CN105154452B - Medicago ruthenica dehydrin gene and its application - Google Patents
Medicago ruthenica dehydrin gene and its application Download PDFInfo
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- CN105154452B CN105154452B CN201510697514.1A CN201510697514A CN105154452B CN 105154452 B CN105154452 B CN 105154452B CN 201510697514 A CN201510697514 A CN 201510697514A CN 105154452 B CN105154452 B CN 105154452B
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Abstract
The present invention relates to a kind of Medicago ruthenica dehydrin gene, which isMrDHN3Encoding gene derives from Medicago ruthenica, it has nucleotide sequence shown in SEQ ID NO.1 in sequence table；Or with the allele with functional activity formed in SEQ ID NO.1 by the missing of one or more bases, insertion or replacement in sequence table；Or the protein with amino acid residue sequence shown in SEQ ID NO.2 in sequence table；Or by the S- segment for replacing, missing or adding but containing a series of 1 serine (Ser) residue of one or several amino acid residues and 2 K- segments (EKKGIMDKIKEKLPG or derivatives thereof) rich in lysine residue and there is the active protein as derived from SEQ ID NO.2 identical as SEQ ID NO.2 amino acid residue sequence with amino acid residue sequence shown in SEQ ID NO.2 in sequence table.Present invention gene obtained can be used for improveing the salt resistance and heat resistance of plant and microorganism.
Description
Technical field
The present invention relates to technical field of biological genetic engineering more particularly to Medicago ruthenica dehydrin gene and its applications.
Background technique
Dehydrated protein belongs to late embryo and Abundant protein (late embryogenesis abundant protein) occurs
LEAII family.Dehydrated protein initially finds in seed, it is subsequent research shows that dehydrated protein be distributed widely in algae, yeast,
In each tissue and organ of nematode, cyanobacteria and higher plant.Arid, low temperature and the environmental factor for causing dehydration such as with high salt
And abscisic acid (ABA) can induce its expression in cell.It is conservative rich in lysine that dehydrated protein contains at least one
Motif K- segment (EKKGIMDKIKEKLPG or derivatives thereof), the spiral knot of amphiphilic (hydrophilelipophile) is commonly formed in they
Structure.Some dehydrated proteins usually also include Y- segment and S- segment, and Y- segment is usually in the form of 1-3 repeats (T/VDEYGNP)
The end N- of dehydrated protein is appeared in, S segment is made of a series of serines (Ser) residue.Based on Y, S, the type that K segment occurs
Dehydrated protein can be divided into 5 subfamilies: YnSKn, SKn, Kn, YnKn and KnS with quantity.
The function of dehydrated protein in the cell is related with cell dehydration protection.By transgenic technology, barley is dehydrated egg
White gene ZmDHN2b is transferred to tobacco, and the anti-seismic design of transgenic plant improves；Wheat DH N-5 gene is overexpressed in arabidopsis
Its resistance to salt and osmotic stress can be enhanced；Wheat dehydrin gene WCOR410, which is overexpressed, can enhance the anti-of strawberry
Jelly property.Therefore, dehydrated protein may be including plant, bacterium and lower animal cell obtain dehydration tolerance it is decisive because
One of element, although its specific mechanism is unclear.Meanwhile dehydrin gene is also in the non-life such as plant arid, low temperature, with high salt
There is important potential using value in the improvement of object stress resistance.
Other than Genetic Transformation in Higher Plants test, the heterologous expression systems such as Escherichia coli and yeast are also widely used for being dehydrated
The identification of albumen.The PM2 albumen and KS type dehydrated protein SLT1629 being overexpressed in soybean LEA3 family can significantly improve
Escherichia coli saline-alkaline tolerance can be improved to Escherichia coli；Barley dehydrated protein, which is introduced after yeast, can be such that it obtains to the side of body with high salt
Urgent tolerance.Therefore, it can be used as the effective system of identification dehydrated protein using yeast, escherichia expression system.
Medicago ruthenicaMedicago ruthenica (L.)It is distributed widely in Siberia, the high latitude in the north of Mongolia and China
Extremely frigid zones are spent, are grown at patana more, there is extremely strong tolerance to adverse circumstance, being uniquely can be extremely high in High aititude
The cold normal overwintering clover category Perennial legume forages in area.There was only the report of fragmentary Medicago ruthenica dehydrin gene clone at present
Road, but there is no the report to its degeneration-resistant Function Identification.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of improvement plant and microorganism salt resistance and the flat Mus of heat resistance
Beans dehydrin gene.
Another technical problem to be solved by this invention is to provide the gene in improvement plant and microorganism salt resistance and resistance to
The application of hot aspect.
