；
(5) bacterium is coated on the yep culture plate containing corresponding antibiotic, be inverted culture 48h for 28 DEG C.
The pcr checking of 1.16 conversion Agrobacteriums
Reaction system is following (20 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
Embodiment 2, express the functional verification of holy No. 9 myb transcription factor family gene gmmyb84 of bean in arabidopsiss
2.1 flower infestation method arabidopsis thaliana transformations
(1), when arabidopsiss (col-0 wild type) grow to bolting 1cm, top is cut to induce the generation of the raw inflorescence in side；
(2) in conversion the previous day, the Agrobacterium gv3101 containing expression vector plasmid that 1ml activated is taken to be added to containing corresponding
In the 40ml yep culture medium of antibiotic and 50 μ g/ml rifampicin, 28 DEG C of concussion and cultivates to od600It is about 1.0-1.2；
(3) room temperature, 4,200rpm, it is centrifuged 10min, collects thalline, with dip-dyeing solution (5% sucrose, 0.05%silwet l-
77) resuspended thalline, makes od600It is about 0.8；
(4) with pipettor, Agrobacterium is dripped to and contaminated on inflorescence, after all inflorescences are all infected, arabidopsiss are put
Enter evacuation 1min in vacuum desiccator；
(5) cover inflorescence with freshness protection package, cut off top within one day as 20-22 DEG C of lucifuge culture and expose inflorescence, be further cultured for one
Throw off freshness protection package after it, cultivate to seed maturity.
The surface sterilization of 2.2 arabidopsiss seeds
Arabidopsiss neutron subject to sterilization in right amount is put in 1.5ml centrifuge tube, adds the ethanol of 1ml 75% (to contain
The tritonx-100 of 0.03% volume ratio) concussion sterilization 1min, then shake sterilization 1min (twice) with 70% ethanol, finally
With suction nozzle, seed is drawn onto on aseptic filter paper and dries up, then clicked and entered in culture medium with aseptic toothpick.
The screening of 2.3 transfer-gen plants
For seed, surface sterilization is carried out to the t0 harvesting, is then spread evenly across afterwards on 1/2ms flat board (containing corresponding antibiosis
Plain baste).Phjytotron growth is moved into after vernalization treatment 3d.Sprout about 10 days, the bottle-green plant of cotyledon is planted for transgenic
Strain, and cotyledon is sent out light green or even the plant of yellow is nontransgenic plants.Transfer-gen plant is proceeded to growth in soil until harvesting
Obtain t1 for transgenic seed, t1 continues screening for plant individual plant sowing, the seed that every plant is collected, and offspring's segregation ratio is 3:
The positive plant of 1 (positive: negative) grows to results t2 for transgenic seed after transplanting, after individual plant sowing, every plant of kind collected
Son can obtain being sheerly t2 for transgenic seed through screening, sees Fig. 5.
The extraction of 2.4 plant rna
(1) vegetable material is put in mortar, using liquid nitrogen be ground into powder (directly apply to following experiment or
Person freeze save backup in -80 DEG C of ultra cold storage freezers)；
Etc. (2), after liquid nitrogen volatilization, immediately 100-200mg plant powder is transferred in 1.5ml centrifuge tube, then rapidly
Add 1ml trizol extracting solution, vortex concussion makes sample be fully dissolved in extracting solution, room temperature places 5min；
(3) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.9ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.2ml chloroform and acutely vibrate mixing 15sec, room temperature places 2-5min；
(4) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.4ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.4ml isopropanol, spin upside down 15 mixing solution, room temperature places 15mim；
(5) 4 DEG C, 12,000rpm, it is centrifuged 10min, abandons supernatant, precipitate twice with the washing with alcohol of 1ml 75%, 4 DEG C, 8,
000rpm, is centrifuged 5min；
(6) abandon supernatant, uncap and rna about 2-5min is dried in the superclean bench, add 40 μ l rnase-free water,
Fully dissolving rna 10min in 60 DEG C；
(7) od value and the concentration of rna sample, a is surveyed with ultraviolet spectrophotometer260/a280Reach 1.7-2.0 to be preferred；Agar
The quality of sugared detected through gel electrophoresis.
The reverse transcription of 2.5 rna
(1) sequentially add following material (40 μ l reaction system) in centrifuge tube:
(2) after gently mixing, 65 DEG C of degeneration 5min, it is inserted on ice immediately, ice bath at least 1min；
(3) sequentially add following material in centrifuge tube
(4) after gently mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of degeneration 10min, -20 DEG C save backup.
