CN105154452A - Dehydrated protein gene for medicago ruthenica (L.) and application thereof - Google Patents
Dehydrated protein gene for medicago ruthenica (L.) and application thereof Download PDFInfo
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Abstract
The invention relates to a dehydrated protein gene for medicago ruthenica (L.). The gene is an MrDHN3 encoding gene and comes from the medicago ruthenica (L.). The gene has a nucleotide sequence shown as SEQ ID NO. 1 in a sequence table, or has an allele with functional activity, or has protein of an amino acid residue sequence shown as SEQ ID NO. 2 in the sequence table, or has protein which comprises an S-fragment including a series of serine (Ser) residues and two K-fragments (EKKGIMDKIKEKLPG or derivatives of the fragments) rich in lysine residues, wherein the allele is formed by deficiency or insertion or replacement of one or more basic groups in the SEQ ID NO. 1 of the sequence table, the protein is formed in the way that an amino acid residue sequence shown in the SEQ ID NO. 2 of the sequence table are subjected to replacement or deficiency or addition through one or more amino acid residues, and the protein has the same activity as the amino acid residue sequence shown in the SEQ ID NO. 2 and is derived through the SEQ ID NO. 2. The obtained gene can be used for improving the salt resistance and heat resistance of plants and microorganisms.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, particularly relate to Medicago ruthenica dehydrin gene and application thereof.
Background technology
Dehydrated protein belongs to late embryo generation Abundant protein (lateembryogenesisabundantprotein) LEAII family.Dehydrated protein finds at first in seed, and research subsequently shows that dehydrated protein is distributed widely in algae, yeast, nematode, each tissue of cyanobacteria and higher plant and organ.The expression that arid, low temperature and high salt etc. cause the environmental factors of dehydration and dormin (ABA) all can induce it in cell.Dehydrated protein contains at least one conservative motif K-fragment (EKKGIMDKIKEKLPG or derivatives thereof) being rich in Methionin, and they often form the spirane structure of amphiphilic (hydrophilelipophile).Some dehydrated protein also comprises Y-fragment and S-fragment usually, and Y-fragment appears at the N-end of dehydrated protein usually with 1-3 repetition form (T/VDEYGNP), S fragment is made up of a series of Serine (Ser) residue.Dehydrated protein can be divided into 5 subfamilies by the type occurred based on Y, S, K fragment and quantity: YnSKn, SKn, Kn, YnKn and KnS.
Dehydrated protein is protected relevant in intracellular function with cell dehydration.By transgenic technology, barley dehydrin gene ZmDHN2b is proceeded to tobacco, the anti-seismic design of transfer-gen plant improves; In Arabidopis thaliana, overexpression Wheat DH N-5 gene can strengthen its resistance to salt and osmotic stress; Wheat dehydrin gene WCOR410 process LAN can strengthen the frost resistance of strawberry.Therefore, dehydrated protein may be comprise plant, bacterium and lower animal cell to obtain one of deciding factor of dehydration tolerance, although its concrete mechanism it be unclear that.Meanwhile, dehydrin gene also has important potential using value in the abiotic stress resistance improvement such as plant arid, low temperature, high salt.
Except Genetic Transformation in Higher Plants test, the heterologous expression system such as intestinal bacteria and yeast is also widely used in the qualification of dehydrated protein.A PM2 albumen in process LAN soybean LEA3 family and KS type dehydrated protein SLT1629 can significantly improve intestinal bacteria and can improve intestinal bacteria saline-alkaline tolerance; Barley dehydrated protein is introduced it can be made after yeast to obtain tolerance to high-salt stress.Therefore, utilize yeast, escherichia expression system can as qualification dehydrated protein effective system.
Medicago ruthenica
medicagoruthenica (L.)be distributed widely in siberian, the northern high latitude extremely frigid zones of Mongolia and China, being grown on patana place more, having extremely strong tolerance to adverse circumstance, is the Medicago Perennial legume forages uniquely normally can survived the winter at the extreme extremely frigid zones of High aititude.Only have the report that fragmentary Medicago ruthenica dehydrin gene is cloned at present, but there is no the report to its degeneration-resistant Function Identification.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Medicago ruthenica dehydrin gene improveing plant and the anti-salt of microorganism and thermotolerance.
Another technical problem to be solved by this invention be to provide this gene improvement plant and the anti-salt of microorganism and heat-resisting in application.
For solving the problem, Medicago ruthenica dehydrin gene of the present invention, is characterized in that: this gene is
mrDHN3encoding gene, it has SEQ ID NO.1 the 1st to the nucleotide sequence shown in 890.
Described gene has the allelotrope with functionally active formed because of the disappearance of one or more base, insertion or replacement in SEQ ID NO.1 the 1st to 890.
Described gene has SEQ ID NO.2 the 1st protein to the amino acid residue sequence shown in 221.
Described gene has SEQ ID NO.2 the 1st and has the protein that by SEQIDNO.2 derived of with SEQIDNO.2 amino acid residue sequence identical activity containing the S-fragment of 1 a series of Serine (Ser) residue with 2 K-fragments (EKKGIMDKIKEKLPG or derivatives thereof) being rich in lysine residue through the replacement of one or several amino-acid residue, disappearance or interpolation to the amino acid residue sequence shown in 221; The sequence of the described protein derived by SEQIDNO.2 is corresponding with allelic sequence.
