CN109913478A - A kind of sorghum E3 ubiquitin ligase SbBAG4 gene and its recombinant vector and expression - Google Patents
A kind of sorghum E3 ubiquitin ligase SbBAG4 gene and its recombinant vector and expression Download PDFInfo
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Abstract
The invention discloses a kind of sorghum E3 ubiquitin ligase SbBAG4 genes, and nucleotide sequence is as shown in SEQ ID NO.1.Invention additionally discloses a kind of protein of sorghum E3 ubiquitin ligase SbBAG4 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 E3 ubiquitin ligase SbBAG4 gene.The present invention passes through the protein sequence of rice BAG4 gene, sorghum homologous gene SbBAG4 is obtained from sorghum genome database, full length gene 780bp, 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 E3 ubiquitin ligase SbBAG4 gene and its recombinant vector and expressions, belong to life
Object 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.
Ubiquitin is separated in the thymus gland of 1975 Nian Congniu, and the molecule has then been had been found that in most of eucaryotes
Presence, coding albumen about 8.5kDa, be made of 76 highly conserved amino acid in sequence, because it is widely present
And referred to as ubiquitin.Ubiquitin is referred to as protein ubiquitination by the process that covalently bound mode is applied to target protein, leads to
Crossing this process makes target protein be hydrolyzed (Smalle and Vierstra 2004) in 26S proteasome.- 26 egg of ubiquitin
White enzyme system is by ubiquitin kinase E1 (ubiquitin activation enzyme, E1), ubiquitin binding enzyme E2 (ubiquitin
Conjugating enzyme, E2), ubiquitin ligase (ubiquitin ligase, E3) and 26S proteasome composition.?
In the presence of ATP, ubiquitin kinase E1 is formed by the sulfydryl of its active site cysteine and the glycine end of ubiquitin
Thioester bond and activate free ubiquitin, the ubiquitin of activation is transferred to ubiquitin binding enzyme E2, then in the work of ubiquitin ligase E3
Under, ubiquitin is transferred to target protein by being combined as bridge with E2, finally, target protein quilt in 26S proteasome
Polypeptide, amino acid are decomposed into for reusing
Ubiquitin -26S proteasome degradation pathway (Ubiquitin Proteasome System, UPS) is to adjusting plant
The balance of internal protein and enzyme plays a key role, it was reported that about 6% gene encodes ubiquitin -26S in arabidopsis
Proteasome component.The effect of this proteolytic pathway be related to phytomorph occur (plant morphogenesis),
It blooms, photoperiod, hormone regulating and controlling and host plant the reply entire life process such as biotic and abiotic stress.
In recent years studies have shown that ubiquitin kinase E1 and ubiquitin binding enzyme E2 are than more conservative, type is less, most of true
Only 1 or 2 E1, dozens of E2 in core biology, but the structure change multiplicity of E3 ubiquitin ligase, and have more type
And type, it is that substrate identifies multifarious main factor.Therefore, carrying out further investigation to plant E3 ubiquitin ligase seems outstanding
It is important.
Bcl-2 correlation anti-apoptotic (Bcl-2associated athanogene, BAG) family protein is a kind of multi-functional E3
Ubiquitin ligase protein family member, function is extensive, all exists in yeast, plant and animal.Existing research shows that planting
BAG albumen intervention plant in object is dead to the response of the environment stresses such as temperature, salt and pathogen infection, plant programmed cell
The biological processes such as die.But so far, both at home and abroad there is not yet the gene 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 E3 ubiquitin ligase SbBAG4 gene and
Its recombinant vector and expression.
The technical scheme is that a kind of sorghum E3 ubiquitin ligase SbBAG4 gene, nucleotide sequence such as SEQ
Shown in ID NO.1.
The present invention also provides the protein of sorghum E3 ubiquitin ligase SbBAG4 gene coding, and amino acid sequence is such as
Shown in SEQ ID NO.2.
Correspondingly, the present invention provides the recombinant vector comprising above-mentioned sorghum E3 ubiquitin ligase SbBAG4 gene and expression side
Method.
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 SbBAG4 gene；By SbBAG4 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-SbBAG4 is obtained.
Preferably, using cDNA as template, using following primer amplification SbBAG4 gene, primer sequence is as follows:
Upstream primer: SbBAG4-F:GCGGATCCATGATGAGCGGCAGATCGG underscore is BamHI restriction enzyme site；
Downstream primer: SbBAG4-R:CGCTCGAGCTAGTCGAACTGCTCCCAGTCA 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 E3 ubiquitin ligase SbBAG4 gene is expressed, comprising the following steps:
(1) recombinant vector described in claim 3 or 4 converts Bacillus coli cells, obtains the recombination of expression SbBAG4
Prokaryotic 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 Liquid Culture again
The expression of IPTG induction recombination SbBAG4 is added in base, after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed SbBAG4 recombinant protein.