The realtime-pcr detection of 2.6 transfer-gen plants
Pcr method screening transgenic positive plant: will have the cdna gmmyb84 gene rt-pcr primer of the plant of resistance
Carry out pcr detection.
Gm myb84 gene rt-pcr primer sequence:
Gm myb84rt-f:5 '-aaccaggcaactcctatgcc-3 '
Gm myb84rt-r:5 '-cgtccaaaatgctgaggcac-3 '
The reaction that common taq enzyme carries out realtime-pcr amplification is following (15 μ l system):
Amplification condition is as follows:
Note: internal standard gene selects gmtub.
Result is shown in Fig. 6.
2.7 treatment with mannitol arabidopsiss
(1) surface sterilization (with 2.2) of arabidopsiss seed.
(2) Mannitol and nacl process
Col-0, gmmyb84-1, gmmyb84-2 seed of bacterium of having gone out is spread evenly across on 1/2ms flat board respectively (containing phase
The antibiotic baste answering).Vernalization treatment moves into phjytotron growth after 3 days.Sprouting about 4d, root length to 1cm about, will intend
Southern mustard seedling move to add variable concentrations Mannitol (0,100,200,300mmol/l) 1/2ms culture medium flat plate on.Treat little
Seedling length, to 11d, observes seedling upgrowth situation.
Result is shown in Fig. 7.
Embodiment 3, express the functional verification of holy No. 9 myb transcription factor family gene gmmyb84 of bean in Semen sojae atricolor
The gene transformation method soybean transformation of 3.1 Semen sojae atricolor Germinating Embryo vacuum infiltration auxiliary
(1) seed disinfection and preculture are processed
By soybean seed with 70% ethanol disinfection 5min, after removing ethanol, use 0.1%hgcl2Sterilization 10min, sterilized water rushes
Wash 5-6 time, sterilized water seed soaking 12h at 25 DEG C -28 DEG C.
Embryo after seed soaking is expanded the Semen sojae atricolor sprouting and puts into precultivation medium, 28 DEG C, optical culture 1d, wherein said preculture
Culture medium prescription is: ms+3.0mg/l 6-ba (6- benzyl aminoadenine)+20g/l sucrose+7g/l agar, ph5.8.
(2) through cutting after seed embryo expands and sprouts, expose or damage Germinating Embryo position
Integrally expand when pre-incubated Semen sojae atricolor embryo, radicle length to 0.5 ± 0.1mm, do not dash forward when breaking in the seed coat, peel off kind of a skin, cut
Remove radicle, retain the cotyledon of plumule, plumular axis and 1/2, use dissecting needle pricker at plumule point simultaneously, make pinprick be covered with plumule point.
(3) Agrobacterium containing genes of interest is infected Germinating Embryo by vacuum infiltration auxiliary law
The Agrobacterium with plant expression vector of 4 DEG C of preservations with the addition of the yep solid culture of 50mg/l rifampicin
Rule on base, 28 DEG C of dark culturing picking single bacterium colony after 3 days, access the yep fluid medium containing 50mg/l rifampicin, dark
Lower 28 DEG C, 200r/min shaken cultivation transfer again after 24 hours, under the same terms cultivate 16h.By bacterium solution be placed in sterilizing from
In heart pipe, 5,000rpm 5 minutes collects thallines of centrifugation, with infecting the resuspended thalline of liquid, adjust to od600It is worth for 0.6, add 30mg/
L acetosyringone is standby.The wherein said formula of liquid that infects is: a large amount of+0.1ms micro+b5 vitamin+0.5mes+1% of 0.1ms
Glucose+2% sucrose, ph5.4.
By in the Semen sojae atricolor immersion Agrobacterium bacterium solution of step (2), evacuation simultaneously maintains 0.05mpa pressure 5-8min, under dark
28 DEG C, 200r/min shaken cultivation, infect 15-20min.
(4) co-culture
The Semen sojae atricolor of step (3) is taken out, sucks unnecessary bacterium solution with aseptic filter paper, cut and be placed face down on co-cultivation culture medium
On, light culture 4 days at 25 DEG C -28 DEG C.Wherein said co-cultivation culture medium prescription is: ms+30mg/l acetosyringone+20g/l
Sucrose+7g/l agar, ph5.8.
(5) grow thickly shoot regeneration, screening and little seedling rooting, screening
Semen sojae atricolor sterile water wash 4 times after co-culturing, then blotted with aseptic filter paper, it is forwarded to inducing clumping bud culture
On base, at 25 DEG C and under the conditions of daily illumination 14h, shift to new culture medium every two weeks, continuously cultivate 20 ± 2d, differentiate clump
Sprout.Wherein said inducing clumping bud screening and culturing based formulas are: ms+3.0mg/l 6-ba (6- benzyl aminoadenine)+
0.2mg/l iaa (heteroauxing)+0.125 μ l/ml baste+200mg/l Cefotaxime Sodium+20g/l sucrose+7g/l agar,
ph5.8.
