CN109912705A - A kind of sorghum 14-3-3 Protein G F14c gene and its recombinant vector and expression - Google Patents
A kind of sorghum 14-3-3 Protein G F14c gene and its recombinant vector and expression Download PDFInfo
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Abstract
The invention discloses a kind of sorghum 14-3-3 Protein G F14c genes, and nucleotide sequence is as shown in SEQ ID NO.1.Invention additionally discloses a kind of protein of sorghum 14-3-3 Protein G F14c 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 14-3-3 Protein G F14c gene.The present invention passes through the protein sequence of corn GF14c gene, sorghum homologous gene GF14c is obtained from sorghum genome database, full length gene 771bp, 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 resistance for improving sorghum by gene editing method.
Description
Technical field
The present invention relates to a kind of sorghum 14-3-3 Protein G F14c gene and its recombinant vector and expressions, belong to biology
Technical 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.
14-3-3 albumen is found in brain tissue earliest, is considered unique related to neuronal tissue, and name comes from pair
Starch gel mobility and the isolated component number of brain total protein.Currently, the study found that 14-3-3 albumen is prevalent in
It is highly conserved eukaryotic protein in all types of eukaryocytes, can be in conjunction with target protein, in most cases, this
Kind, which combines, needs to occur phosphorylation modification, and then the activity, stability, subcellular localization and the participation that adjust them form albumen
Compound.In plant growth and development process, 14-3-3 albumen participates in various plants by the interaction with other albumen and swashs
The regulation processes such as plain signal transduction, various metabolic regulations, matter transportation and optical signal response.In arabidopsis, tobacco and barley etc.
14-3-3 protein gene is had been found that in many plants.But so far, both at home and abroad there is not yet 14-3-3 protein gene is in sorghum
Research report.
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 14-3-3 Protein G F14c gene and its
Recombinant vector and expression.
The technical scheme is that a kind of sorghum 14-3-3 Protein G F14c gene, nucleotide sequence such as SEQ ID
Shown in NO.1.
The present invention also provides the protein of sorghum 14-3-3 Protein G F14c 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 14-3-3 Protein G F14c 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 GF14c gene；By GF14c amplified production after BamHI and XhoI double digestion, by DNA ligase be inserted into through
In the pET-28a expression vector of same double digestion, recombinant plasmid pET-28a-GF14c is obtained.
Preferably, using cDNA as template, using following primer amplification GF14c gene, primer sequence is as follows:
Upstream primer: GF14c-F:GCGAATTCATGGCATCAGCAGAGCTTT underscore is BamHI restriction enzyme site；
Downstream primer: GF14c-R:CGCTCGAGTTACTGCCCCTCGCTCGA 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 14-3-3 Protein G F14c gene is expressed, comprising the following steps:
(1) recombinant vector described in claim 3 or 4 converts Bacillus coli cells, and the recombination for obtaining expression GF14c is former
Nuclear expression bacterial strain；
(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 GF14c is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed GF14c recombinant protein.
Preferably, when earthquake culture is to OD600=0.5 in step (3), the IPTG of final concentration of 1.2mM is added, 30
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: for fewer to the gene studies in sorghum at present, especially 14-3-3 albumen
GF14c gene does not have relevant report, and the present invention passes through the protein sequence of corn GF14c gene, from sorghum genome database
Obtain sorghum homologous gene GF14c, gene C DS overall length 771bp, according to the amino acid sequence that the gene is derived, in NCBI
The GF14c gene in other species is downloaded in database, unrooted chadogram is constructed with adjacent method, the results showed that its amino acid sequence
Gathering with corn GF14c is one, and affiliation is closer, illustrates that GF14c gene is sorghum 14-3-3 protein gene.