Medicago ruthenica dehydrin gene as above improvement plant and the anti-salt of microorganism and heat-resisting in application.
The present invention compared with prior art has the following advantages:
1, the present invention is analyzed by transcription group, in conjunction with RT-PCR technology, is separated to a new dehydrin gene in Medicago ruthenica
mrDHN3.Will
mrDHN3after gene coding region is connected to coli expression carrier; this prokaryotic expression carrier is proceeded to E. coli expression strains; the overexpression of MrDHN3 albumen in intestinal bacteria is achieved with IPTG induction; result shows; MrDHN3 albumen overexpression has protected effect to the intestinal bacteria under high salt and heat stress; therefore, this gene can be used for anti-salt and the thermotolerance improvement of plant and microorganism by biotechnological means.
2, by after carrying out RT-PCR amplification to the Medicago ruthenica dehydrin gene in the present invention, carry out agarose gel electrophoresis (as Suo Shi Fig. 1, Fig. 5 ~ 8), the amplified production of about 900bp can be detected.
3, pass through the Medicago ruthenica dehydrated protein in the present invention
mrDHN3the coding region of gene is connected to coli expression carrier, cuts (as shown in Figure 9) after qualification, transformation of E. coli expression vector through enzyme, containing, for example above-mentioned structure
mrDHN3induce through IPTG in the intestinal bacteria of the prokaryotic expression carrier of gene, SDS-PAGE electrophoresis is carried out to culture bacterial protein, have in culture of Escherichia coli after result display induction one with expect the close protein band (as shown in Figure 10) of molecular weight, therefore Medicago ruthenica dehydrated protein MrDHN3 can in intestinal bacteria overexpression.
4, by the intestinal bacteria of the Medicago ruthenica dehydrated protein MrDHN3 in overexpression the present invention through heat stress process with containing after high salt culture medium is cultivated; observe colibacillary growth survival condition described above; result display MrDHN3 albumen overexpression can improve the tolerance (as Suo Shi Figure 11 ~ 12) of escherichia coli host to high-salt stress and heat stress process, and therefore MrDHN3 albumen has protected effect to cell under high-salt stress and heat stress process.
5, gene structure feature of the present invention:
The present invention is according to Medicago ruthenica transcript profile sequencing result, and design PCR primer, wherein primer P1+P2 is used for amplification containing 5 '-and 3 '-non-translational region and coding region
mrDHN3gene order (Fig. 1 ~ 2).Primer P3+P4 increases
mrDHN3genes encoding region nucleotide sequence, for the functionally active of prokaryotic expression qualification clone gene.
Use primer P1+P2, Medicago ruthenica cDNA is that template increases, and obtains the band (Fig. 1) of about 900bp.Obtain 890bp(after order-checking as Fig. 2) as shown in sequence table SEQ IDNO.1 containing 5 '-and 3 '-non-translational region and coding region
mrDHN3gene order, wherein 173-839 position is translated region.The aminoacid sequence derived by SEQIDNO.1 sequence is as shown in Fig. 2 (S fragment underscore marks, and 2 conservative K fragment shades mark) and sequence table SEQ IDNO.2.This aminoacid sequence shows, and protein is made up of 221 amino-acid residues, and predicted molecular weight (Mw) is 24.84kD, and theoretical iso-electric point (pI) is 5.56, is acidic protein.Overall average hydrophobicity index Grandaverageofhydropathicity (GRAVY) is-1.444, has higher wetting ability.Carry out search at Pfam database (http://pfam.janelia.org) database to the structural domain of the aminoacid sequence that MrDHN3 encodes to show, MrDHN3 albumen belongs to LEAII protein family, there is the K-fragment (dotted line collimation mark goes out) (Fig. 3) that a S-fragment (solid box marks) and 2 are rich in lysine residue, is SK2 type dehydrated protein.Utilize in ncbi database BLAST to the aminoacid sequence of MrDHN3 coded protein and other species protein amino acid sequence compare (Fig. 3) find with M. truncatula (Medicagotruncatula) and Trifolium repense (Trifoliumrepens) Amino acid sequence identity (identity) the highest, reach more than 81%.Find SKn type dehydrated protein aminoacid sequence constructing system evolutionary tree (Fig. 4) of 6 species that NCBI downloads, the sibship of MrDHN3 and leguminous plants pea SK2 albumen is nearest.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is Medicago ruthenica dehydrin gene of the present invention
mrDHN3rT-PCR clone products electrophoretogram.
Fig. 2 is the present invention
mrDHN3the aminoacid sequence of gene cDNA sequence and coded protein thereof.
Fig. 3 is the compare of analysis of Medicago ruthenica dehydrated protein MrDHN3 of the present invention and other species dehydrated protein aminoacid sequence.Wherein TrDHN3 (ADD09573.1) is from Trifolium repense (Trifoliumrepens); MtDHN3 (XP_003603987.1) is from M. truncatula (Medicagotruncatula); PsDHN3 (CAA78515.1) is from pea (Pisumsativum); CaDHN3 (XP_004500781.1) is from garbanzo (Cicerarietinum).1 solid box shows conservative S-fragment, and 2 dotted line frames are the conservative K-fragment being rich in lysine residue.