Grow to the Multiple Buds of 1-2cm after choosing screening, budlet is individually cut, transfers to and take root in screening culture medium, 25
DEG C and daily illumination 14 ± 1h under the conditions of, continuously cultivate 14d.Wherein said screening and culturing based formulas of taking root are: ms+0.4mg/l
Iba (second diindyl butanoic acid)+0.125 μ l/ml baste+200mg/l Cefotaxime Sodium+20g/l sucrose+7g/l agar, ph5.8.
(6) seedling strong sprout and transplanting
The seedling selecting root growth good is transferred on strong seedling culture base, at 25 DEG C and under the conditions of daily illumination 14 hours,
Culture 7-10d.When seedling is after screening is survived and root growth is good, it is not injured root and take out, remove remaining culture medium
Move into soil, cover flowerpot to improve humidity with thin film.Wherein said strong seedling culture based formulas are: ms+2.0mg/l kt is (exciting
Element)+0.4mg/l naa (α-naphthaleneacetic acid)+20g/l sucrose+7g/l agar, ph5.8, is shown in Fig. 8.
3.2 ctab methods extract transfer-gen plant genome dna
Weigh the regeneration plant blade of 0.5g, after shredding, use liquid nitrogen grinding, rapidly powder is transferred to 1.5ml centrifuge tube
In, it is subsequently adding 700 μ l and is preheated to 65 DEG C of 2xctab Extraction buffer and 0.1% thin base ethanol, gently overturn centrifuge tube
Mix, be subsequently placed in 65 DEG C of water bath heat preservation 2h, frequently overturn and mix.Mixture adds isopyknic phenol: atmosphere after being cooled to room temperature
Imitative: isoamyl alcohol (25: 24: 1), 10,000g centrifugation 10min at 4 DEG C.Aqueous phase is transferred in a clean centrifuge tube, the body such as addition
Long-pending chloroform: isoamyl alcohol (24:1), 10000g centrifugation 10min at 4 DEG C.Aqueous phase is transferred in a clean centrifuge tube, adds two
Times volume dehydrated alcohol, gentle mix, place 30min precipitation dna for -20 DEG C.At 4 DEG C, 10,000g centrifugation 10min, discards liquid,
And with 70% washing with alcohol twice, after drying at room temperature dna, add the dissolving of 100ul te liquid, 37 DEG C of water-bath 30min.Add 200 μ l
Dehydrated alcohol, gentle mixing, place 30min precipitation dna for -20 DEG C.At 4 DEG C, 10,000g centrifugation 10min, discards liquid, is used in combination
70% washing with alcohol twice, after drying at room temperature dna, adds the aseptic water dissolution dna of 40 μ l, 4 DEG C of preservations are stand-by.
3.3 pcr and sothern blot detection transfer-gen plants
3.3.1 pcr method detection positive transgenic plant
The total dna of resistant plant is respectively with 35s promoter primer, gmmyb84 gene primer be connected with gmmyb84 genetic fragment
Carry out pcr detection with the aligning primer of plant expression vector fragment.
35s fragment promoter primer sequence:
35s-f:5 '-gcagaggcatcttcaacg-3 '
35s-r:5 '-gacgatctacccgagcaa-3 '
Gmmyb84 gene primer sequence:
Gmmyb84 ox-f:5 '-aaaaagcaggctcaatgtctacttcaaagagcgtcag-3 '
Gmmyb84 ox-r:5 '-agaaagctgggttttatttttgtaacttgctaaattgc-3 '
35s promoter f+gmmyb84 gene r primer sequence:
35s-f:5 '-gcagaggcatcttcaacg-3 '
Gmmyb84 ox-r:5 '-agaaagctgggttttatttttgtaacttgctaaattgc-3 '
Reaction system is following (20 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies, sees Fig. 9.
3.3.2 sothern blot hybridization verification
Choose 3 plants of positive plants (gm myb84ox-1, gm myb84ox-2, gm myb84ox-3) and carry out sothern
Blot hybridization verification.Step is as follows:
(1) ctab method extracts positive plant, WT lines dna
(2) ecori enzyme action positive plant and WT lines dna, electrophoresis
(3) transferring film
1. alkaline denaturation: under room temperature, gel is immersed 30min in the denaturing liquid of several times volume.Denaturing liquid: 0.5mol/l
naoh；1.5mol/l nacl.
