CN109913471A - A kind of sorghum transcription factor SbGRF4 gene and its recombinant vector and expression - Google Patents
A kind of sorghum transcription factor SbGRF4 gene and its recombinant vector and expression Download PDFInfo
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Abstract
The invention discloses a kind of sorghum transcription factor SbGRF4 genes, and nucleotide sequence is as shown in SEQ ID NO.1.Invention additionally discloses a kind of protein of sorghum transcription factor SbGRF4 gene coding, and amino acid sequence is as shown in SEQ ID NO.2.The invention also discloses recombinant vectors and expression comprising above-mentioned sorghum transcription factor SbGRF4 gene.The present invention passes through the protein sequence of rice GRF4 gene, sorghum homologous gene SbGRF4 is obtained from sorghum genome database, full length gene 921bp, and according to the gene constructed prokaryotic expression carrier, it is purified with protein purification system, as a result further research albumin crystal structure and biological characteristics lay the foundation, and then foundation is provided for the disease resistance for improving sorghum by gene editing method.
Description
Technical field
The present invention relates to a kind of sorghum transcription factor SbGRF4 gene and its recombinant vector and expressions, belong to biological skill
Art field.
Background technique
Sorghum (Sorghum bicolor (L.) Moench) also known as reed broomcorn millet, sorgo are a kind of important cereal crops,
With stronger degeneration-resistant border ability and feed, wine brewing, the important source material for producing bio-fuel, papermaking and industrial starch, together
When be also a kind of herding forage crops widely used in recent years.Cultivation history of the sorghum in China is long, before foundation
Not large-scale planting, until 1970s, with the development of economy and society, China just gradually payes attention to and scale is planted
Train sorghum.Sorghum belongs to short-day C4 plant, and compared to other energy crops, sorghum has more obvious photosynthetic efficiency
And therefore higher yield heterosis is known as " high energy crop ".In recent years, as global environmental problem and the energy are endangered
Machine is got worse, and people constantly seek a kind of safe and pollution-free, efficient biomass energy again.Wherein sorghum is as one
The good energy-source plant of kind causes the attention and research of scientific circles.In order to more further investigate sorghum, U.S. Department of Energy biology and
Environmental Research Center was smoothly sequenced and was assembled to sorghum genome in 2009, this indicate the research work to sorghum into
Enter gene level, while also providing convenience for the clone of sorghum important gene and functional analysis.
Be otherwise known as transcription factor (transcription factor TF) trans-acting factor, can open with gene
Mover combines, so that the expression of gene is activated or inhibited, to cope with the signal inside and outside from organism.It is reported that turning
Factor regulatory function multiplicity is recorded, is all played a significant role in the entire growth cycle of plant, is sprouted in the suspend mode of regulation plant, seed
The various aspects such as hair, aging, hormone response and the abiotic and biotic of reply all play an important role.
Growth regulatory factor GRF (growth-regulating factors) be in plant it is special containing transcription factor,
Its major regulatory plant cell size participates in the plant growth and developments mistakes such as chloroplaset proliferation, Stamen development, regulation osmotic stress
Journey.GRF transcription factor mainly includes two conservative structural domains of QLQ and WRC.And GRF transcription factor is deposited in various plants
, including rice, tomato, arabidopsis, rape, potato, corn etc., but so far, both at home and abroad there is not yet GRF gene is in height
Report is studied in fine strain of millet.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of sorghum transcription factor SbGRF4 gene and its again
Group carrier and expression.
The technical scheme is that a kind of sorghum transcription factor SbGRF4 gene, nucleotide sequence such as SEQ ID
Shown in NO.1.
The present invention also provides the protein of sorghum transcription factor SbGRF4 gene coding, amino acid sequence such as SEQ
Shown in ID NO.2.
Correspondingly, the present invention provides recombinant vector and expression comprising above-mentioned sorghum transcription factor SbGRF4 gene.
Specifically, the recombinant vector the preparation method is as follows: extracting sorghum RNA, and reverse transcription goes out cDNA；It is with cDNA
Template amplification SbGRF4 gene；By SbGRF4 amplified production after BamHI and XhoI double digestion, it is inserted by DNA ligase
In pET-28a expression vector through same double digestion, recombinant plasmid pET-28a-SbGRF4 is obtained.