Fig. 4 is the systematic evolution tree that Medicago ruthenica dehydrated protein MrDHN3 of the present invention and other species SKn type dehydrated protein build.Wherein OsDHN3 (ABS44866.1) is from japonica rice (OryzasativaJaponicaGroup); TaDHN3 (AAB18202.1) is from wheat (Triticumaestivum); MaDHN3 (AEI54683.1) is from plantain (MusaABBGroup); CsDHN3 (ACT10283.1) is from tea (Camelliasinensis); PpDHN3 (AAZ83586.1) is from peach (Prunuspersica); PsDHN3 (AAL50315.1) is from pea (Pisumsativum).Filled triangle symbols instruction Medicago ruthenica dehydrated protein MrDHN3.
Fig. 5 is Medicago ruthenica dehydrated protein of the present invention
mrDHN3the semi-quantitative RT-PCR analysis of gene gene expression dose under the process of simulation desiccation stress,
actinfor for interior target Medicago ruthenica actin gene.
Fig. 6 is Medicago ruthenica dehydrated protein of the present invention
mrDHN3the semi-quantitative RT-PCR analysis of gene gene expression dose under NaCl Stress treatment,
actinfor for interior target Medicago ruthenica actin gene.
Fig. 7 is Medicago ruthenica dehydrated protein of the present invention
mrDHN3the semi-quantitative RT-PCR analysis of gene gene expression dose under ABA process,
actinfor for interior target Medicago ruthenica actin gene.
Fig. 8 is Medicago ruthenica dehydrated protein of the present invention
mrDHN3the semi-quantitative RT-PCR analysis of gene gene expression dose under subzero treatment,
actinfor for interior target Medicago ruthenica actin gene.
Fig. 9 is Medicago ruthenica dehydrated protein of the present invention
mrDHN3the enzyme of prokaryotic expression carrier pET30a-MrDHN3 cut qualification electrophoretogram.Wherein " BamHI+SacI " is the pET30a-MrDHN3 carrier of the double digested process of BamHI and SacI, " BamHI " is the pET30a-MrDHN3 carrier of BamHI single endonuclease digestion digestion process, " SacI " is the pET30a-MrDHN3 carrier not doing digestions process for the pET30a-MrDHN3 carrier of SacI single endonuclease digestion digestion process, " Plasmid ".
Figure 10 is the SDS-PAGE electroresis appraisal of Medicago ruthenica dehydrated protein MrDHN3 of the present invention abduction delivering in e. coli bl21 (DE3).Wherein " 1 " is the total protein before IPTG induction, and " 2 " are the total protein after IPTG induction, and " 3 " are the albumen of abduction delivering in supernatant liquor, and " 4 " are supernatant liquor soluble proteins after 100 DEG C of process 10min.
Figure 11 is that Medicago ruthenica dehydrated protein MrDHN3 of the present invention tests the bacterium drop plate of the environment stress protected effect of Host Strains after expression in escherichia coli.Wherein BL/pET30a is BL21 (DE3) intestinal bacteria containing pET30a (+) plasmid; BL/MrDHN3 is BL21 (DE3) intestinal bacteria containing pET30a-MrDHN3 plasmid; LB is that Luria-Bertani (LB) solid medium is added with 0.5mmol/LIPTG; LB+500mMNaCl is that LB solid medium adds 0.5mmol/LIPTG and 0.5mol/LNaCl; LB+500mMKCl is that LB solid medium adds 0.5mmol/LIPTG and 0.5mol/LKCl; LB (55 DEG C, 30min) is 55 DEG C of thermal treatment 30min.
Figure 12 be Medicago ruthenica dehydrated protein MrDHN3 of the present invention after expression in escherichia coli to the enumeration statistics of the environment stress protected effect of Host Strains.Wherein BL/pET30a is BL21 (DE3) intestinal bacteria containing pET30a (+) plasmid; BL/MrDHN3 is BL21 (DE3) intestinal bacteria containing pET30a-MrDHN3 plasmid; NaCl (0.5M) and KCl (0.5M) is high-salt stress, Heat be (55 DEG C, heat stress process 30min.
Embodiment
embodiment 1medicago ruthenica dehydrin gene, this gene is
mrDHN3encoding gene, it has SEQ ID NO.1 the 1st to the nucleotide sequence shown in 890.
One, Medicago ruthenica dehydrated protein
mrDHN3the clone of gene.
1, the process of Medicago ruthenica seedling low temperature stress and transcript profile check order:
(1) Medicago ruthenica low temperature stress process:
By Medicago ruthenica seed with after dense sulfuric acid treatment 10min, remove remaining sulfuric acid for several times with distilled water flushing.Above-mentioned treated seed is placed in and is covered with in double-layer filter paper culture dish, 21 DEG C, germinate under 16h/8h photoperiod condition; After 3d by germinate seedling replanting in vermiculite: continue to cultivate under these conditions in the mixed-matrix of Nutrition Soil (3:1), weekly with containing 1/2MS nutritive medium pouring once; After 3 weeks, seedling is transferred to-4 DEG C of illumination boxs and carries out subzero treatment 24h, before subzero treatment and after process, round strain seedling, clean up root, with liquid nitrogen flash freezer after thieving paper exhaustion residual moisture ,-80 DEG C of preservations.