2. neutralize: gel is transferred to neutralizer 15min.Neutralizer: 1mol/l tris-hcl (ph 7.4)；1.5mol/
l nacl；
3. shift: cut out nc film or nylon membrane by the size of gel and cut off one jiao as labelling, after water-soaked, immersion turns
5min in liquid relief.Cut a strip whatman 3mm filter paper more slightly wider than film as salt bridge, then cut 3-5 by the size of gel and open
Filter paper and substantial amounts of napkin are standby.Shifted.(transfer process generally requires 8-24h, every a few hours change wet fall
Napkin.Shift liquid 20 × ssc.Note can not having bubble between film and glue.It is infected with film to be prevented during whole operation
Other dirts) transfer liquid (20 × ssc): nacl 175.3g；Trisodium citrate 82.2g, naoh adjust ph to 7.0, plus ddh2o is extremely
1,000ml.
4. take out nc film after transfer terminates, immerse 6 × ssc solution for several minutes, wash away the gel particle of contamination on film, be placed in
Between two filter paper, 80 DEG C of baking 2h, then nc film is clipped between two layers of filter paper, is stored at being dried.
(4) according to roche dig high prime dna labeling and detection starter kit ii
Description labelling gmmyb84, and hybridized and detect.
Detection finds selected gmmyb84 transfer-gen plant (gmmyb84ox-1, gmmyb84ox-2, gmmyb84ox-3)
Genome all incorporate genes of interest, see Figure 10.
The Drought Stress Tolerance Analysis of A of 3.4 Transgenic soybean plants
Tl for genetically engineered soybean seed with compare seed after presoaking and germinating, be transplanted to polypots (1/2hogland respectively
Fluid medium) in, with the pouring of 1/2hogland fluid medium so as to growth 2 girths are to two leaves under the conditions of 25 DEG C of room temperature
After one heart stage, stop pouring nutritional solution, cultivate 20 days under drought condition, nutrient solution again again after 20 days, to transfer-gen plant
Observed with WT lines phenotype.
1/2hogland liquid culture based formulas:
Sequence table
<110>Shandong University
<120>Semen sojae atricolor sage No. 9 myb transcription factor family gene gmmyb84 of bean and its application
<141> 2016-11-22
<160> 3
<210> 1
<211> 954
<212> cdna
<213>Semen sojae atricolor (glycine max?(l.) merr)
<221>Semen sojae atricolor sage No. 9 myb transcription factor family gene gmmyb84 of bean
<222>(1) ... (954)
<400>1
atgtctactt caaagagcgt cagcagttct agtgaagatg acaatgaact tagaagaggg 60
ccttggactc ttgaagagga taatttgctc tcccaatata tttctagtca tggagaaggg 120
cgatggaatt tgctagctaa acgttcagga ttaaagcgaa ctgggaaaag ttgcagatta 180
aggtggctaa attatctaaa gccagatgta aaacggggaa atttaacccc acaagagcaa 240
cttataatcc tcgaactcca ctcaaagtgg ggaaacaggt ggtcaaaaat tgcacaaaat 300
ttgccaggca gaacagacaa tgaaatcaag aactattgga gaactaggat tcagaaacaa 360
gcaagacatt tgaaaattga cactgacagc agagagtttc aggaacttgt taggcgtttc 420
tggatgccta gattgcttca aaaagccaaa gaatcatctt cttcagccat gtcaattcaa 480
aaccaggcaa ctcctatgcc ttttgatggt gtttctcagc attcaactgt tgggaccata 540
ccatcacatt cacacacccc ttggcaggga ccttgtatga atgaagctgg tcccacttac 600
atggaccaac atgagcagaa ctcagactct gaacacaaca atggttcatg catctccttg 660
tctgagtcag caaattttcc aaaagtgcct cagcattttg gacgcaccac catcacccaa 720
tatcatgcct tgaataacaa tgactttggc accttcacat atgacggcta caatgtaagc 780
aacaatgtct atgagatgga caacttcaaa acgcctacta caagggtggc tgaggatgcg 840
caatacccaa ctggtgattg tcaaatggta ggaagcaatt gggtaaacag cgattttgca 900
tgtaacatgt ggaacatgga tgaattgtgg caatttagca agttacaaaa ataa 954
<210> 2
<211> 10442
<212> dna
<213>artificial sequence
<221>the plant expression vector pk2gw7::gmmyb84 containing gene gmmyb84
<222>(1) ... (10442)
<400> 2
ctcccatatg gtcgactaga gccaagctga tctcctttgc cccggagatc accatggacg 60
actttctcta tctctacgat ctaggaagaa agttcgacgg agaaggtgac gataccatgt 120
tcaccaccga taatgagaag attagcctct tcaatttcag aaagaatgct gacccacaga 180
tggttagaga ggcctacgcg gcaggtctca tcaagacgat ctacccgagt aataatctcc 240
aggagatcaa ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaact 300
gcatcaagaa cacagagaaa gatatatttc tcaagatcag aagtactatt ccagtatgga 360
cgattcaagg cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaagaaag 420
tagttcctac tgaatcaaag gccatggagt caaaaattca gatcgaggat ctaacagaac 480
tcgccgtgaa gactggcgaa cagttcatac agagtctttt acgactcaat gacaagaaga 540
aaatcttcgt caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata 600
cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc 660
tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag 720
gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcctctg 780
ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 840
ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 900
acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt 960
tggagaggac tccggtattt ttacaacaat accacaacaa aacaaacaac aaacaacatt 1020
acaatttact attctagtcg acctgcaggc ggccgcacta gtgatatcgg ggacaagttt 1080
gtacaaaaaa gcaggctatg tctacttcaa agagcgtcag cagttctagt gaagatgaca 1140
atgaacttag aagagggcct tggactcttg aagaggataa tttgctctcc caatatattt 1200
ctagtcatgg agaagggcga tggaatttgc tagctaaacg ttcaggatta aagcgaactg 1260
ggaaaagttg cagattaagg tggctaaatt atctaaagcc agatgtaaaa cggggaaatt 1320
taaccccaca agagcaactt ataatcctcg aactccactc aaagtgggga aacaggtggt 1380
caaaaattgc acaaaatttg ccaggcagaa cagacaatga aatcaagaac tattggagaa 1440
ctaggattca gaaacaagca agacatttga aaattgacac tgacagcaga gagtttcagg 1500
aacttgttag gcgtttctgg atgcctagat tgcttcaaaa agccaaagaa tcatcttctt 1560
cagccatgtc aattcaaaac caggcaactc ctatgccttt tgatggtgtt tctcagcatt 1620
caactgttgg gaccatacca tcacattcac acaccccttg gcagggacct tgtatgaatg 1680
aagctggtcc cacttacatg gaccaacatg agcagaactc agactctgaa cacaacaatg 1740
gttcatgcat ctccttgtct gagtcagcaa attttccaaa agtgcctcag cattttggac 1800
gcaccaccat cacccaatat catgccttga ataacaatga ctttggcacc ttcacatatg 1860
acggctacaa tgtaagcaac aatgtctatg agatggacaa cttcaaaacg cctactacaa 1920