Preferably, using cDNA as template, using following primer amplification SbGRF4 gene, primer sequence is as follows:
Upstream primer: SbGRF4-F:GCGGATCCATGTCGTTCCACTACCCCGA underscore is BamHI restriction enzyme site；
Downstream primer: GCCTCGAGTTACCTTTTCTTCTTAGTCTTTTCA underscore is XhoI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
Specifically, the method for sorghum transcription factor SbGRF4 gene is expressed, comprising the following steps:
(1) Bacillus coli cells are converted with the recombinant vector, obtains the recombined pronucleus expression bacterial strain of expression SbGRF4；
(2) it by recombined pronucleus expression strain inoculated to kanamycins or kanamycins+chloramphenicol fluid nutrient medium, stays overnight
Cultivate activated strains；
(3) the recombined pronucleus expression bacterial strain of activation is transferred to kanamycins or kanamycins+chloramphenicol fluid nutrient medium
In, the expression of IPTG induction recombination SbGRF4 is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed SbGRF4 recombinant protein.
Preferably, when earthquake culture is to OD600=0.5 in step (3), the IPTG of final concentration of 0.8mM is added, 25
DEG C Fiber differentiation 10h.
Kanamycins is 50ug/mL in the kanamycins fluid nutrient medium.
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL in the kanamycins+chloramphenicol fluid nutrient medium.
The beneficial effects of the present invention are: especially GRF transcription factor does not have for fewer to the gene studies in sorghum at present
There is relevant report, the present invention passes through the protein sequence of rice GRF gene, the homologous base of sorghum is obtained from sorghum genome database
Because SbGRF4, gene C DS overall length 1221bp download it in ncbi database according to the amino acid sequence that the gene is derived
GRF gene in its species constructs unrooted chadogram with adjacent method, the results showed that it is one that its amino acid sequence gathers with corn GRF
Branch, affiliation is closer, illustrates that SbGRF4 gene is sorghum transcription factor.
The prokaryotic expression of gene is one of the common technology for studying gene function, but in prokaryotic expression, albumen usually with
The form of inclusion body is present in precipitating, and how to allow albumen is face in prokaryotic expression with soluble form expression in cell conditioned medium
The technical problem faced.The present invention is to obtain SbGRF4 soluble protein, constructs the prokaryotic expression carrier of SbGRF4 gene, and
A series of optimization is carried out for protein expression condition, discovery SbGRF4 can be in BL21 (DE3), JM109 (DE3), Rosetta
(DE3), it is expressed in Rosetta-gami 2 (DE3), Tuner (DE3) bacterial strain, wherein expression quantity is most in Tuner (DE3)；
When inducing temperature is 25 DEG C, expression quantity is best；With the increase of IPTG concentration, expressing quantity is gradually increased, and is most preferably lured
Leading concentration is 0.8mM；At 25 DEG C, when IPTG concentration is 0.8, inducing expression 10h, expression quantity is best.Through SDS-PAGE electrophoresis
Detection, obtains the purpose band that molecular weight is about 44kDa, for the expected size of SbGRF4 albumen, finally obtains SbGRF4 solubility
Albumen.Meanwhile the present invention purifies it with protein purification system, as a result further research albumin crystal structure and life
Object characteristic lays the foundation, and then provides foundation for the resistance for improving sorghum by gene editing method.
Detailed description of the invention
Fig. 1 is the systematic evolution tree of sorghum SbGRF4 gene；
Fig. 2 is pET-28a Vector map；
Fig. 3 is digestion result schematic diagram, and 1 is the Plasmid DNA of digestion, and 2 be EcoRI and SalI digestion as a result, 3 be DNA
Marker；
Fig. 4 is the albumen peak figure that SbGRF4 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 5 is the electrophoresis result figure after SbGRF4 protein purification；
Fig. 6 is the result figure that SDS-PAGE electrophoresis tests optimum expression bacterial strain；
Fig. 7 is the result figure that SDS-PAGE electrophoresis tests best IPTG induced concentration；
Fig. 8 is the result figure that SDS-PAGE electrophoresis tests best inducing temperature；
Fig. 9 is western blot detection figure.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
Embodiment 1: the acquisition and analysis of sorghum transcription factor SbGRF4 gene
According to the protein sequence of reported rice GRF4 gene (gene number is LOC_Os02g47280), blastP search
Sorghum genome database (https: //phytozome) finds sorghum homologous gene SbGRF4, and (gene number is
Sb04g030770, nucleotide sequence is as shown in SEQ ID NO.1).With SbGRF4 albumen (its amino acid sequence such as SEQ ID
Shown in NO.2) search ncbi database, the GRF gene in other species is downloaded, with 7.0 software of MEGA, with adjacent method
(Neighbor-Joining, NJ) (bootstrap=1000) constructs unrooted chadogram, as seen from Figure 1, SbGRF4 and jade
It is one that rice GRF, which gathers, and affiliation is nearest, it is known that SbGRF4 gene is sorghum transcription factor.