To solve the above problems, Medicago ruthenica dehydrin gene of the present invention, it is characterised in that: the gene isMrDHN3Encoding gene, it has nucleotide sequence shown in SEQ ID NO.1 the 1st to 890 in sequence table.
The gene have missing in sequence table in SEQ ID NO.1 the 1st to 890 because of one or more bases,
The allele with functional activity of insertion or replacement and formation.
The gene has the albumen of amino acid residue sequence shown in SEQ ID NO.2 the 1st to 221 in sequence table
Matter.
The gene has in sequence table amino acid residue sequence shown in SEQ ID NO.2 the 1st to 221 through one
Or the S- segment and 2 richnesses of several amino acid residues replaced, missed or added but contain a series of 1 serine (Ser) residue
K- segment (EKKGIMDKIKEKLPG or derivatives thereof) containing lysine residue and have and SEQ ID NO.2 amino acid residue
The identical active protein as derived from SEQ ID NO.2 of sequence；The sequence of the protein as derived from SEQ ID NO.2 with
The sequence of allele is corresponding.
Medicago ruthenica dehydrin gene as described above is in improvement plant and microorganism salt resistance and the application of heat-resisting aspect.
Compared with the prior art, the present invention has the following advantages:
1, the present invention is analyzed by transcription group, and in conjunction with RT-PCR technology, a new dehydration is separated in Medicago ruthenica
Protein geneMrDHN3.It willMrDHN3After gene coding region is connected to coli expression carrier, which is turned
Enter E. coli expression strains, realizes overexpression of the MrDHN3 albumen in Escherichia coli with IPTG induction, the results show that
MrDHN3 albumen overexpression has protecting effect to the Escherichia coli under with high salt and heat stress, therefore, by biotechnology side
The gene can be used for the salt resistance and heat resistance improvement of plant and microorganism by method.
2, after by carrying out RT-PCR amplification to the Medicago ruthenica dehydrin gene in the present invention, Ago-Gel electricity is carried out
It swims (such as Fig. 1, shown in Fig. 5 ~ 8), can detecte the amplified production of about 900bp.
3, by by the Medicago ruthenica dehydrated protein in the present inventionMrDHN3The code area of gene is connected to Bacillus coli expression
Carrier converts coli expression carrier, containing such as above-mentioned building after digestion is identified (as shown in Figure 9)MrDHN3Base
It is induced in the Escherichia coli of the prokaryotic expression carrier of cause through IPTG, SDS-PAGE electrophoresis, knot is carried out to culture bacterial protein
There is one and protein band (as shown in Figure 10) similar in expected molecular weight in culture of Escherichia coli after fruit display induction,
Therefore Medicago ruthenica dehydrated protein MrDHN3 can in Escherichia coli overexpression.
4, it is handled by the Escherichia coli to the Medicago ruthenica dehydrated protein MrDHN3 in the overexpression present invention through heat stress
With after containing cultivating on high salt culture medium, the growth survival condition of Escherichia coli as described above is observed, as the result is shown MrDHN3 egg
White overexpression can be improved the tolerance (as shown in Figure 11 ~ 12) that escherichia coli host handles high-salt stress and heat stress,
Therefore MrDHN3 albumen has protecting effect to cell under high-salt stress and heat stress processing.
5, gene structure feature of the invention:
The present invention designs PCR primer, wherein primer P1+P2 contains for expanding according to Medicago ruthenica transcript profile sequencing result
There are 5 '-and 3 '-non-translational regions and code areaMrDHN3Gene order (Fig. 1 ~ 2).Primer P3+P4 amplificationMrDHN3Gene coding
Region nucleotide sequence, the functional activity for prokaryotic expression identification clone gene.
It is that template is expanded with primer P1+P2, Medicago ruthenica cDNA, obtains the band (Fig. 1) of about 900bp.It is obtained after sequencing
890bp(such as Fig. 2) contain such as sequence table SEQ ID NO.1 shown in 5 '-with 3 '-non-translational regions and code areaMrDHN3Base
Because of sequence, wherein 173-839 are translated region.The amino acid sequence such as Fig. 2 derived by SEQ ID NO.1 sequence (use by S segment
Underscore marks, and 2 conservative K segments are marked with shade) and sequence table SEQ ID NO.2 shown in.The amino acid sequence shows,
Protein product is made of 221 amino acid residues, and predicted molecular weight (Mw) is 24.84 kD, and theoretical isoelectric point (pI) is
5.56, it is acidic protein.Overall average hydrophobicity index Grand average of hydropathicity (GRAVY) be-
1.444 hydrophily with higher.MrDHN3 is compiled in Pfam database (http://pfam.janelia.org) database
The structural domain of the amino acid sequence of code scans for showing that MrDHN3 albumen belongs to LEAII protein family, and there are a S- segments
(solid box marks) and 2 K- segments (dotted line frame marks) (Fig. 3) rich in lysine residue, are SK2 type dehydrated protein.?