gggtggctga ggatgcgcaa tacccaactg gtgattgtca aatggtagga agcaattggg 1980
taaacagcga ttttgcatgt aacatgtgga acatggatga attgtggcaa tttagcaagt 2040
tacaaaaata aggggaccac tttgtacaag aaagctgggt gatatcccgc ggccatgcta 2100
gagtccgcaa aaatcaccag tctctctcta caaatctatc tctctctatt tttctccaga 2160
ataatgtgtg agtagttccc agataaggga attagggttc ttatagggtt tcgctcatgt 2220
gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 2280
aatttctaat tcctaaaacc aaaatccagt gacctgcagg catgcgacgt cgggcccaag 2340
cttagcttga gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt 2400
gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taacggatat 2460
ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg 2520
gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct 2580
tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc taagttacgc 2640
gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa 2700
agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg 2760
gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg 2820
aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg 2880
accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga 2940
ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc gagcgcatcc 3000
aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc accacgccgg 3060
ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt tccctaatca 3120
tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc 3180
gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac caggaaggcc 3240
gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac 3300
ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg 3360
acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag 3420
catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag agatcgaggc 3480
ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct 3540
gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg ccagcttggc 3600
cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt 3660
gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac gcaaggggaa 3720
cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac gaccatcgca 3780
acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt cgattccgat 3840
ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc 3900
ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta 3960
gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac 4020
ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc cgacctggtg 4080
gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg 4140
tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac 4200
gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg cactgccgcc 4260
gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg 4320
ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag aaaatgagca 4380
aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt 4440
tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg ccggcggagg 4500
atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc gagctgctat 4560
ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag tagatgaatt 4620
ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga cgccgtggaa 4680
tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg 4740
ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc 4800
cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt tgaaggccgc 4860
gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc 4920
ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat 4980
taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc tctatgacgt 5040
gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga 5100
ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag aggtttccgc 5160
agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg cggtttccca 5220
tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg gccgcgtgtt 5280
ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg gaaagcagaa 5340
agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca tgcagcgtac 5400
gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct tgattagccg 5460
ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg agctagctga 5520
ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg ttcaccccga 5580
ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac gccgcgccgc 5640
aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg 5700
agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg acctgccgga 5760
gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa 5820
cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc tagggcaaat 5880
tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca cgtacattgg 5940
gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa agccgtacat 6000
tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg atttttccgc 6060
ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg cataactgtc 6120
tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct 6180
acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct 6240
acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg accgccggcg 6300
cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 6360
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 6420
gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 6480
gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 6540
gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 6600
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6660
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 6720
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 6780
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 6840
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 6900
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6960
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7020
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7080
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7140
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7200
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7260
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7320