Embodiment 2: the building and identification of sorghum transcription factor SbGRF4 gene recombined vector
1, sorghum RNA is extracted, and reverse transcription goes out cDNA
Sorghum BTx623 material is taken, extracts seedling using RNA extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.)
Phase total serum IgE obtains cDNA with reverse transcription reagent box (Promega) reverse transcription.
2, using cDNA as template amplification SbGRF4 gene；
Design primer expands SbGRF4 gene using cDNA as template.
Primer is as follows:
Upstream primer: SbGRF4-F:GCGAATTCATGGCGATGCCGTATGCC underscore is EcoRI restriction enzyme site；
Downstream primer: SbGRF4-R:CGGTCGACTTAGTCATCGTTGGGCGACT underscore is SalI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
PCR amplification system used in gene magnification for vector construction is as follows: 5 × buffer 10 μ L, FastPfu
4 μ L, cDNA template of Fly DNA Polymerase 1 μ L (Quan Shijin), 2.5mmol dNTPs, 0.5 μ L, 10 μm of ol upstream and downstream are drawn
Object each 1 μ L, ddH2O 32.5μL。
The following are PCR amplification conditions: 95 DEG C of 2min, 35 circulations (95 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 30s-1min (according to
DNA fragmentation length determines), 72 DEG C of 5min.
3, SbGRF4 gene recombined vector is constructed
After PCR product is purified, it is connected to pET-28a carrier.PCR product is detected using 1% agarose.PCR
Product is recycled with reference to Nanjing Vazyme Biotechnology Co., Ltd.'s Ago-Gel QIAquick Gel Extraction Kit specification.It is after the recovery
Product is inserted into the pET-28a expression vector through same double digestion after EcoRI and SalI double digestion by DNA ligase
(expression vector map is as shown in Figure 2), 16 DEG C connect 16 hours, construct pET-28a-SbGRF4 recombinant plasmid.
By recombinant plasmid transformed to coli strain DH5a, trained on 50ug/mL kanamycins solid plate in 37 DEG C
It supports, picking individual colonies hold up the limited public affairs of section's biotechnology after digestion identification is correct (digestion result is as shown in Figure 3), then through Beijing
Take charge of its correct sequence of sequence verification.Sequencing result shows that SbGRF4 coding sequence obtained fulfills the expectation, and says
Bright construction of recombinant plasmid success is simultaneously named as pET-28a-SbGRF4, i.e. recombinant vector.
The inducing expression of embodiment 3:SbGRF4 albumen
1, the recombined pronucleus expression bacterial strain of SbGRF4 is obtained
It selects and successful monoclonal is sequenced in embodiment 2 is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight, according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit by pET-28a-
SbGRF4 recombinant expression carrier extracts, and recombinant expression carrier plasmid is taken to convert E. coli expression strains BL21 (DE3),
JM109 (DE3), Rosetta (DE3), Rosetta-gami 2 (DE3), Tuner (DE3), NovaBlue (DE3), detection
The expression of SbGRF4 albumen.
2, activated strains are incubated overnight
It is incubated overnight and activates above-mentioned recombined pronucleus expression bacterial strain.For example, by BL21 (DE3), JM109 (DE3), Tuner
(DE3), NovaBlue (DE3) bacterial strain is transferred in 50ug/mL kanamycins fluid nutrient medium, by Rosetta (DE3),
RosettagamiB (DE3) bacterial strain is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight activated strains.