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.In addition, the present invention is to obtain GF14c soluble protein, sorghum 14-3-3 Protein G F14c gene is constructed
Prokaryotic expression carrier, and a series of optimization is carried out for protein expression condition, discovery GF14c can be at BL21 (DE3)
PLysS, JM109 (DE3), Rosetta (DE3), BL21 (DE3), Tuner (DE3) are expressed in this 5 bacterial strains, wherein in BL21
(DE3) expression quantity is most in；When inducing temperature is 30 DEG C, expression quantity is best；With the increase of IPTG concentration, protein expression
Amount gradually increases, and best induced concentration is 1.2mM.Through SDS-PAGE electrophoresis detection, the purpose item that molecular weight is about 36kDa is obtained
Band finally obtains GF14c soluble protein for the expected size of GF14c albumen.Meanwhile present invention protein purification system is to it
It is purified, as a result further research albumin crystal structure and biological characteristics lay the foundation, and then to be compiled by gene
The resistance that the method for collecting improves sorghum provides foundation.
Detailed description of the invention
Fig. 1 is the systematic evolution tree of sorghum GF14c gene；
Fig. 2 is digestion result schematic diagram, and 1 is the Plasmid DNA of digestion, and 2 be the digestion of BamHI and XhoI inscribe as a result, 3 are
DNA Marker；
Fig. 3 is the albumen peak figure that GF14c albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 4 is the electrophoresis result figure after GF14c 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 14-3-3 Protein G F14c gene
According to the protein sequence of reported corn GF14c gene, blastP searches for sorghum genome database
(https: //phytozome) finds sorghum homologous gene GF14c (gene number is Sb03g028430).With GF14c protein searches
Ncbi database downloads the 14-3-3 gene in other species, with 7.0 software of MEGA, with adjacent method (Neighbor-
Joining, NJ) (bootstrap=1000) building unrooted chadogram, as seen from Figure 1, GF14c gathers with corn GF14c is
One, affiliation is nearest.
Embodiment 2: the building and identification of sorghum 14-3-3 Protein G F14c 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 GF14c gene；
Design primer expands GF14c gene using cDNA as template.
Primer is as follows:
Upstream primer: GF14c-F:GCGAATTCATGGCATCAGCAGAGCTTT underscore is BamHI restriction enzyme site；
Downstream primer: GF14c-R:CGCTCGAGTTACTGCCCCTCGCTCGA underscore is XhoI 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, GF14c 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 XhoI double digestion by DNA ligase,
16 DEG C connect 16 hours, construct pET-28a-GF14c 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 GF14c coding sequence obtained fulfills the expectation, and says
Bright construction of recombinant plasmid success is simultaneously named as pET-28a-GF14c, i.e. recombinant vector.
The inducing expression of embodiment 3:GF14c albumen
1, the recombined pronucleus expression bacterial strain of GF14c is obtained
It chooses the successful monoclonal of sequencing to be inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm trains overnight
It supports, pET-28a-GF14c is recombinantly expressed according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit and is carried
Body extracts, and recombinant expression carrier conversion Escherichia coli is taken to convert respectively e. coli bl21 (DE3), JM109 (DE3),
Rosetta (DE3), BL21 (DE3) pLysS, Tuner (DE3), NovaBlue (DE3) detect the expression of GF14c 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), BL21
(DE3) pLysS bacterial strain is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C, 200rpm it is overnight
Cultivate 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), 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 1.2mM is added, is trained in 30 DEG C of inductions
It supports 10 hours, obtains the recombinant protein of GF14c.
4, the purifying of GF14c recombinant protein
After the completion of induction, expressed GF14c recombinant protein is recycled and purified, is carried out using AKTA protein purification system pure
Change 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-GF14c recombinant expression carrier obtains
The albumen taken can use Ni-NTA and carry out affinitive layer purification.Affinity chromatography is with protein or large biological molecule and to be incorporated in
Based on special affinity on medium between aglucon, because being connected to a NTA in the matrix of chromatography gel used in His-tags
[(nitrilotriacetic acid) nitrilotriacetic acid], can with Ni ions binding, and the 6 of Ni ion and fusion protein ×
Attraction is generated between His amino acid, so that the fusion protein with His-tags histidine tag be distinguished with other albumen
It comes, five yuan of imidazole rings of histidine residues are the key that albumen and Ni ionization, therefore when we use the imidazoles of high concentration
(Imidazole) solution elution when marquis (such as 400mM), imidazoles be convenient for protein His-tags imidazole ring competitive binding, most
Fusion protein is eluted from gel at last.
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.GF14c albumen is purified by AKTA protein purification system
The albumen peak figure arrived.GF14c purifying figure ultraviolet (UV) absorption value when being purifying GF14c albumen.