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7380
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7440
ggtcatgcat gatatatctc ccaatttgtg tagggcttat tatgcacgct taaaaataat 7500
aaaagcagac ttgacctgat agtttggctg tgagcaatta tgtgcttagt gcatctaatc 7560
gcttgagtta acgccggcga agcggcgtcg gcttgaacga atttctagct agacattatt 7620
tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg 7680
cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga 7740
cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg 7800
cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct 7860
catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa 7920
atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa 7980
cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct 8040
cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag 8100
ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga 8160
gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc 8220
gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct 8280
tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat 8340
ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt 8400
cccccatgat gtttaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc 8460
tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca 8520
tagactgtac cccaaaaaaa catgtcataa caagaagcca tgaaaaccgc cactgcgccg 8580
ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgacggcag ttacgctact 8640
tgcattacag cttacgaacc gaacgaggct tatgtccact gggttcgtgc ccgaattgat 8700
cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 8760
gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 8820
agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 8880
atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 8940
gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 9000
tttaatgtac tgaattaacg ccgaattgaa ttatcagctt gcatgccggt cgatctagta 9060
acatagatga caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta 9120
tcgcgtatta aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg 9180
tcatgcatta catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa 9240
tcatcgcaag accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac 9300
gatctgcttg actctagcta gagtccgaac cccagagtcc cgctcagaag aactcgtcaa 9360
gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga 9420
agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt 9480
cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat 9540
tttccaccat gatattcggc aagcaggcat cgccgtgggt cacgacgaga tcctcgccgt 9600
cgggcatccg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt 9660
cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc 9720
gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca 9780
ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct 9840
gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca 9900
cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgga 9960
gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg 10020
acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga 10080
atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc 10140
ctcgatcgag ttgagagtga atatgagact ctaattggat accgagggga atttatggaa 10200
cgtcagtgga gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt 10260
ttgaacgcgc aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg 10320
cggctgagtg gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc 10380
ccgcgtcatc ggcgggggtc ataacgtgac tcccttaatt ctcatgtatg ataattcgag 10440
ct 10442
<210> 3
<211> 10189
<212> dna
<213>artificial sequence
<221>the plant expression vector pb2gw7::gmmyb84 containing gene gmmyb84
<222>(1) ... (10189)
<400> 3
ctcccatatg gtcgactaga gccaagctga tctcctttgc cccggagatc accatggacg 60
actttctcta tctctacgat ctaggaagaa agttcgacgg agaaggtgac gataccatgt 120
tcaccaccga taatgagaag attagcctct tcaatttcag aaagaatgct gacccacaga 180
tggttagaga ggcctacgcg gcaggtctca tcaagacgat ctacccgagt aataatctcc 240
aggagatcaa ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaact 300
gcatcaagaa cacagagaaa gatatatttc tcaagatcag aagtactatt ccagtatgga 360
cgattcaagg cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaagaaag 420
tagttcctac tgaatcaaag gccatggagt caaaaattca gatcgaggat ctaacagaac 480
tcgccgtgaa gactggcgaa cagttcatac agagtctttt acgactcaat gacaagaaga 540
aaatcttcgt caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata 600
cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc 660
tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag 720
gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcctctg 780
ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 840
ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 900
acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt 960
tggagaggac tccggtattt ttacaacaat accacaacaa aacaaacaac aaacaacatt 1020
acaatttact attctagtcg acctgcaggc ggccgcacta gtgatatcgg ggacaagttt 1080
gtacaaaaaa gcaggctatg tctacttcaa agagcgtcag cagttctagt gaagatgaca 1140
atgaacttag aagagggcct tggactcttg aagaggataa tttgctctcc caatatattt 1200
ctagtcatgg agaagggcga tggaatttgc tagctaaacg ttcaggatta aagcgaactg 1260
ggaaaagttg cagattaagg tggctaaatt atctaaagcc agatgtaaaa cggggaaatt 1320
taaccccaca agagcaactt ataatcctcg aactccactc aaagtgggga aacaggtggt 1380
caaaaattgc acaaaatttg ccaggcagaa cagacaatga aatcaagaac tattggagaa 1440
ctaggattca gaaacaagca agacatttga aaattgacac tgacagcaga gagtttcagg 1500
aacttgttag gcgtttctgg atgcctagat tgcttcaaaa agccaaagaa tcatcttctt 1560
cagccatgtc aattcaaaac caggcaactc ctatgccttt tgatggtgtt tctcagcatt 1620
caactgttgg gaccatacca tcacattcac acaccccttg gcagggacct tgtatgaatg 1680
aagctggtcc cacttacatg gaccaacatg agcagaactc agactctgaa cacaacaatg 1740
gttcatgcat ctccttgtct gagtcagcaa attttccaaa agtgcctcag cattttggac 1800
gcaccaccat cacccaatat catgccttga ataacaatga ctttggcacc ttcacatatg 1860
acggctacaa tgtaagcaac aatgtctatg agatggacaa cttcaaaacg cctactacaa 1920
gggtggctga ggatgcgcaa tacccaactg gtgattgtca aatggtagga agcaattggg 1980