3, inducing expression
The recombined pronucleus expression bacterial strain of activation is transferred in the fluid nutrient medium containing corresponding antibiotic, for example, will activation
BL21 (DE3), JM109 (DE3), Tuner (DE3), NovaBlue (DE3) bacterial strain be transferred to 50ug/mL kanamycins liquid
In culture medium, Rosetta (DE3), RosettagamiB (DE3) bacterial strain are transferred to 50ug/mL kanamycins+50ug/mL chlorine
In mycin fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0.8mM is added, is lured at 25 DEG C
Culture 10 hours is led, the recombinant protein of SbGRF4 is obtained.
4, the purifying of SbGRF4 recombinant protein
After the completion of induction, expressed SbGRF4 recombinant protein is recycled and purified, is carried out using AKTA protein purification system
Purifying and desalination, it is preferable that protein purification is carried out using AKTA purifier10 fast protein purification system, steps are as follows:
Since pET28a has 6 His coded sequences before terminator, so pET28a-SbGRF4 recombinant expression carrier
The albumen of acquisition can use Ni-NTA and carry out affinitive layer purification.Affinity chromatography is with protein or large biological molecule and combination
Based on special affinity between aglucon on medium, because being connected to one in the matrix of chromatography gel used in His-tags
NTA [(nitrilotriacetic acid) nitrilotriacetic acid], can be with Ni ions binding, and the 6 of Ni ion and fusion protein
Attraction is generated between × His amino acid, thus by the fusion protein for having His-tags histidine tag and other protein regions
It separates, five yuan of imidazole rings of histidine residues are the key that albumen and Ni ionization, therefore when we use the miaow of high concentration
Azoles (Imidazole) solution elution when marquis (such as 400mM), imidazoles be convenient for protein His-tags imidazole ring competitive binding,
Finally fusion protein is eluted from gel.
Concrete operations: taking out the thallus of -80 DEG C of preservations, and Lysis buffer is added and is resuspended, the bacterium solution after resuspension is in ice bath
Middle carry out ultrasonic disruption, power 350W, duty ratio 50%, each circulation 30s, total time 35min.It is broken even
Slurry, 4 DEG C, 1,2000rpm centrifugation 30min.Supernatant is adsorbed on Ni-NTA adsorption column with peristaltic pump, 5mL/min, repeats one
It is secondary, retain 20 μ L by the sample of adsorption column and carries out protein adhesive electrophoresis；Ni-NTA adsorption column is connected to fast protein purifying system
On system, eluted；Albumen is collected according to UV absorption curve, and samples and carries out protein adhesive electrophoresis detection.
Fig. 4 is AKTA protein purification system purifying protein peak figure.SbGRF4 albumen is purified by AKTA protein purification system
Obtained albumen peak figure.SbGRF4 purifying figure ultraviolet (UV) absorption value when being purifying SbGRF4 albumen.
Fig. 5 is the electrophoretogram after SbGRF4 protein purification.It is the expression and purification at 25 DEG C, IPTG concentration 0.8mM
SbGRF4 albumen result.1 is Supernatant samples after cell ultrasonication；2 be the efflux after Ni column is affine after purification；3 are
Deposit sample after cell ultrasonication；4 be the affine rear efflux of Ni column；M is albumen marker；Arrow meaning is shown as SbGRF4 egg
It is white.Fig. 5 shows that SbGRF4 albumen often has in supernatant under this condition, after purification by AKTA protein purification system
To SbGRF4 purity of protein reach requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 4:SbGRF4
1, the recombinant strains of SbGRF4 albumen are obtained
It selects and successful monoclonal is sequenced in embodiment 2 is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight, according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit by pET-28a-
SbGRF4 recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), JM109 respectively
(DE3), Rosetta (DE3), Rosetta-gami 2 (DE3), Tuner (DE3), NovaBlue (DE3) bacterial strain are coated on and contain
It is incubated overnight on 50ug/mL kanamycins plate in 37 DEG C, picking single colonie is inoculated into the training of 50ug/mL kanamycins liquid
Support base in, 37 DEG C, 200rpm be incubated overnight, in bacterium solution be added sterilizing 50% glycerol, frozen after mixing in -80 DEG C of refrigerators, obtained
To the prokaryotic expression bacterial strain of expression SbGRF4.
