CN109971768A - A kind of sorghum transcription factor SbSBP5 gene and its recombinant vector and expression - Google Patents
A kind of sorghum transcription factor SbSBP5 gene and its recombinant vector and expression Download PDFInfo
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Abstract
The invention discloses a kind of sorghum transcription factor SbSBP5 genes, and nucleotide sequence is as shown in SEQ ID NO.1.Invention additionally discloses a kind of protein of sorghum transcription factor SbSBP5 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 SbSBP5 gene.The present invention passes through the protein sequence of rice SBP gene, sorghum homologous gene SbSBP5 is obtained from sorghum genome database, full length gene 1227bp, 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 SbSBP5 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.
SBP (SQUAMOSA promoter-binding-protein, SQUA promoter binding protein)-box gene family
Encoding the distinctive transcription factor of a kind of plant-SBP transcription factor is one kind transcription factor specific to plant, it can be identified
And MADSbox gene SQUAMOSA (SQUA) promoter is combined, so the SQUA promoter binding protein that is otherwise known as.SBP family
Member has the characteristics that common in structure: each member contains the very conservative DNA knot being made of 76 or so amino acid
It closes domain (i.e. SBP binding domain).Transcription factor containing SBP binding domain is widely present in plant, and participates in plant growth, hair
The many aspects educated.Such as formation of floral organ etc..In arabidopsis, clover, corn, Chinese cabbage, tomato, rice etc. at present
It was found that having SBP transcription factor, but so far, both at home and abroad there is not yet SBP transcription factor studies report in sorghum.
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 SbSBP5 gene and its again
Group carrier and expression.
The technical scheme is that a kind of sorghum transcription factor SbSBP5 gene, nucleotide sequence such as SEQ ID
Shown in NO.1.
The present invention also provides the protein of sorghum transcription factor SbSBP5 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 SbSBP5 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 SbSBP5 gene；By SbSBP5 amplified production after BamHI and EcoRI double digestion, it is inserted by DNA ligase
In pET-28a expression vector through same double digestion, recombinant plasmid pET-28a-SbSBP5 is obtained.
Preferably, using cDNA as template, using following primer amplification SbSBP5 gene, primer sequence is as follows:
Upstream primer: SbSBP5-F:GCGGATCCATGGAGTCCGGTGGCG underscore is BamHI restriction enzyme site；
Downstream primer: SbSBP5-R:CGGAATTCCTAGAGTGACCAGTCCATTGTGT underscore is EcoRI digestion position
Point；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
Specifically, the method for sorghum transcription factor SbSBP5 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 SbSBP5；
(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 SbSBP5 is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed SbSBP5 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.
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 SBP 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 SBP gene (gene LOC_Os08g39890), from sorghum genome
Sorghum homologous gene SbSBP5, gene C DS overall length 1227bp, the amino acid sequence derived according to the gene are obtained in database
It is listed in the SBP gene downloaded in other species in ncbi database, unrooted chadogram is constructed with adjacent method, the results showed that its amino
It is one that acid sequence and millet (Setaria italica) SBP, which gather, and affiliation is closer, illustrates that SbSBP5 gene is that sorghum turns
Record the 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 SbSBP5 soluble protein, constructs the prokaryotic expression carrier of SbSBP5 gene, and
A series of optimization is carried out for protein expression condition, discovery SbSBP5 can be in BL21 (DE3), JM109 (DE3), Rosetta
(DE3), it is expressed in this 5 bacterial strains of BL21 (DE3) pLysS, Tuner (DE3), wherein expression quantity is most in Rosetta (DE3)
It is more；When inducing temperature is 25 DEG C, expression quantity is best；With the increase of IPTG concentration, expressing quantity is gradually increased, most preferably
Induced concentration is 0.8mM；At 25 DEG C, when IPTG concentration is 0.8, inducing expression 10h, expression quantity is best.Through SDS-PAGE electricity
Swimming detection, obtains the purpose band that molecular weight is about 55kDa, and for the expected size of SbSBP5 albumen, it is solvable to finally obtain SbSBP5
Property albumen.Meanwhile the present invention purifies it with protein purification system, as a result further research albumin crystal structure and
Biological characteristics lay the foundation, and then provide 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 SbSBP5 gene；
Fig. 2 is digestion result schematic diagram, and 1 is the Plasmid DNA of digestion, and 2 be BamHI and EcoRI digestion as a result, 3 be DNA
Marker；
Fig. 3 is the albumen peak figure that SbSBP5 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 4 is the electrophoresis result figure after SbSBP5 protein purification；
Fig. 5 is the result figure that SDS-PAGE electrophoresis tests optimum expression bacterial strain；
Fig. 6 is the result figure that SDS-PAGE electrophoresis tests best IPTG induced concentration；
Fig. 7 is the result figure that SDS-PAGE electrophoresis tests best inducing temperature；
Fig. 8 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 SbSBP5 gene
According to the protein sequence of reported rice SBP gene, blastP search for sorghum genome database (https: //
Phytozome) finding sorghum homologous gene SbSBP5, (gene number is Sb08g005080, nucleotide sequence such as SEQ ID
Shown in NO.1).Ncbi database is searched for SbSBP5 albumen (its amino acid sequence is as shown in SEQ ID NO.2), is downloaded other
SBP gene in species, with 7.0 software of MEGA, with adjacent method (Neighbor-Joining, NJ) (bootstrap=1000)
Unrooted chadogram is constructed, as seen from Figure 1, it is one that SbSBP5 and millet (Setaria italica) SBP, which gathers, and relationship is closed
System is nearest, it is known that SbSBP5 gene is sorghum transcription factor.