taaacagcga ttttgcatgt aacatgtgga acatggatga attgtggcaa tttagcaagt 2040
tacaaaaata aggggaccac tttgtacaag aaagctgggt gatatcccgc ggccatgcta 2100
gagtccgcaa aaatcaccag tctctctcta caaatctatc tctctctatt tttctccaga 2160
ataatgtgtg agtagttccc agataaggga attagggttc ttatagggtt tcgctcatgt 2220
gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 2280
aatttctaat tcctaaaacc aaaatccagt gacctgcagg catgcgacgt cgggcccaag 2340
cttagcttga gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt 2400
gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taacggatat 2460
ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg 2520
gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct 2580
tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc taagttacgc 2640
gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa 2700
agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg 2760
gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg 2820
aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg 2880
accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga 2940
ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc gagcgcatcc 3000
aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc accacgccgg 3060
ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt tccctaatca 3120
tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc 3180
gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac caggaaggcc 3240
gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac 3300
ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg 3360
acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag 3420
catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag agatcgaggc 3480
ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct 3540
gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg ccagcttggc 3600
cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt 3660
gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac gcaaggggaa 3720
cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac gaccatcgca 3780
acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt cgattccgat 3840
ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc 3900
ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta 3960
gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac 4020
ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc cgacctggtg 4080
gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg 4140
tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac 4200
gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg cactgccgcc 4260
gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg 4320
ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag aaaatgagca 4380
aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt 4440
tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg ccggcggagg 4500
atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc gagctgctat 4560
ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag tagatgaatt 4620
ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga cgccgtggaa 4680
tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg 4740
ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc 4800
cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt tgaaggccgc 4860
gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc 4920
ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat 4980
taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc tctatgacgt 5040
gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga 5100
ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag aggtttccgc 5160
agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg cggtttccca 5220
tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg gccgcgtgtt 5280
ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg gaaagcagaa 5340
agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca tgcagcgtac 5400
gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct tgattagccg 5460
ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg agctagctga 5520
ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg ttcaccccga 5580
ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac gccgcgccgc 5640
aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg 5700
agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg acctgccgga 5760
gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa 5820
cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc tagggcaaat 5880
tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca cgtacattgg 5940
gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa agccgtacat 6000
tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg atttttccgc 6060
ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg cataactgtc 6120
tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct 6180
acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct 6240
acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg accgccggcg 6300
cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 6360
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 6420
gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 6480
gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 6540
gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 6600
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6660
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 6720
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 6780
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 6840
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 6900
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6960
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7020
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7080
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7140
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7200
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7260
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7320
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7380