Fig. 4 is the electrophoretogram after GF14c protein purification.It is the expression and purification GF14c at 30 DEG C, IPTG concentration 1.2mM
Albumen result.1 is the albumen after Ni column is affine after purification；2 be the affine rear albumen efflux of Ni column；M is albumen marker；
Arrow meaning is shown as GF14c albumen.Fig. 5 shows that GF14c albumen often has in supernatant under this condition, passes through AKTA egg
The GF14c purity of protein that white purification system obtains after purification reaches requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 4:GF14c
1, the recombinant strains of GF14c 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-
GF14c recombinant expression carrier extracts, and recombinant expression carrier is taken to convert respectively e. coli bl21 (DE3), JM109 (DE3),
Rosetta (DE3), BL21 (DE3) pLysS, Tuner (DE3), NovaBlue (DE3) bacterial strain, be coated on card containing 50ug/mL that
It is incubated overnight on mycin (Rosetta (DE3), BL21 (DE3) pLysS need to add 50ug/mL chloramphenicol) plate in 37 DEG C,
Picking single colonie is inoculated into 50ug/mL kanamycins, and (Rosetta (DE3), it is mould that BL21 (DE3) pLysS need to add 50ug/mL chlorine
Element) in fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight, 50% glycerol of sterilizing is added in bacterium solution, freezes after mixing in -80
DEG C refrigerator obtains the prokaryotic expression bacterial strain of expression GF14c.
2, the determination of GF14c 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 inoculated with Rosetta (DE3), BL21 (DE3) into 50ug/mL kanamycins fluid nutrient medium
In pLysS to 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.
The BL21 (DE3) of activation, JM109 (DE3), Tuner (DE3), NovaBlue (DE3) bacterial strain are transferred to 50ug/
In mL kanamycins fluid nutrient medium, Rosetta (DE3), BL21 (DE3) pLysS bacterial strain are transferred to 50ug/mL kanamycins
In+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 1mM is added,
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,
It collects thallus, bacterial precipitation and negative control 2mL and PBS buffer (with preceding addition 1mM PMSF) suspension is pre-chilled, it is broken with ultrasound
Broken instrument progress clasmatosis, 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, GF14c can in BL21 (DE3) pLysS,
JM109 (DE3), Rosetta (DE3), BL21 (DE3), Tuner (DE3) are expressed in this 5 bacterial strains, wherein at BL21 (DE3)
Middle expression quantity is most.
3, the determination of the best IPTG concentration of GF14c 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 0.4,0.6,0.8,1.0,1.2mM
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 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 IPTG induced concentration, as a result as shown in fig. 6, when IPTG concentration is 1.2mM,
GF14c expression is most.
4, the determination of GF14c Primary structure optimum temperature
Above-mentioned recombined pronucleus expression bacterial strain BL21 (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 50ug/mL kanamycins liquid again
In culture medium, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0.8mM is added, respectively 16,20,25,
30 and 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 sees Fig. 7, and in 30 DEG C of Fiber differentiations, GF14c expression quantity is most
It is more.