Preferably, when earthquake culture is to OD600=0.5 in step (3), the IPTG of final concentration of 0.8mM 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 E3 ubiquitin ligase
SbBAG4 gene does not have relevant report, and the present invention passes through the protein sequence of rice BAG4 gene, from sorghum genome database
Obtain sorghum homologous gene SbBAG4, gene C DS overall length 780bp, according to the amino acid sequence that the gene is derived, in NCBI
The BAG gene in other species is downloaded in database, with adjacent method construct unrooted chadogram, the results showed that its amino acid sequence with
It is one that corn BAG, which gathers, and affiliation is closer, illustrates that SbBAG4 gene is sorghum E3 ubiquitin ligase 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 SbBAG4 soluble protein, sorghum E3 ubiquitin ligase SbBAG4 base is constructed
The prokaryotic expression carrier of cause, and a series of optimization is carried out for protein expression condition, find SbTCP14 at JM109 (DE3)
Expression quantity in bacterial strain is most；It was found that expression quantity is best when inducing temperature is 30 DEG C；With the increase of IPTG concentration, egg
White expression quantity gradually increases, and best induced concentration is 0.8mM；At 30 DEG C, when IPTG concentration is 0.8mM, expression quantity is best.Through
SDS-PAGE electrophoresis detection obtains the purpose band that molecular weight is about 35kDa, for the expected size of SbBAG4 albumen, finally obtains
SbBAG4 soluble protein.Meanwhile the present invention purifies it with protein purification system, as a result further studying albumen
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 SbBAG4 gene；
Fig. 2 is pET-28a-SbBAG4 recombinant vector map；
Fig. 3 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, M is
DNA Marker；
Fig. 4 is the albumen peak figure that SbBAG4 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 5 is the electrophoresis result figure after SbBAG4 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 E3 ubiquitin ligase SbBAG4 gene
According to the protein sequence of reported rice BAG4 gene (gene number is LOC_Os01g61500), blastP search
Sorghum genome database (https: //phytozome) finds sorghum homologous gene SbBAG4, and (gene number is
Sb03g038830).With SbBAG4 protein searches ncbi database, the BAG gene in other species is downloaded, it is soft with MEGA 7.0
Part constructs unrooted chadogram with adjacent method (Neighbor-Joining, NJ) (bootstrap=1000), can be seen by Fig. 1
Out, it is one that SbBAG4 and corn BAG, which gathers, and affiliation is nearest.
Embodiment 2: the building and identification of sorghum E3 ubiquitin ligase SbBAG4 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 SbBAG4 gene；
Design primer expands SbBAG4 gene using cDNA as template.
Primer is as follows:
Upstream primer: SbBAG4-F:GCGGATCCATGATGAGCGGCAGATCGG underscore is BamHI restriction enzyme site；
Downstream primer: SbBAG4-R:CGCTCGAGCTAGTCGAACTGCTCCCAGTCA 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, SbBAG4 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
(expression vector map is as shown in Figure 2), 16 DEG C connect 16 hours, construct pET-28a-SbBAG4 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 SbBAG4 coding sequence obtained fulfills the expectation, and says
Bright construction of recombinant plasmid success is simultaneously named as pET-28a-SbBAG4, i.e. recombinant vector.
The inducing expression of embodiment 3:SbBAG4 albumen
1, the recombined pronucleus expression bacterial strain of SbBAG4 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-
SbBAG4 recombinant expression carrier extracts, take recombinant expression carrier plasmid conversion E. coli expression strains BL21 (DE3),
Rosetta (DE3) and JM109 (DE3) detects the expression of SbBAG4 albumen.
2, activated strains are incubated overnight
It is inoculated with above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3), JM109 (DE3) respectively to the liquid containing kanamycins
Culture medium；It is inoculated with recombined pronucleus expression bacterial strain Rosetta (DE3) and arrives the fluid nutrient medium containing kanamycins and chloramphenicol (wherein
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL) 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) bacterial strain be transferred in 50ug/mL kanamycins fluid nutrient medium, by Rosetta (DE3) bacterium
Strain is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol 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, 30 DEG C Fiber differentiation 10 hours, obtain the recombinant protein of SbBAG4.
4, the purifying of SbBAG4 recombinant protein
After the completion of induction, expressed SbBAG4 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-SbBAG4 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.SbBAG4 albumen is purified by AKTA protein purification system
Obtained albumen peak figure.SbBAG4 purifying figure ultraviolet (UV) absorption value when being purifying SbBAG4 albumen.