Embodiment 2: the building and identification of sorghum transcription factor SbSBP5 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 SbSBP5 gene；
Design primer expands SbSBP5 gene using cDNA as template.
Primer is as follows:
Upstream primer: SbSBP5-F:GCGGATCCATGGAGTCCGGTGGCG underscore is BamHI restriction enzyme site；
Downstream primer: SbSBP5-R:CGGAATTCCTAGAGTGACCAGTCCATTGTGT underscore is EcoRI digestion position
Point；
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 circulation (95 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 1min, 72 DEG C
5min。
3, SbSBP5 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 BamHI and EcoRI double digestion by DNA ligase,
16 DEG C connect 16 hours, construct pET-28a-SbSBP5 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 2), then through Beijing
Take charge of its correct sequence of sequence verification.Sequencing result shows that SbSBP5 coding sequence obtained fulfills the expectation, and says
Bright construction of recombinant plasmid success is simultaneously named as pET-28a-SbSBP5, i.e. recombinant vector.
The inducing expression of embodiment 3:SbSBP5 albumen
1, the recombined pronucleus expression bacterial strain of SbSBP5 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-
SbSBP5 recombinant expression carrier extracts, take recombinant expression carrier plasmid conversion E. coli expression strains BL21 (DE3),
JM109 (DE3), BL21 (DE3) pLysS, Tuner (DE3), Rosetta (DE3) detect the expression of SbSBP5 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) bacterial strain is transferred in 50ug/mL kanamycins fluid nutrient medium, by Rosetta (DE3), BL21 (DE3) pLysS bacterial strain
Be transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C, 200rpm be 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) bacterial strain be transferred in 50ug/mL kanamycins fluid nutrient medium, will
Rosetta (DE3), BL21 (DE3) pLysS bacterial strain are transferred to 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium
In, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0.8mM is added, 25 DEG C Fiber differentiation 10 hours, obtain
Obtain the recombinant protein of SbSBP5.
4, the purifying of SbSBP5 recombinant protein
After the completion of induction, expressed SbSBP5 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-SbSBP5 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. 3 is AKTA protein purification system purifying protein peak figure.SbSBP5 albumen is purified by AKTA protein purification system
Obtained albumen peak figure.
Fig. 4 is the electrophoretogram after SbSBP5 protein purification.It is the expression and purification at 25 DEG C, IPTG concentration 0.8mM
SbSBP5 albumen result.1 and 2 be the albumen after Ni column is affine after purification；3 be Supernatant samples after cell ultrasonication；M is
Albumen marker；Arrow meaning is shown as SbSBP5 albumen.Fig. 4 shows that SbSBP5 albumen often has in supernatant under this condition
In, the SbSBP5 purity of protein obtained after purification by AKTA protein purification system reaches requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 4:SbSBP5
1, the recombinant strains of SbSBP5 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-
SbSBP5 recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), JM109 respectively
(DE3), Rosetta (DE3), BL21 (DE3) pLysS, Tuner (DE3) bacterial strain, is coated on the plate of kanamycins containing 50ug/mL
It is incubated overnight on (Rosetta (DE3), BL21 (DE3) pLysS need to add 50ug/mL chloramphenicol) in 37 DEG C, picking single bacterium
It falls and is inoculated into 50ug/mL kanamycins (Rosetta (DE3), BL21 (DE3) pLysS need to add chloramphenicol) fluid nutrient medium,
37 DEG C, 200rpm be incubated overnight, in bacterium solution be added sterilizing 50% glycerol, frozen after mixing in -80 DEG C of refrigerators, expressed
The prokaryotic expression bacterial strain of SbSBP5.