Using BLAST to the amino acid sequence and other species protein amino acid sequences of MrDHN3 coding protein in ncbi database
Compare (Fig. 3) discovery and M. truncatula (Medicago truncatula) and Trifolium repense (Trifolium repens) amino acid
Sequence identity (identity) highest, up to 81% or more.To the SKn type dehydrated protein amino acid sequence of 6 species of NCBI downloading
Column building systematic evolution tree (Fig. 4) discovery, the affiliation of MrDHN3 and leguminous plant pea SK2 albumen are nearest.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is Medicago ruthenica dehydrin gene of the present inventionMrDHN3RT-PCR clone products electrophorogram.
Fig. 2 is the present inventionMrDHN3The amino acid sequence of gene cDNA sequence and its coded protein.
Fig. 3 is Medicago ruthenica dehydrated protein MrDHN3 of the present invention and the comparison point of other species dehydrated protein amino acid sequences
Analysis.Wherein TrDHN3 (ADD09573.1) comes from Trifolium repense (Trifolium repens)；MtDHN3(XP_003603987.1)
From M. truncatula (Medicago truncatula)；PsDHN3 (CAA78515.1) comes from pea (Pisum sativum)；
CaDHN3 (XP_004500781.1) comes from chick-pea (Cicer arietinum).1 solid box shows conservative S- segment, 2
Dotted line frame is the conservative K- segment rich in lysine residue.
Fig. 4 is the phyletic evolution that Medicago ruthenica dehydrated protein MrDHN3 of the present invention and other species SKn type dehydrated proteins construct
Tree.Wherein OsDHN3 (ABS44866.1) comes from japonica rice (Oryza sativa Japonica Group)；TaDHN3
(AAB18202.1) wheat (Triticum aestivum) is come from；MaDHN3 (AEI54683.1) comes from plantain (Musa ABB
Group)；CsDHN3 (ACT10283.1) comes from tea (Camellia sinensis)；PpDHN3 (AAZ83586.1) comes from peach
(Prunus persica)；PsDHN3 (AAL50315.1) comes from pea (Pisum sativum).Filled triangle symbols instruction is flat
Mu beans dehydrated protein MrDHN3.
Fig. 5 is Medicago ruthenica dehydrated protein of the present inventionMrDHN3Gene gene expression dose under simulation desiccation stress processing
Semi-quantitative RT-PCR analysis,ActinFor for interior target Medicago ruthenica actin gene.
Fig. 6 is Medicago ruthenica dehydrated protein of the present inventionMrDHN3The semidefinite of gene gene expression dose under NaCl Stress treatment
RT-PCR analysis is measured,ActinFor for interior target Medicago ruthenica actin gene.
Fig. 7 is Medicago ruthenica dehydrated protein of the present inventionMrDHN3The sxemiquantitative RT- of gene gene expression dose under ABA processing
PCR analysis,ActinFor for interior target Medicago ruthenica actin gene.
Fig. 8 is Medicago ruthenica dehydrated protein of the present inventionMrDHN3The sxemiquantitative of gene gene expression dose under low-temperature treatment
RT-PCR analysis,ActinFor for interior target Medicago ruthenica actin gene.
Fig. 9 is Medicago ruthenica dehydrated protein of the present inventionMrDHN3Prokaryotic expression carrier pET30a- MrDHN3 digestion identification
Electrophorogram.Wherein " BamH I+Sac I " is the pET30a- MrDHN3 carrier of the double digested processing of BamH I and Sac I,
" BamH I " is the pET30a- MrDHN3 carrier of BamH I single endonuclease digestion digestion process, and " Sac I " is at Sac I single endonuclease digestion digestion
The pET30a- MrDHN3 carrier of reason, " Plasmid " are the pET30a- MrDHN3 carrier for not doing digestions processing.
Figure 10 is the SDS- of Medicago ruthenica dehydrated protein MrDHN3 of the present invention inducing expression in e. coli bl21 (DE3)
PAGE electroresis appraisal.Wherein " 1 " is the total protein before IPTG induction, and " 2 " are the total protein after IPTG induction, and " 3 " are supernatant
The albumen of middle inducing expression, " 4 " are supernatant soluble protein after 100 DEG C of 10 min of processing.