2, the determination of SbGRF4 Primary structure optimum strain
Be inoculated with respectively above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3), JM109 (DE3), Tuner (DE3),
NovaBlue (DE3) bacterial strain is into 50ug/mL kanamycins fluid nutrient medium, bacterial strain Rosetta (DE3), Rosetta-gami
2 (DE3) into 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activation bacterium
Strain.
By the recombined pronucleus expression bacterial strain BL21 (DE3) of activation, JM109 (DE3), Tuner (DE3), NovaBlue (DE3)
It is transferred to 50ug/mL kanamycins, bacterial strain Rosetta (DE3), Rosetta-gami2 (DE3) are transferred to 50ug/mL card that are mould
In element+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is added final concentration of 1mM's
IPTG, 25 DEG C Fiber differentiation 12 hours (pET-28a bacterium is taken to make negative control).4 DEG C after the completion of induction, 1,2000rpm centrifugation
2min collects thallus, bacterial precipitation and negative control 2mL and PBS buffer (with preceding addition 1mM PMSF) suspension is pre-chilled, uses
Ultrasonic Cell Disruptor progress clasmatosis, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes
Clearly, SDS-PAGE electrophoresis detection, test optimum expression bacterial strain, as a result as shown in fig. 6, SbGRF4 can at BL21 (DE3),
JM109 (DE3), Rosetta (DE3), Rosetta-gami2 (DE3) are expressed in Tuner (DE3) bacterial strain, wherein in Tuner
(DE3) expression quantity is most in.
3, the determination of the best IPTG concentration of SbGRF4 Primary structure
Above-mentioned recombined pronucleus expression bacterial strain Tuner (DE3) is inoculated with into 50ug/mL kanamycins fluid nutrient medium, 37
DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to the training of 50ug/mL kanamycins liquid
It supports in base, when 37 DEG C of shake cultures are to OD600=0.5, is separately added into final concentration of 0,0.2,0.4,0.6,0.8,1.0,1.2mM
IPTG, 25 DEG C Fiber differentiation 10 hours (bacterium not induced is taken to make negative control).4 DEG C after the completion of induction, 1,2000rpm from
Heart 2min collects thallus, bacterial precipitation and negative control 2mL and PBS buffer (with preceding addition 1mM PMSF) suspension is pre-chilled,
Clasmatosis is carried out with Ultrasonic Cell Disruptor, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes
Supernatant, SDS-PAGE electrophoresis detection test best IPTG induced concentration, as a result as shown in fig. 7, when IPTG concentration is 0.8mM,
SbGRF4 expression is most.
4, the determination of SbGRF4 Primary structure optimum temperature
Above-mentioned recombined pronucleus expression bacterial strain Tuner (DE3) is inoculated with into 50ug/mL kanamycins fluid nutrient medium, 37
DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to the training of 50ug/mL kanamycins liquid
Support in base, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0.8mM be added, respectively 16,20,25,30,
With 37 DEG C Fiber differentiation 8-16 hours (pET-28a bacterium is taken to make negative control).4 DEG C after the completion of induction, 1,2000rpm centrifugation
2min collects thallus, bacterial precipitation and negative control 2mL and PBS buffer (with preceding addition 1mM PMSF) suspension is pre-chilled, uses
Ultrasonic Cell Disruptor progress clasmatosis, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes
Clearly, SDS-PAGE electrophoresis detection tests best inducing temperature, as a result as shown in figure 8, in 25 DEG C of Fiber differentiations, SbGRF4 table
It is most up to amount.
The western blot of embodiment 5:SbGRF4 recombinant protein is detected
(1) it extracts by SbGRF4 albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-SbGRF4 plasmid, IPTG inducing expression；
2. protein lysate (50mM Tris pH 8.0,120mM NaCl, 1mM DTT, 0.5%NP-40, before is added
1mM PMSF, 1mM DTT is added), after ultrasonic disruption cell, supernatant is collected by centrifugation, places 20min on ice；
3.15,000g, 4 DEG C of centrifugation 5min abandon precipitating；
4. taking 200 μ L supernatants that 5 × Loading Buffer is added, concussion is mixed, and -80 DEG C of remaining sample freeze；
5. boiling water boiling 5min, ice bath, 12,000rpm centrifugation 5min draw 15 μ L of supernatant and carry out protein electrophoresis, remaining sample
- 20 DEG C of product freeze；
6. prepare SDS-PAGE glue, first carry out electrophoresis 20min with 90V, when sample enters separation gel, with 150V voltage into
Row electrophoresis, until bromophenol blue stops electrophoresis when running out of separation gel.