2, the determination of SbSBP5 Primary structure optimum strain
BL21 (DE3), JM109 (DE3), Tuner (DE3) bacterial strain are transferred to 50ug/mL kanamycins fluid nutrient medium
In, Rosetta (DE3), BL21 (DE3) pLysS bacterial strain are transferred to the training of 50ug/mL kanamycins+50ug/mL chloramphenicol liquid
Support base in, 37 DEG C, 200rpm be incubated overnight activated strains.
The BL21 (DE3) of activation, JM109 (DE3), Tuner (DE3) bacterial strain are transferred to 50ug/mL kanamycins liquid
In culture medium, Rosetta (DE3), BL21 (DE3) pLysS bacterial strain are transferred to 50ug/mL kanamycins+50ug/mL chloramphenicol
In fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 1mM is added, in 25 DEG C of Fiber differentiations
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, and bacterium is heavy
It forms sediment and negative control 2mL is pre-chilled PBS buffer (with preceding addition 1mM PMSF) and suspends, it is broken to carry out cell with Ultrasonic Cell Disruptor
It is broken, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-PAGE electrophoresis detection,
Test optimum expression bacterial strain, as a result as shown in figure 5, SbSBP5 can at BL21 (DE3), JM109 (DE3), Rosetta (DE3),
It is expressed in this 5 bacterial strains of BL21 (DE3) pLysS, Tuner (DE3), wherein expression quantity is most in Rosetta (DE3).
3, the determination of the best IPTG concentration of SbSBP5 Primary structure
Above-mentioned recombined pronucleus expression bacterial strain Rosetta (DE3) is inoculated with to 50ug/mL kanamycins+50ug/mL chloramphenicol
In fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to 50ug/
In mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is separately added into dense eventually
Degree is the IPTG of 0.2,0.4,0.6,0.8,1.0mM, 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 centrifugation 2min collects thallus, bacterial precipitation and negative control with 2mL and PBS is pre-chilled
Buffer (with preceding addition 1mM PMSF) suspends, and carries out clasmatosis with Ultrasonic Cell Disruptor, each 10sec, and totally 10 times, between each
Every 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-PAGE electrophoresis detection tests best IPTG induced concentration, as a result
As shown in fig. 6, SbSBP5 expression is most when IPTG concentration is 0.8mM.
4, the determination of SbSBP5 Primary structure optimum temperature
Above-mentioned recombined pronucleus expression bacterial strain Rosetta (DE3) is inoculated with to 50ug/mL kanamycins+50ug/mL chloramphenicol
In fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to again
In 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is added dense eventually
Degree is the IPTG of 0.8mM, (takes pET-28a bacterium to make negative right within Fiber differentiation 8-16 hours at 16,20,25,30 and 37 DEG C respectively
According to).4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min collects thallus, bacterial precipitation and negative control with 2mL and PBS is pre-chilled
Buffer (with preceding addition 1mM PMSF) suspends, and carries out clasmatosis with Ultrasonic Cell Disruptor, each 10sec, and totally 10 times, between each
Every 30sec；1,2000rpm centrifugation 10min, takes supernatant, and SDS-PAGE electrophoresis detection tests best inducing temperature, as a result such as Fig. 7
Shown, in 25 DEG C of Fiber differentiations, SbSBP5 expression quantity is most.
The western blot of embodiment 5:SbSBP5 recombinant protein is detected
(1) it extracts by SbSBP5 albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-SbSBP5 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 8) on 5200 chemiluminescence detectors in day, and succeed table as can be seen from the results
Up to SbSBP5 albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of sorghum transcription factor SbSBP5 gene and its recombinant vector and expression