The western blot of embodiment 5:GF14c recombinant protein is detected
(1) it extracts by GF14c albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-GF14c 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 GF14c albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of sorghum 14-3-3 Protein G F14c gene and its recombinant vector and expression
<160> 4
<210>1
<211>771
<212> DNA
<213>sorghum 14-3-3 Protein G F14c
<400>1
ATGTCGAGGG AGGAGAATGT TTACATGGCC AAGCTGGCTG AGCAGGCTGA AAGGTATGAG 60
GAGATGGTTG AGTATATGGA GAAGGTGGCC AAGACTGTAG ATGTTGAAGA GCTCACTGTT 120
GAGGAGCGGA ACCTCCTGTC TGTCGCATAC AAGAATGTCA TTGGGGCTCG CCGTGCTTCA 180
TGGCGCATTG TCTCTTCCAT TGAACAGAAG GAGGAGTCTC GTAAGAACGA AGAGCATGTG 240
AACCTTATCA AGGAATACCG TGGGAAGATT GAGGCTGAAC TGAGCAACAT CTGTGATGGT 300
ATCCTGAAGC TACTTGACTC CCACCTAGTG CCTTCTTCTA CTGCTGCCGA ATCAAAGGTC 360
TTCTACCTCA AGATGAAGGG TGACTATCAC AGGTATCTTG CGGAATTTAA GACTGGTGCT 420
GAGAGGAAGG AATCTGCTGA GAGCACGATG GTAGCCTACA AGGCTGCTCA GGACATTGCT 480
CTGGCTGAGC TGGCACCAAC CCATCCGATA AGGCTTGGGC TTGCTCTTAA CTTCTCGGTG 540
TTCTATTATG AGATTCTGAA CTCCCCAGAC AAAGCTTGCA ACCTTGCAAA GCAGGCGTTT 600
GATGAAGCCA TCTCTGAGTT GGATACCCTT GGGGAGGAGT CATACAAAGA TAGCACTCTG 660
ATCATGCAGC TCCTGAGGGA CAACTTGACC CTTTGGACCT CTGACCTCAC GGAGGACGGT 720
GCTGATGAGT GCAAAGAAGC CTCGAAAGGC GATGCTGGCG AGGGACAGTA A 771
<210>2
<211>256
<212>PRT
<213>sorghum 14-3-3 Protein G F14c
<400>2
MSREENVYMA KLAEQAERYE EMVEYMEKVA KTVDVEELTV EERNLLSVAY KNVIGARRAS 60
WRIVSSIEQK EESRKNEEHV NLIKEYRGKI EAELSNICDG ILKLLDSHLV PSSTAAESKV 120
FYLKMKGDYH RYLAEFKTGA ERKESAESTM VAYKAAQDIA LAELAPTHPI RLGLALNFSV 180
FYYEILNSPD KACNLAKQAF DEAISELDTL GEESYKDSTL IMQLLRDNLT LWTSDLTEDG 240
ADECKEASKG DAGEGQ 256
<210>3
<211>27
<212>DNA
<213>artificial sequence
<400>3
GCGAATTCAT GGCATCAGCA GAGCTTT 27
<210>4
<211>26
<212>DNA
<213>artificial sequence
<400>4
CGCTCGAGTT ACTGCCCCTC GCTCGA 26
Claims (9)
1. a kind of sorghum 14-3-3 Protein G F14c gene, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
2. the protein of sorghum 14-3-3 Protein G F14c 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 GF14c gene；By GF14c amplified production through BamHI and XhoI
After double digestion, it is inserted into the pET-28a expression vector through same double digestion by DNA ligase, obtains recombinant plasmid pET-
28a-GF14c。
5. recombinant vector according to claim 4, which is characterized in that using cDNA as template, utilize following primer amplification
GF14c gene, primer sequence are as follows:
Upstream primer: GF14c-F:GCGAATTCATGGCATCAGCAGAGCTTT underscore is BamHI restriction enzyme site；
Downstream primer: GF14c-R:CGCTCGAGTTACTGCCCCTCGCTCGA underscore is XhoI 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 14-3-3 Protein G F14c gene, which comprises the following steps:
(1) recombinant vector described in claim 3 or 4 converts Bacillus coli cells, obtains the recombination protokaryon table of expression GF14c
Up to 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 GF14c is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed GF14c recombinant protein.
7. the method for expression sorghum 14-3-3 Protein G F14c 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 1.2mM is added, in 30 DEG C of Fiber differentiations.
8. the method for expression sorghum 14-3-3 Protein G F14c gene according to claim 6, which is characterized in that the card
Kanamycins is 50ug/mL in that mycin fluid nutrient medium.
9. the method for expression sorghum 14-3-3 Protein G F14c gene according to claim 6, which is characterized in that the card
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL in that mycin+chloramphenicol fluid nutrient medium.
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| CN109943574A (en) * | 2019-04-09 | 2019-06-28 | 贵州大学 | A kind of sorghum 14-3-3 Protein G F14b gene and its recombinant vector and expression |
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| CN102643831A (en) * | 2012-04-13 | 2012-08-22 | 昆明理工大学 | Torch pear drought resistance gene Pp 14-3-3 and application of torch pear drought resistance gene Pp 14-3-3 |
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