(2) transcript profile order-checking:
By the seedling before subzero treatment and after process, BGI Technology Solutions Co., Ltd. is sent to carry out Total RNAs extraction and transcript profile order-checking.
2, design of primers:
According to transcript profile sequencing result design PCR primer, specific as follows:
P15’-CGTGTCATTATGTGTAGTAGTGAAG-3’
P25’-TAAACAAAGCACCCTCCA-3’
3, Total RNAs extraction and cDNA first chain synthesize:
The sample of above-mentioned low temperature stress process is pulverized last in liquid nitrogen, extracts total serum IgE with Trizol reagent (Invitrogen Products) according to its specification sheets.The total serum IgE water dissolution of 20 μ LDEPC process, adjustment concentration is 500ng/ μ L.
CDNA first chain synthesis reaction uses PrimeScript RTreagentKit (PerfectRealTime) (Takara product) test kit to carry out according to its specification sheets.
4,
mrDHN3gene cDNA sequence increases:
PCR reaction system is by 10 μ L2 × PCRBufferI, 1.6 μ LdNTP, 0.4 μ LP1 (10 μMs), 0.4 μ LP2 (10 μMs), 0.05 μ LLA
?taq DNA polymerase (5U/ μ L) (Takara product), 0.05 μ LPyrobest
?archaeal dna polymerase (5U/ μ L) (Takara product), 1 μ LcDNA first chain synthetic product composition, add ddH
2o(distilled water) to final volume be 20 μ L.
Wherein: 2 × PCRBufferI is and LA
?the supporting damping fluid of Taq DNA polymerase.
DNTP is by dATP(triphosphoric acid adenyl-deoxyribonucleotide), dTTP(deoxythymidine triphosphate), dGTP(deoxyguanosine triphosphate trisodium) and dCTP(deoxycytidine triphosphate) form; DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is undertaken by follow procedure:
After reaction terminates, the sepharose with 0.7% detects PCR primer, obtains the band of about 900bp as shown in Figure 1.
5, PCR primer reclaims:
PCR primer reclaims test kit (the raw chemical product in Shanghai) with UNIQ-10 pillar DNA glue and reclaims.
6, the clone of target DNA fragment:
Linked system:
(1) linked system total amount is 5 μ L, by 0.5 μ LpGEM
?-TEasy carrier (Promega product), 0.5 μ LT
4dNA ligase, 2.5 μ L2 × Rapid connect damping fluid and 1.5 μ L target DNA fragments composition.Wherein ligase enzyme is that pGEM-TEasy carrier carries with being connected damping fluid.
(2) 5 μ L linked systems are connected at 4 DEG C and spend the night.
(3) 5 μ L linked systems are all added in 50 μ L bacillus coli DH 5 alpha competent cells, after mixing, ice bath 30min.
(4) the mixed system of step (3) gained after heat shock 90s, is placed in ice bath 2min at once in 42 DEG C of water-baths.
(5) in the mixed system of step (4) gained, add 400 μ LLB liquid nutrient mediums, with the rate oscillation 1h of 160rpm at 37 DEG C, obtain the DH5 α bacterium liquid after activating.
(6) get the DH5 α bacterium liquid after 200 μ L activation, mix with 40uLx-gal (20mg/ml) with 5 μ LIPTG (1M), coated plate on the LB plate culture medium containing penbritin (Amp) (100 μ g/ml), cultivates 12 ~ 16h at 37 DEG C.
7. the qualification of positive colony and order-checking:
(1), with the positive bacterium colony of the anti-penbritin of the white that toothpick picking LB plate culture medium grows, contain Amp(100 μ g/ml in another) LB plate culture medium on streak culture 8 ~ 14h, obtain single bacterium colony.
(2), with single bacterium colony that the setting-out of toothpick picking is cultivated, be applied to the bottom of PCR pipe.
(3) PCR reaction system:
PCR reaction system is by 1.6 μ LdNTP, 2 μ L10 × PCRBuffer, 0.4 μ LP1 (10 μMs), 0.4 μ LP2 (10 μMs), 0.1 μ LTaq
?enzyme (Takara product) forms, and uses ddH
2it is 20 μ L that O adds to final volume.
Wherein: 10 × PCRBuffer is Taq
?the supporting damping fluid of archaeal dna polymerase.
DNTP is made up of dATP, dTTP, dGTP and dCTP; DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is undertaken by follow procedure:
After reaction terminates, the sepharose with 0.7% detects, and determines that goal gene fragment has been inserted in pGEM-TEasy carrier.
(4) (3) identify that single bacterium colony of acquisition is to containing Amp(100 μ g/ml by toothpick picking step) LB liquid nutrient medium in, with the speed of 200rpm concussion overnight incubation at 37 DEG C.
(5) draw 700 μ L bacterium liquid in the 1.5mL centrifuge tube of sterilizing, add 300 μ L50% glycerine, mixing, send Sangon Biotech (Shanghai) Co., Ltd. to check order.
Obtain after order-checking
mrDHN3the complete nucleotide sequence as shown in Fig. 2 and sequence table SEQ IDNO.1 of gene 890bp, includes the opening code-reading frame of 666bp, 3 ' the UTR district of 51bp and 5 ' the UTR district of 173bp.
Two, Medicago ruthenica dehydrated protein
mrDHN3the prokaryotic expression of gene.