Fig. 5 is the electrophoretogram after SbBAG4 protein purification.It is the expression and purification at 30 DEG C, IPTG concentration 0.8mM
SbBAG4 albumen result.1 is the affine rear efflux of Ni column；2 be Supernatant samples after cell ultrasonication；3 be cell ultrasonication
Deposit sample afterwards；4, it is albumen marker for the albumen M after Ni column is affine after purification；Red arrow meaning is shown as SbBAG4
Albumen.Fig. 5 shows that SbBAG4 albumen often has in supernatant under this condition, after purification by AKTA protein purification system
Obtained SbBAG4 purity of protein reaches requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 4:SbBAG4
1, the recombinant strains of SbBAG4 are obtained
It chooses and chooses the successful monoclonal of sequencing in embodiment 2 and be 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-
SbBAG4 recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), Rosetta respectively
(DE3), JM109 (DE3) bacterial strain is coated on kanamycins containing 50ug/mL (Rosetta (DE3) need to add 50ug/mL chloramphenicol)
It is incubated overnight on plate in 37 DEG C, picking single colonie is inoculated into 50ug/mL kanamycins, and (Rosetta (DE3) need to add chlorine
Mycin) in fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight, 50% glycerol of sterilizing is added in bacterium solution, frozen after mixing in-
80 DEG C of refrigerators obtain the prokaryotic expression bacterial strain of expression SbBAG4.
2, the determination of SbBAG4 Primary structure optimum strain
BL21 (DE3), JM109 (DE3) bacterial strain are transferred in 50ug/mL kanamycins fluid nutrient medium, by Rosetta
(DE3) bacterial strain is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, and 37 DEG C, 200rpm is incubated overnight
Activated strains.
The recombined pronucleus expression bacterial strain BL21 (DE3) of activation, JM109 (DE3) are transferred to 50ug/mL kanamycins, bacterium
Strain Rosetta (DE3) is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, and 37 DEG C of shake cultures are extremely
When OD600=0.5, the IPTG of final concentration of 1mM is added, 25 DEG C Fiber differentiation 10 hours.4 DEG C after the completion of induction, 1,
2000rpm is centrifuged 2min, collects thallus, bacterial precipitation and negative control 2mL and PBS buffer is pre-chilled (with preceding addition 1mM
PMSF it) suspends, carries out clasmatosis with Ultrasonic Cell Disruptor, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm from
Heart 10min, takes supernatant, and SDS-PAGE electrophoresis detection tests optimum expression bacterial strain, as a result as shown in fig. 6, SbBAG4 can be in BL21
(DE3), Rosetta (DE3), express in JM109 (DE3) bacterial strain, wherein the expression quantity highest in JM109 (DE3) bacterial strain.
3, the determination of the best IPTG concentration of SbBAG4 Primary structure
Above-mentioned recombined pronucleus expression bacterial strain JM109 (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 0.2,0.4,0.6,0.8,1.0,1.2mM
IPTG, 30 DEG C Fiber differentiation 8 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. 7, when IPTG concentration is 0.8mM,
SbBAG4 expression is most.
4, the determination of SbBAG4 Primary structure optimum temperature
Above-mentioned recombined pronucleus expression bacterial strain JM109 (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 1mM is added, is lured respectively 16,20,25,30
Lead 8-16 hours (pET-28a bacterium is taken to make negative control) of culture.4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min collects bacterium
Body, bacterial precipitation and negative control with 2mL be pre-chilled PBS buffer (with preceding addition 1mM PMSF) suspension, with Ultrasonic Cell Disruptor into
Row clasmatosis, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-PAGE
Electrophoresis detection tests best inducing temperature, as a result sees Fig. 8, and in 30 DEG C of Fiber differentiations, SbBAG4 expression quantity is most.