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7440
ggtcatgcat gatatatctc ccaatttgtg tagggcttat tatgcacgct taaaaataat 7500
aaaagcagac ttgacctgat agtttggctg tgagcaatta tgtgcttagt gcatctaatc 7560
gcttgagtta acgccggcga agcggcgtcg gcttgaacga atttctagct agacattatt 7620
tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg 7680
cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga 7740
cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg 7800
cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct 7860
catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa 7920
atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa 7980
cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct 8040
cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag 8100
ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga 8160
gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc 8220
gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct 8280
tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat 8340
ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt 8400
cccccatgat gtttaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc 8460
tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca 8520
tagactgtac cccaaaaaaa catgtcataa caagaagcca tgaaaaccgc cactgcgccg 8580
ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgacggcag ttacgctact 8640
tgcattacag cttacgaacc gaacgaggct tatgtccact gggttcgtgc ccgaattgat 8700
cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 8760
gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 8820
agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 8880
atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 8940
gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 9000
tttaatgtac tgaattaacg ccgaattgaa ttatcagctt gcatgccggt cgatctagta 9060
acatatagat gacaccgcgc gcgataattt atcctagttt gcgcgctata ttttgttttc 9120
tatcgcgtat taaatgtata attgcgggac tctaatcata aaaacccatc tcataaataa 9180
cgtcatgcat tacatgttaa ttattacatg cttaacgtaa ttcaacagaa attatatgat 9240
aatcatcgca agaccggcaa caggattcaa tcttaagaaa ctttattgcc aaatgtttga 9300
acgatctgct tgactctagg ggtcatcaga tttcggtgac gggcaggacc ggacggggcg 9360
gcaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc ccgtgcttga 9420
agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc atgcgcacgc 9480
tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt gcctccaggg 9540
acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg cggggggaga 9600
cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag ggacccgcgt 9660
aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag cgctcccgca 9720
gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg aagttgaccg 9780
tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc gcctcggtgg 9840
cacggcggat gtcggccggg cgtcgttctg ggctcatggt agatcccctc gatcgagttg 9900
agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt cagtggagca 9960
tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg aacgcgcaat 10020
aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg ctcagtggct 10080
ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc 10140
gggggtcata acgtgactcc cttaattctc atgtatgata attcgagct 10189
Claims (5)
1. in a kind of Semen sojae atricolor sage bean 9 myb transcription factor gene it is characterised in that: described unnamed gene be Semen sojae atricolor myb transcription because
The nucleotide sequence of sub-family gene gmmyb84, this gene cdna is as shown in seq id no.1.
2. a kind of plant expression vector containing Semen sojae atricolor myb transcription factor gene described in claim 1 it is characterised in that: described
Plant expression vector is named as expression vector pk2gw7::gmmyb84, this expression vector cloning region nucleotide sequence such as seq
Shown in id no.2.
3. a kind of plant expression vector containing myb transcription factor family gene in Semen sojae atricolor sage bean 9 described in claim 1, its
It is characterised by: described plant expression vector is named as expression vector pb2gw7::gmmyb84, this expression vector cloning region nucleoside
Acid sequence is as shown in seq id no.3.
4. myb transcription factor gene answering in improving plant drought stress tolerance in Semen sojae atricolor sage bean 9 described in claim 1
With.
5. as claimed in claim 4 application it is characterised in that: described plant is Semen sojae atricolor or arabidopsiss.
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| CN107164404A (en) * | 2017-06-30 | 2017-09-15 | 浙江农林大学 | Alpine ash EgrZFP6 adapts to the purposes of stress improving plant under osmotic stress |
| CN108047320A (en) * | 2018-02-13 | 2018-05-18 | 淮阴工学院 | The application of Protein G mMYB12 and encoding gene in isoflavones accumulation and salt tolerance is improved |
| CN109517846A (en) * | 2018-11-21 | 2019-03-26 | 华中农业大学 | Method based on CRISPR/Cas9 system high flux construction cotton mutant library |
| CN111087458A (en) * | 2019-12-30 | 2020-05-01 | 上海交通大学 | Alfalfa MYB transcription factor and aluminum toxin resistant application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107164404A (en) * | 2017-06-30 | 2017-09-15 | 浙江农林大学 | Alpine ash EgrZFP6 adapts to the purposes of stress improving plant under osmotic stress |
| CN107164404B (en) * | 2017-06-30 | 2020-10-20 | 浙江农林大学 | Application of E.grandis EgrZFP6 in improving stress adaptation of plants under osmotic stress |
| CN108047320A (en) * | 2018-02-13 | 2018-05-18 | 淮阴工学院 | The application of Protein G mMYB12 and encoding gene in isoflavones accumulation and salt tolerance is improved |
| CN109517846A (en) * | 2018-11-21 | 2019-03-26 | 华中农业大学 | Method based on CRISPR/Cas9 system high flux construction cotton mutant library |
| CN111087458A (en) * | 2019-12-30 | 2020-05-01 | 上海交通大学 | Alfalfa MYB transcription factor and aluminum toxin resistant application thereof |
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