1, prokaryotic expression design of primers:
According to sequencing result design primer P3 and P4, in order to amplification
mrDHN3the coding region DNA sequence dna of gene, and introduce BamH I and Sac I two restriction enzyme sites (underscore) at DNA sequence dna 5 '-end and 3 '-end respectively.
P3：5’-GC
GGATCCATGGCTGATCAGGAGAATCAGAAC-3’
P4：5’-CGC
GAGCTCTCAATGAGTAGTAGTCTCATCCTT-3’
2, recombinant plasmid and expression vector pET-30a (+) plasmid (Novagen product) is extracted with the little extraction reagent kit of plasmid (the raw chemical product in Shanghai).
3,
mrDHN3coding sequence pcr amplification and clone:
PCR reaction system is by 12.5 μ L2 × LABufferI, 2 μ LdNTP, 0.5 μ LP3 (10 μMs), 0.5 μ LP4 (10 μMs), 0.05 μ LPyrobest
?archaeal dna polymerase (Takara product) (5U/ μ L), 1 μ L contain
mrDHN3pGEM-TEasy recombinant plasmid (the diluting 50 times) composition of gene, and use ddH
2it is 25 μ L that O adds to final volume.
Wherein dNTP is made up of dATP, dTTP, dGTP and dCTP; DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is undertaken by follow procedure:
After reaction terminates, obtain
mrDHN3the PCR primer of gene coding region.
4, reclaim test kit with UNIQ-10 pillar DNA glue to reclaim
mrDHN3coding region PCR primer.
5,
mrDHN3coding region PCR primer rTaq
?after archaeal dna polymerase (Takara product) tailing process, carry out order-checking after being connected to pGEM-TEasy carrier and confirm.
6, the double digestion of goal gene fragment and expression vector pET-30a:
To contain
mrDHN3the pGEM-TEasy carrier of coding region DNA sequence dna and prokaryotic expression carrier pET-30a(Novagen product) with BamH I (NEB product) and SacI(NEB product) carry out double digestion, the digestion products sepharose of 0.7% is separated, and reclaims test kit reclaim goal gene fragment and carrier segments respectively with UNIQ-10 pillar DNA glue.
7, connect and identify:
The ratio of the double digestion product of the goal gene double digestion product and pET-30a (+) that are inserted into fragment in mole number 10:1 is mixed, adds T4DNA ligase enzyme (Promega product), connect at 4 DEG C and spend the night.
Get 5 μ L to connect products and be added in 50 μ L bacillus coli DH 5 alpha competent cells and transform, adopt kantlex (Kan) resistance screening, the bacterium colony grown directly is identified with PCR, the positive colony identified is shaken bacterium, extract plasmid, then carry out list/double digestion qualification (Fig. 9) with BamHI and Sac I.
8, conversion and protein expression:
By PET-30a plasmid transformation escherichia coli BL21 (DE3) (Novagen product) competent cell containing target DNA fragment, with kantlex (Kan
+) carry out resistance screening.Picking positive colony is inoculated in containing 50mg/LKan
+lB liquid nutrient medium in.Shake after bacterium spends the night with the speed of 250rpm at 37 DEG C, by 1:100(v/v) be transferred in fresh LB substratum and cultivate after 2 ~ 3h to OD600 reaches 0.6 ~ 0.8, take out 1mL bacterium liquid as sample before induction, adding isopropylthiogalactoside (IPTG) to final concentration in all the other bacterium liquid is 0.5mmol/L, with the speed inducing culture 3h of 250rpm at 37 DEG C.
9, expressing protein extracts and Analysis of Heat Tolerance:
After inducing culture terminates, draw the bacterium liquid after 1mL induction in 1.5mL centrifuge tube, together with the 1mL bacterium liquid before induction with the centrifugal 30s of 2000rpm, abandon supernatant liquor, add 1mLddH
2o, abundant vortex.All the other thalline of collected by centrifugation, 100 μ L2 × SDS-PAGE sample-loading buffer (2 × SDS-PAGE sample-loading buffers: 100mmol/LTrispH6.8,4% (w/v) SDS, 0.2% (w/v) tetrabromophenol sulfonphthalein, 20% (v/v) glycerine, 2% (v/v) beta-mercaptoethanol) resuspended thalline, after 100 DEG C of water-bath 10min, be cooled to room temperature, with the centrifugal 10min of the speed of 10000rpm, get supernatant liquor and carry out 12%SDS-PAGE electrophoresis.Simultaneously by thalline PBS damping fluid (the PBS damping fluid (pH8.0): 0.2mol/LNaH after induction
2pO4,0.2mol/LNa
2hPO
4) resuspended thalline, the broken thalline of ultrasonic 5min, the centrifugal 15min of 12000r/min, collects supernatant, and a point supernatant is 2 parts, and portion processes 10min in 100 ° of C boiling water baths, gets supernatant liquor for electrophoresis after centrifugal; Another portion of supernatant liquor is directly used in electrophoretic analysis.As shown in Figure 10, after IPTG induction, occurred in culture of Escherichia coli total protein one with expect the close protein band of molecular weight, after boiling water bath process, still have target protein to exist (Figure 10) in supernatant liquor, show
mrDHN3gene can have a thermostability at E. coli.
Three, Medicago ruthenica dehydrin gene
mrDHN3expression characterization analysis.