(2) transferring film
Cut with a paper cutter out the pvdf membrane more somewhat larger than glue (for pvdf membrane, first with methanol impregnate 30 seconds, then
ddH23-5min is impregnated in O, is then placed in transferring film buffer) rubber plate clamp is put into electrophoretic blotting groove.Transferring film buffer is poured into,
4 DEG C, 100V 90min.
(3) it closes
After the completion of transferring film, pvdf membrane is taken out, is put in the PBST solution with 5% skimmed milk power, 2h is closed on shaking table.
(4) primary antibody is incubated for
Confining liquid is discarded, is added Anti His antibody (1:5000), shaking table is incubated for 12h.
(5) secondary antibody is incubated for
Primary antibody is outwelled, washs pvdf membrane 3 times with 1 × PBST, each 5min；Corresponding secondary antibody (sheep anti-Mouse) is added, shakes
Bed is incubated for 1h.
(6) ECL exposes
Secondary antibody is abandoned, washs pvdf membrane 3 times with 1 × PBST, each 5min；HRP chemiluminescent substrate is added on pvdf membrane
Liquid can detect protein expression signal (as shown in Figure 9) on 5200 chemiluminescence detectors in day, and succeed table as can be seen from the results
Up to SbGRF4 albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of sorghum transcription factor SbGRF4 gene and its recombinant vector and expression
<160> 4
<210>1
<211>1221
<212>DNA
<213>sorghum transcription factor SbGRF4
<400>1
ATGGCGATGC CGTATGCCTC TCTTTCCCCG GCAGGCGCCG ACCACCGCTC CTCCACGGCC 60
ACGGCGGCGT CGCTCCTCCC CTTCTGCCGC TCCACCCCGC TCTCCGCGGG CGGCGGCGGC 120
GGCCTGGGGG AGGACGCCCA GTTGAGCTCG CGGTGGCCGG CCGCGAGGCC GGTGGTGCCG 180
TTCACGCCGG CGCAGTACGA GGAGCTGGAG CAGCAGGCGC TCATATACAA GTACCTGGTG 240
GCCGGCGTGC CCGTCCCGCC GGATCTCGTG GTTCCAATCC GCCGCGGTCT CGACTCCCTC 300
GCAACCCGCT TCTACGGCCA TCCCACACTT GGTGGGTACG GGACGTACTA CTTAGGCAAG 360
AAACTGGATC CGGAGCCGGG GCGGTGCCGG CGTACGGACG GCAAGAAGTG GCGGTGCTCC 420
AAGGAGGCCG CCCCAGACTC CAAGTACTGC GAGCGCCACA TGCACCGCGG CCGCAACCGT 480
TCAAGAAAGC CTGTGGAAAC GCAGCTCGTG CCCCAGTCCC AACCGCCCGC CACCGCCGCT 540
GCCGTCTCCG CCGCTCCGCC CTTGGCCTTG GCCGCCGCCA CCACCACCAC CAACGGCAGC 600
TGCTTCCAGA ATCACTCTCT TTACCCGGCC ATTGCAGGCA GCACCGGTGG AGGTGGCGGG 660
GCCAGCAATA TCTCTACCCC GTTCTCCTCG TCGATGGGGT CGTCTCAGCT GCACATGGAC 720
AATGCTGCCA GCTACGCAGC TCTTGGTGGT GGAACTGCAA AGGATCTCAG GTACAACGCC 780
TACGGAATAA GATCTTTGGC GGAGGAGCAC AACCAGCTGA TTGCAGAAGC CATTGACTCA 840
TCAATGGAGA ACCAGTGGCG CCTCCCGCCA TCCCAAACCT CTTCGTTTCC GCTCTCGAGC 900
TACCCCCAGC TTGGGGCGCT GAGCAACCTG GGTCAGAGCA CAGTCACCTC GCTGTCGAAG 960
ATGGAGCGGC AGCAGCCACT CTCCTTCCTA GGGAACTCCG AGTTCGGGGC CATGGAATCC 1020
GCCGCCAAGC AGCAGGAGAA CCAGACGCTG CGGCCCTTCT TCGACGAGTG GCCCAAGGCG 1080
AGGGACTCCT GGCCGGGCCT CTCCGACGAC AACGCCGCAA GCCTCGCTCC GTCGTTCCCG 1140
GCGACCCAGC TGTCGATGTC CATACCGATG GCGTCCTCGG ACTTCTCCGT GGCCAGCTCC 1200
CAGTCGCCCA ACGATGACTA A 1221
<210>2
<211>406
<212>PRT
<213>sorghum transcription factor SbGRF4
<400>2
MAMPYASLSP AGADHRSSTA TAASLLPFCR STPLSAGGGG GLGEDAQLSS RWPAARPVVP 60
FTPAQYEELE QQALIYKYLV AGVPVPPDLV VPIRRGLDSL ATRFYGHPTL GGYGTYYLGK 120
KLDPEPGRCR RTDGKKWRCS KEAAPDSKYC ERHMHRGRNR SRKPVETQLV PQSQPPATAA 180