The western blot of embodiment 5:SbBAG4 recombinant protein is detected
(1) it extracts by SbBAG4 albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-SbBAG4 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 SbBAG4 albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of sorghum E3 ubiquitin ligase SbBAG4 gene and its recombinant vector and expression
<160> 4
<210>1
<211>780
<212> DNA
<213>sorghum E3 ubiquitin ligase SbBAG4
<400>1
ATGATGAGCG GCAGATCGGG CGGGAGGGAC GCCGAGGGCG AGTGGGAGGT CCGGCCCGGC 60
GGGATGCTGG TGCAGCGCAG GGACGGCGAG GCGGCCGGCC CCGTCATCAG GATCAGGGTC 120
TCCCACGGCG CCAGCTTCCG CGAGGTCCTC GTCCCCGCGC AGGCTACCTT CGGTGAGTTG 180
AAGAGTATCC TTGCTCAGAC TACTGGCTTG GAGCCTGAGA GGCAGAGGCT CTTCTTCCGT 240
GGGAAGGAGA AGAGTGACAG AGAGTTCCTG CACACGGCAG GTGTTAAGGA TGGAGCAAAG 300
TTGCTTCTGC TTGAGAAGCC TGCCCCTGCC AACATAGAGC AGAAGGTGGA GCCTGTGGTT 360
ATGGATGAGA GCATGATGAA GGCATGTGAG GCTGTTGCTC GTGTTCGAGC TGAAGTTGAT 420
AAGCTGTCTG CTAAGGTGTG TGACTTGGAG AAGAATGTGC TTGGAGGGAG AAAGGTTGAG 480
GATAAAGAAT TTGTCGTCTT GACGGAGCTG CTTATGATGC AACTGCTGAA ACTCGATGGC 540
ATAGAAGCTG AAGGAGAAGC AAGGGCACAG AGGAAGGCTG AGGTGCGCCG AGTTCAGTCC 600
CTTGTGGAGA CCTTGGACAA GCTGAAAGCA AGAAATGCAA ACCCCTTCAG CGACCATAAC 660
AAAGCTGTTT CAGTAACAAC GCAGTGGGAG ACGTTTGAGA ACGGCATGGG CAGCTTGAGC 720
GCTCCCCCTC CCCGTGTTTC TTCGACACAA GTTAACACTG ACTGGGAGCA GTTCGACTAG 780
<210>2
<211>259
<212> PRT
<213>sorghum E3 ubiquitin ligase SbBAG4
<400>2
MMSGRSGGRD AEGEWEVRPG GMLVQRRDGE AAGPVIRIRV SHGASFREVL VPAQATFGEL 60
KSILAQTTGL EPERQRLFFR GKEKSDREFL HTAGVKDGAK LLLLEKPAPA NIEQKVEPVV 120
MDESMMKACE AVARVRAEVD KLSAKVCDLE KNVLGGRKVE DKEFVVLTEL LMMQLLKLDG 180
IEAEGEARAQ RKAEVRRVQS LVETLDKLKA RNANPFSDHN KAVSVTTQWE TFENGMGSLS 240
APPPRVSSTQ VNTDWEQFD 259
<210>3
<211>27
<212> DNA
<213>artificial sequence
<400>3
GCGGATCCAT GATGAGCGGC AGATCGG 27
<210>4
<211>30
<212> DNA
<213>artificial sequence
<400>4
CGCTCGAGCT AGTCGAACTG CTCCCAGTCA 30
Claims (9)
1. a kind of sorghum E3 ubiquitin ligase SbBAG4 gene, which is characterized in that its nucleotide sequence such as SEQ ID NO.1 institute
Show.
2. the protein of sorghum E3 ubiquitin ligase SbBAG4 gene coding as described in claim 1, amino acid sequence such as SEQ
Shown in ID 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 SbBAG4 gene；By SbBAG4 amplified production through BamHI and
After XhoI double digestion, it is inserted into the pET-28a expression vector through same double digestion by DNA ligase, obtains recombinant plasmid
pET-28a-SbBAG4。
5. recombinant vector according to claim 4, which is characterized in that using cDNA as template, utilize following primer amplification
SbBAG4 gene, primer sequence are as follows:
Upstream primer: SbBAG4-F:GCGGATCCATGATGAGCGGCAGATCGG underscore is BamHI restriction enzyme site；
Downstream primer: SbBAG4-R:CGCTCGAGCTAGTCGAACTGCTCCCAGTCA 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 E3 ubiquitin ligase SbBAG4 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 SbBAG4
Express bacterial strain；
(2) by recombined pronucleus expression strain inoculated to kanamycins or kanamycins+chloramphenicol culture medium, it is incubated overnight activation
Bacterial strain；
(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 SbBAG4 is added after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed SbBAG4 recombinant protein.
7. the method for expression sorghum E3 ubiquitin ligase SbBAG4 gene according to claim 6, which is characterized in that step
(3) when earthquake culture is to OD600=0.5 in, the IPTG of final concentration of 0.8mM is added, in 30 DEG C of Fiber differentiations.
8. the method for expression sorghum E3 ubiquitin ligase SbBAG4 gene according to claim 6, which is characterized in that described
Kanamycins is 50ug/mL in kanamycins fluid nutrient medium.
9. the method for expression sorghum E3 ubiquitin ligase SbBAG4 gene according to claim 6, which is characterized in that described
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL in kanamycins+chloramphenicol fluid nutrient medium.
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Application publication date: 20190621 |
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