1, Medicago ruthenica seedling is coerced and ABA process:
(1) Medicago ruthenica seed is pressed
embodiment 1(mono-)described method, after dense sulfuric acid treatment, sprouting, transplanting grow 3 weeks, is carried out coercing and ABA process, is rounded strain seedling with different treatment time point, and after liquid nitrogen flash freezer ,-80 DEG C of preservations, in order to extracting total serum IgE.
(2) subzero treatment: carry out at-4 DEG C of illumination cultivation incubators, respectively at 0h, 8h, 24h, 3d, 7d and 14d, rounds strain seedling.
(3) NaCl Stress treatment: water bottom flowerpot with the 150mmol/LNaCl aqueous solution weekly, saturated to culture medium, respectively at process 0h, 8h, 24h, 3d, 7d and 14d, round strain seedling.
(4) during desiccation stress process: shifted out from culture medium by seedling, clean root matrix with distilled water, after sucking excessive moisture with thieving paper, be placed in culture dish at room temperature natural-dehydration, round strain seedling respectively at 0h, 2h, 4h, 8h, 12h.
(5) ABA process: seedling is shifted out from culture medium, root matrix is cleaned with distilled water, after sucking residual moisture with thieving paper, be placed in the culture dish being covered with thieving paper, after whole strain is watered with 100 μm of ol/L dormin solution (ABA) sprays containing 0.05%Tween20 (v/v), capping, in case plant natural-dehydration, rounds strain seedling respectively at 0h, 0.5h, 1h, 3h, 6h, 12h.
2, Total RNAs extraction and
mrDHN3the semi-quantitative RT-PCR analysis of genetic transcription:
(1) Total RNAs extraction and cDNA first chain synthesize same
embodiment 1(mono-).
(2) PCR reaction:
PCR reaction system is by 1.6 μ LdNTP, 2 μ L10 × PCRBuffer, 0.4 μ LP1 (10 μMs), 0.4 μ LP2 (10 μMs), 0.1 μ LrTaq
?archaeal dna polymerase (Takara product) forms, and uses ddH
2it is 20 μ l that O adds to final volume.
Wherein dNTP is made up of dATP, dTTP, dGTP and dCTP; DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is undertaken by follow procedure:
Carry out
mrDHN3during gene amplification, under same reaction system and reaction conditions, to Medicago ruthenica Actin gene amplification, as reference gene, Actin primer sequence used is as follows:
MrActinF:5’-TGCTTCTAACTGAGGCTCCACT-3’
MrActinR：5’-AAAGGACTTCTGGGCAACG-3’
Amplified reaction terminates, and after adding 4uL6 × loadingBuffer, draws 5uL sample in reaction system, detects with the sepharose that mass concentration is 0.7%.
(3) in the Medicago ruthenica seedling of Different stress and ABA process
mrDHN3the Semiquatitative RT-PCR assay of genetic transcription detects, and finds
mrDHN3gene is constitutive expression in Medicago ruthenica seedling, and expression level is not coerced to be affected (Fig. 5 ~ 8) with ABA process.
embodiment 2medicago ruthenica dehydrin gene, this gene is
mrDHN3encoding gene, it has the allelotrope with functionally active formed because of the disappearance of one or more base, insertion or replacement in SEQ ID NO.1 the 1st to 890.
Medicago ruthenica dehydrated protein
mrDHN3the clone of gene and Medicago ruthenica dehydrated protein
mrDHN3the prokaryotic expression of gene is same
embodiment 1.
embodiment 3medicago ruthenica dehydrin gene, this gene is
mrDHN3encoding gene, it has shown in Fig. 2 and SEQ ID NO.2 the 1st protein to the amino acid residue sequence shown in 221.
Medicago ruthenica dehydrated protein
mrDHN3the clone of gene and Medicago ruthenica dehydrated protein
mrDHN3the prokaryotic expression of gene is same
embodiment 1.
Right
mrDHN3protein coded by cDNA reading frame is made up of 221 amino-acid residues, molecular weight (
mw) be 24.84kD, theoretical iso-electric point (pI) is 5.56, is acidic protein.Overall average hydrophobicity index Grandaverageofhydropathicity (GRAVY) is-1.444, has higher wetting ability.
The structural domain of Pfam database (http://pfam.janelia.org) database to the aminoacid sequence that MrDHN3 encodes is searched for, show that MrDHN3 albumen belongs to LEAII protein family, there is the K-fragment (dotted line collimation mark goes out) (Fig. 3) that a S-fragment (solid box marks) and 2 are rich in Methionin, is SK2 type dehydrated protein.Utilize in ncbi database BLAST to the aminoacid sequence of MrDHN3 coded protein and other species protein amino acid sequence compare (Fig. 3) find with M. truncatula (Medicagotruncatula) and Trifolium repense (Trifoliumrepens) Amino acid sequence identity (identity) the highest, reach more than 81%.SKn type dehydrated protein aminoacid sequence constructing system evolutionary tree (Fig. 4) of 6 species that NCBI downloads is found, the sibship of MrDHN3 and leguminous plants pea SK2 albumen is nearest, it is one group that pulse family different plant species is gathered, it is one group that Gramineae different plant species is gathered, other section plants are another group, and demonstrating DHN3 albumen has phylogenetic feature.
embodiment 4medicago ruthenica dehydrin gene, this gene is
mrDHN3encoding gene, it has SEQ ID NO.2 the 1st and has the protein that by SEQIDNO.2 derived of with SEQIDNO.2 amino acid residue sequence identical activity containing the S-fragment of 1 a series of Serine (Ser) residue with 2 K-fragments (EKKGIMDKIKEKLPG or derivatives thereof) being rich in lysine residue through the replacement of one or several amino-acid residue, disappearance or interpolation to the amino acid residue sequence shown in 221; The sequence of the protein derived by SEQIDNO.2 is corresponding with allelic sequence.