AVSAAPPLAL AAATTTTNGS CFQNHSLYPA IAGSTGGGGG ASNISTPFSS SMGSSQLHMD 240
NAASYAALGG GTAKDLRYNA YGIRSLAEEH NQLIAEAIDS SMENQWRLPP SQTSSFPLSS 300
YPQLGALSNL GQSTVTSLSK MERQQPLSFL GNSEFGAMES AAKQQENQTL RPFFDEWPKA 360
RDSWPGLSDD NAASLAPSFP ATQLSMSIPM ASSDFSVASS QSPNDD 406
<210>3
<211>26
<212>DNA
<213>artificial sequence
<400>3
GCGAATTCAT GGCGATGCCG TATGCC 26
<210>4
<211>28
<212>DNA
<213>artificial sequence
<400>4
CGGTCGACTT AGTCATCGTT GGGCGACT 28
Claims (9)
1. a kind of sorghum transcription factor SbGRF4 gene, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
2. the protein of sorghum transcription factor SbGRF4 gene coding as described in claim 1, amino acid sequence such as SEQ ID
Shown in NO.2.
3. the recombinant vector comprising gene described in claim 1.
4. recombinant vector according to claim 3, which is characterized in that the recombinant vector the preparation method is as follows: extract
Sorghum RNA, and reverse transcription goes out cDNA；Using cDNA as template amplification SbGRF4 gene；By SbGRF4 amplified production through EcoRI and
After SalI double digestion, it is inserted into the pET-28a expression vector through same double digestion by DNA ligase, obtains recombinant plasmid
pET-28a-SbGRF4。
5. recombinant vector according to claim 4, which is characterized in that using cDNA as template, utilize following primer amplification
SbGRF4 gene, primer sequence are as follows:
Upstream primer: SbGRF4-F:GCGAATTCATGGCGATGCCGTATGCC underscore is EcoRI restriction enzyme site；
Downstream primer: SbGRF4-R:CGGTCGACTTAGTCATCGTTGGGCGACT underscore is SalI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
6. a kind of method for expressing sorghum transcription factor SbGRF4 gene, which comprises the following steps:
(1) recombinant vector described in claim 3 or 4 converts Bacillus coli cells, obtains the recombination protokaryon of expression SbGRF4
Express bacterial strain；
(2) it by recombined pronucleus expression strain inoculated to kanamycins or kanamycins+chloramphenicol fluid nutrient medium, is incubated overnight
Activated strains；
(3) the recombined pronucleus expression bacterial strain of activation is transferred to again in kanamycins or kanamycins+chloramphenicol fluid nutrient medium,
The expression of IPTG induction recombination SbGRF4 is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed SbGRF4 recombinant protein.
7. the method for expression sorghum transcription factor SbGRF4 gene according to claim 6, which is characterized in that step (3)
When middle earthquake culture is to OD600=0.5, the IPTG of final concentration of 0.8mM is added, in 25 DEG C of Fiber differentiations.
8. the method for expression sorghum transcription factor SbGRF4 gene according to claim 6, which is characterized in that described to block that
Kanamycins is 50ug/mL in mycin fluid nutrient medium.
9. the method for expression sorghum transcription factor SbGRF4 gene according to claim 6, which is characterized in that described to block that
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL in mycin+chloramphenicol fluid nutrient medium.
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