<160> 4
<210>1
<211>1227
<212>DNA
<213>sorghum transcription factor SbSBP5
<400>1
ATGGAGTCCG GTGGCGCCAG TGGCGGCGGC GGCGGCGGCG GCGGGGACGA CCAGCTGCAC 60
GGCCTCAAGT TCGGCAAGAA GATCTACTTC GAGGACGCCG CCGCGGCCGG CTCCAGCAGC 120
GGTGGCGGTG GCGGTGGCAG TGGCAATGCT AGCGCGACCA CCGCGGCTCC AGCGACGCAG 180
CAGCCGTCCC CGCCGCAGGC CGCTTCGCCC AGGGCAGCCA GCGGCGGCGG CGGCAGGAGG 240
GGCAGGGCCG CGGGCGGCCC CTCCCCGGCG CCCGCGCCCG CGCGCTGCCA GGTCGACGGC 300
TGCAACGTGG ACCTCACCGA CGTCAAGCCC TACTACTGCC GCCACAAGGT CTGCAAAATG 360
CACTCCAAGG AGCCCCGCGT CGTCGTCAAC GGCCTCGAGC AGCGCTTCTG CCAGCAGTGC 420
AGCAGGTTCC ACCAGCTGCC TGAATTTGAC CAACTAAAAA AAAGCTGCCG CAGACGTCTC 480
GCAGGCCACA ATGAACGCCG GAGGAGGCCA CCTCCTGGAC CTCTTGCAAC ACGATATGGT 540
CGCCTTGCTG CATCATTCGG TGAGCCCGGC AGGTTCCGAA GCTTTATGTT GGATTTCTCA 600
TACCCAAGGG TTCCAACCGC CATGAGGGAT GGGTTTCCAG CAGTTCGACC TGGTGAAAGG 660
GCGCCTGGTA GTATCCAGTG GCAAGCGAGC TTAGATCCTC ATCATCATCA AAGTGCGGTC 720
GCAGGATACA GTGCTCACTC ATATGGGAGC CAGGGTAGCT CGTCGTCAAG GCCACCGGTG 780
TTCCCTGGTC CGGAGATCCC CCCAGGTGGA TGTCTTGCAG GAGTCCCCTC GGACTCTAGC 840
TGTGCTCTCT CTCTTCTGTC AACTCAGCCA TGGGATACTA CCCACAGCTC TGGCCACAGC 900
CATGCTGCAT CAATGCCTGC AACAGCAGGT TTTGACGGCA ACCCTGTGGC ACCCTCCCTC 960
ATGGCGAGTA GCTACATTGC GCCAAGCCCC TGGACTGACT CCCGGGGCCA TGAAGGCGGG 1020
CGGAACGTGC CTCAGTTGCC ACCTGACGTC CCCCTCAGCG AGGTGCACTC TGGCTCAAGC 1080
AGCCATCACG GCCAGTTCTC AGGTGAGCTC GAGCTTGCCC TGCAGGGAAA CAGGCCAGCA 1140
CCAGGGTCAG CGCCAGCGCC GCGCAATGAT CAGGGCTCCA CAGGCGCATT CGACCAGTCC 1200
GGCAACACAA TGGACTGGTC ACTCTAG 1227
<210>2
<211>408
<212 PRT
<213>sorghum transcription factor SbSBP5
<400>2
MESGGASGGG GGGGGDDQLH GLKFGKKIYF EDAAAAGSSS GGGGGGSGNA SATTAAPATQ 60
QPSPPQAASP RAASGGGGRR GRAAGGPSPA PAPARCQVDG CNVDLTDVKP YYCRHKVCKM 120
HSKEPRVVVN GLEQRFCQQC SRFHQLPEFD QLKKSCRRRL AGHNERRRRP PPGPLATRYG 180
RLAASFGEPG RFRSFMLDFS YPRVPTAMRD GFPAVRPGER APGSIQWQAS LDPHHHQSAV 240
AGYSAHSYGS QGSSSSRPPV FPGPEIPPGG CLAGVPSDSS CALSLLSTQP WDTTHSSGHS 300
HAASMPATAG FDGNPVAPSL MASSYIAPSP WTDSRGHEGG RNVPQLPPDV PLSEVHSGSS 360
SHHGQFSGEL ELALQGNRPA PGSAPAPRND QGSTGAFDQS GNTMDWSL 408
<210>3
<211>24
<212>DNA
<213>artificial sequence
<400>3
GCGGATCCAT GGAGTCCGGT GGCG 24
<210>4
<211>31
<212>DNA
<213>artificial sequence
<400>4
CGGAATTCCT AGAGTGACCA GTCCATTGTG T 31
Claims (9)
1. a kind of sorghum transcription factor SbSBP5 gene, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
2. the protein of sorghum transcription factor SbSBP5 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 SbSBP5 gene；By SbSBP5 amplified production through BamHI and
After EcoRI double digestion, it is inserted into the pET-28a expression vector through same double digestion by DNA ligase, obtains recombinant plasmid
pET-28a-SbSBP5。
5. recombinant vector according to claim 4, which is characterized in that using cDNA as template, utilize following primer amplification
SbSBP5 gene, primer sequence are as follows:
Upstream primer: SbSBP5-F:GCGGATCCATGGAGTCCGGTGGCG underscore is BamHI restriction enzyme site；
Downstream primer: SbSBP5-R:CGGAATTCCTAGAGTGACCAGTCCATTGTGT underscore is EcoRI 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 SbSBP5 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 SbSBP5
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 in kanamycins or kanamycins+chloramphenicol fluid nutrient medium, is shaken
The expression that IPTG induction recombinates SbSBP5 is added after swinging culture；
(4) it after the completion of inducing, recycles and purifies expressed SbSBP5 recombinant protein.
7. the method for expression sorghum transcription factor SbSBP5 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 SbSBP5 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 SbSBP5 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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