Medicago ruthenica dehydrated protein
mrDHN3the clone of gene and Medicago ruthenica dehydrated protein
mrDHN3the prokaryotic expression of gene is same
embodiment 1.
embodiment 5recombinant expression vector pET30a-MrDHN3 containing Medicago ruthenica dehydrin gene, refers to the encoding gene of this Medicago ruthenica dehydrated protein
mrDHN3opening code-reading frame sequence is inserted into coli expression carrier pET-30a(Novagen product) between BamHI and SacI restriction enzyme site, to obtain final product.
Medicago ruthenica dehydrated protein
mrDHN3the clone of gene and Medicago ruthenica dehydrated protein
mrDHN3the prokaryotic expression of gene is same
embodiment 1.
embodiment 6the recombinant bacterium BL/MrDHN3 of the recombinant expression vector pET30a-MrDHN3 containing Medicago ruthenica dehydrin gene, refers to that this recombinant expression vector pET30a-MrDHN3 is transformed in E. coli expression strains BL21 (DE3), to obtain final product.
This recombinant bacterium passes through cultivation in containing kantlex liquid nutrient medium, after adding IPTG induction, produces the Medicago ruthenica dehydrated protein of described genes encoding.
embodiment 7medicago ruthenica dehydrin gene improvement plant and the anti-salt of microorganism and heat-resisting in application.
(1) bacterium liquid point plate test:
Intestinal bacteria cultivation and IPTG inductive condition are as described in embodiment 3, as Escherichia coli bacteria liquid about the OD600 to 1.0 of IPTG induction, with the LB liquid nutrient medium of the IPTG of the kantlex containing 50mg/L and 0.5mmol/L, bacterium liquid is diluted 10 times, carry out next step Stress treatment.Salt stress is treated to each 10 μ L of bacterium liquid after by original bacteria liquid and dilution and drips on the solid LB media containing 50mg/L kantlex, 0.5mmol/LIPTG and 500mMNaCl or KCl, 37 DEG C of Escherichia coli Growth situations of observing contrast and processing after cultivating 3d; In heat stress test by the bacterium liquid after original bacteria liquid and dilution after 55 DEG C of water bath processing 30min, therefrom get 10 μ L bacterium drops respectively to containing on 50mg/L kantlex solid LB media, observe after 37 DEG C of cultivation 16h.In test simultaneously with containing pET-30a (+) BL21 (DE3) bacterial strain for contrasting.
(2) enumeration analysis:
By the culture (OD600 ≈ 1.0) as (1) described IPTG induction, 10 times are diluted with the fresh LB liquid nutrient medium containing 50mg/L kantlex, 0.5mmol/LIPTG, getting 100 μ L is spread evenly across on the solid LB flat board of the kantlex containing 50mg/L, carry out enumeration after 37 DEG C of cultivations, calculate E. coli clones survival rate.As qualitative analysis test, salt stress is tested, and cultivates 3d; Heat stress is tested, and cultivates 16h.
High-salt stress and high temperature stress treatment condition are tested with bacterium liquid point plate.
(3) result:
MrDHN3 expression can significantly improve intestinal bacteria and grow and survival ability (Figure 11 ~ 12) under above-mentioned high salt and high temperature stress condition.Enumeration result shows, and under 0.5mol/LNaCl and KCl high-salt stress, the survival rate of MrDHN3 expression strain is respectively 16.71% and 20.8%, and containing empty carrier pET-30a(+) Strain survival rate respectively 3.55% and 4.29%; After 55 DEG C of pyroprocessing, the survival rate that MrDHN3 expresses bacterium is 11.22%, and empty carrier Strain survival rate is only 2.4%(Figure 11 ~ 12).
Conclusion:
MrDHN3 albumen overexpression has protected effect to intestinal bacteria under environment stress, therefore, this gene can be used for by biotechnological means the anti-salt and the thermotolerance that improve plant and microorganism.
embodiment 8the recombinant expression vector pET30a-MrDHN3 of Medicago ruthenica dehydrin gene improvement plant and the anti-salt of microorganism and heat-resisting in application.
embodiment 9the recombinant bacterium of the recombinant expression vector pET30a-MrDHN3 of Medicago ruthenica dehydrin gene improvement plant and the anti-salt of microorganism and heat-resisting in application.
SEQIDNO.1
1CGTGTCATTATGTGTAGTAGTGAAGTAGTATAAAGTGCTACCCCATCTCTTTGATTTTCA
61TCATCGAAACAACATAATCTCATACTTTACCTTTAGCATTTCCAGCTACATAAGAATTAA
121TATTATTCCAATTGATTAATCTTGTTAATTTGCTTAGTGATCATCAATTCATCATGGCTG
181ATCAGGAGAATCAGAACAAGTACGAGGAAACCACCGCAACCAACTCTGAGACAGAGATCA
241AAGACAGGGGTGTTTTTGATTTTCTAGGTGGTAAGAAAAAGGATGAAGAACATAAGCCTC
301AAGAGGAAGCTATTGCAACTGATTTTAATCACAAGGTGACTTTGTATGAAGCTCCATCAG
361AGACCAAAGTAGAAGAAGAAGAAAAAGGTGAAAAGAAACACACCAGCCTTTTGGAGAAAC
421TTCACCGATCTGATAGCTCTTCAAGCTCTTCGAGCGAGGAGGAAGTTGATGGAGAGAAGA
481GGAAAAAGAAGAAGAAGGAAAAGAAGGAGAAGAAAGAGGACACATCAGTGCCAGTAGAGA
541AAGTTGATGTTGTTGATGGAACAACAGCAAGTACTGAGGAGAAGAAAGGTTTCCTAGACA
601AAATTAAGGAGAAGCTTCCAGGACACAAGAAAACTGAGGATGTAACAACTCAACCGCCTG
661TTGCTGCTGTTGCTGTGCCATCTGCTGAGACAACAACAACAACAACAACTAGTCATGATC
721AAGGAGAGAAGAAAGGTATTTTGGAAAAGATCAAAGAGAAGATTCCTGGTTATCACCCTA
781AGACTGCTACTGATCATGAAGACAAAGATCATCACAAGGATGAGACTACTACTCATTGAT
841TATGATCGATCCATTGTGTTGTTTTGAGGCTTTGGAGGGTGCTTTGTTTA
SEQIDNO.2
1MADQENQNKYEETTATNSETEIKDRGVFDFLGGKKKDEEHKPQEEAIATDFNHKVTLYEA
61PSETKVEEEEKGEKKHTSLLEKLHRSDSSSSSSSEEEVDGEKRKKKKKEKKEKKEDTSVP
121VEKVDVVDGTTASTEEKKGFLDKIKEKLPGHKKTEDVTTQPPVAAVAVPSAETTTTTTTS
181HDQGEKKGILEKIKEKIPGYHPKTATDHEDKDHHKDETTTH
Claims (5)
1. Medicago ruthenica dehydrin gene, is characterized in that: this gene is
mrDHN3encoding gene, it has SEQ ID NO.1 the 1st to the nucleotide sequence shown in 890.
2. Medicago ruthenica dehydrin gene as claimed in claim 1, is characterized in that: described gene has the allelotrope with functionally active formed because of the disappearance of one or more base, insertion or replacement in SEQ ID NO.1 the 1st to 890.
3. Medicago ruthenica dehydrin gene as claimed in claim 2, is characterized in that: described gene has SEQ ID NO.2 the 1st protein to the amino acid residue sequence shown in 221.
4. Medicago ruthenica dehydrin gene as claimed in claim 3, is characterized in that: described gene has SEQ ID NO.2 the 1st and has the protein that by SEQIDNO.2 derived of with SEQIDNO.2 amino acid residue sequence identical activity containing the S-fragment of 1 a series of serine residue with 2 K-fragments being rich in lysine residue through the replacement of one or several amino-acid residue, disappearance or interpolation to the amino acid residue sequence shown in 221; The sequence of the described protein derived by SEQIDNO.2 is corresponding with allelic sequence.
5. the Medicago ruthenica dehydrin gene as described in claim 1,2,3 or 4 improvement plant and the anti-salt of microorganism and heat-resisting in application.
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Cited By (2)
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| CN107619436A (en) * | 2017-10-19 | 2018-01-23 | 上海交通大学 | A kind of degeneration-resistant albumen and its encoding gene |
| CN110684091A (en) * | 2019-10-31 | 2020-01-14 | 青海民族大学 | Y related to alfalfa bean stress resistance2K4Type dehydrin protein MrY2K4Coding gene and application thereof |
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| CN103305526A (en) * | 2012-03-13 | 2013-09-18 | 中国农业科学院作物科学研究所 | Application of protein TaDREB (triticum aestivum L. dehydration responsive element binding) 6 to preparation of high temperature resistant transgenic plant |
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| CN102140135A (en) * | 2011-01-24 | 2011-08-03 | 北京工商大学 | Corn dehydration responsive element bonding protein ZmDBP3 and coding gene thereof |
| CN103305526A (en) * | 2012-03-13 | 2013-09-18 | 中国农业科学院作物科学研究所 | Application of protein TaDREB (triticum aestivum L. dehydration responsive element binding) 6 to preparation of high temperature resistant transgenic plant |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107619436A (en) * | 2017-10-19 | 2018-01-23 | 上海交通大学 | A kind of degeneration-resistant albumen and its encoding gene |
| CN107619436B (en) * | 2017-10-19 | 2021-07-06 | 上海交通大学 | Stress-resistant protein and coding gene thereof |
| CN110684091A (en) * | 2019-10-31 | 2020-01-14 | 青海民族大学 | Y related to alfalfa bean stress resistance2K4Type dehydrin protein MrY2K4Coding gene and application thereof |
| CN110684091B (en) * | 2019-10-31 | 2022-02-01 | 青海民族大学 | Y related to alfalfa bean stress resistance2K4Type dehydrin protein MrY2K4Coding gene and application thereof |
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