Figure 11 is for Medicago ruthenica dehydrated protein MrDHN3 of the present invention to the environment stress of host strain after expression in escherichia coli
The bacterium solution drop plate test of protecting effect.Wherein BL/pET30a is BL21 (DE3) Escherichia coli containing pET30a (+) plasmid；
BL/MrDHN3 is BL21 (DE3) Escherichia coli containing pET30a- MrDHN3 plasmid；LB is solid for Luria-Bertani (LB)
Body culture medium is added with 0.5 mmol/L IPTG；500 mM NaCl of LB+ is that LB solid medium adds 0.5 mmol/L
IPTG and 0.5 mol/L NaCl；+ 500 mM KCl of LB is that LB solid medium adds 0.5 mmol/L IPTG and 0.5
mol/L KCl；(55 DEG C, 30 min) of LB are 55 DEG C of 30 min of heat treatment.
Figure 12 is for Medicago ruthenica dehydrated protein MrDHN3 of the present invention to the environment stress of host strain after expression in escherichia coli
The bacterium colony counting statistics result of protecting effect.Wherein BL/pET30a is BL21 (DE3) large intestine bar containing pET30a (+) plasmid
Bacterium；BL/MrDHN3 is BL21 (DE3) Escherichia coli containing pET30a- MrDHN3 plasmid；NaCl (0.5M) and KCl
(0.5M) is high-salt stress, Heat is that (55 DEG C, heat stress handles 30min.
Specific embodiment
1 Medicago ruthenica dehydrin gene of embodiment, the gene areMrDHN3Encoding gene, it has SEQ in sequence table
Nucleotide sequence shown in ID NO.1 the 1st to 890.
One, Medicago ruthenica dehydrated proteinMrDHN3The clone of gene.
1, the processing of Medicago ruthenica seedling low temperature stress is sequenced with transcript profile:
(1) Medicago ruthenica low temperature stress is handled:
By Medicago ruthenica seed with after dense sulfuric acid treatment 10min, removing remaining sulfuric acid for several times with distilled water flushing.Above-mentioned process
The seed of processing, which is placed in, to be covered in double-layer filter paper culture dish, is germinateed under the conditions of the photoperiod in 21 DEG C, 16h/8h；By germination after 3d
Seedling replanting is in vermiculite: continuing to cultivate under the above conditions in the mixed-matrix of Nutrition Soil (3:1), uses nutrition containing 1/2MS weekly
Liquid pours primary；Seedling is transferred to -4 DEG C of illumination boxs after 3 weeks and carries out low-temperature treatment for 24 hours, before low-temperature treatment and is located
After reason, it is rounded strain seedling, cleans up root, with liquid nitrogen flash freezer after blotting paper exhaustion residual moisture, -80 DEG C of preservations.
(2) transcript profile is sequenced:
By before low-temperature treatment and treated seedling, send BGI Technology Solutions Co., Ltd. to carry out total serum IgE and mention
It takes and is sequenced with transcript profile.
2, design of primers:
PCR primer is designed according to transcript profile sequencing result, specific as follows:
P1 5’- CGTGTCATTATGTGTAGTAGTGAAG -3’
P2 5’- TAAACAAAGCACCCTCCA -3’
3, Total RNAs extraction is synthesized with the first chain of cDNA:
The sample that above-mentioned low temperature stress is handled is pulverized in liquid nitrogen last, with Trizol reagent (Invitrogen
Products) according to its specification extraction total serum IgE.The water dissolution that 20 μ L DEPC of total serum IgE are handled, adjustment concentration are 500ng/ μ
L。
The first chain synthesis reaction of cDNA uses PrimeScript RT reagent Kit (Perfect Real
Time) (Takara product) kit is carried out according to its specification.
4、MrDHN3Gene cDNA sequence amplification:
PCR reaction system is by 10 μ 2 × PCR of L Buffer I, 1.6 μ L dNTP, 0.4 μ L P1 (10 μM), 0.4 μ L
P2 (10μM)、0.05μL LA™Taq archaeal dna polymerase (5U/ μ L) (Takara product), 0.05 μ L Pyrobest™DNA is poly-
Synthase (5U/ μ L) (Takara product), 1 μ L cDNA the first chain synthetic product composition, add ddH2O(distilled water) to final volume
For 20 μ L.
Wherein: 2 × PCR Buffer I is and LA™The mating buffer of Taq archaeal dna polymerase.
DNTP is by dATP(triphosphoric acid adenyl-deoxyribonucleotide), dTTP(deoxythymidine triphosphate), dGTP(deoxyguanosine
Triphosphoric acid trisodium) and dCTP(deoxycytidine triphosphate) composition；DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is carried out by following procedure:
After reaction, PCR product is detected with 0.7% Ago-Gel, obtains the item of about 900bp as shown in Figure 1
Band.
5, PCR product recycles:
PCR product UNIQ-10 pillar DNA plastic recovery kit (the raw chemical product in Shanghai) recycling.
6, the clone of target DNA fragment:
Linked system:
(1) linked system total amount is 5 μ L, by 0.5 μ L pGEM®- T Easy carrier (Promega product), 0.5 μ L T4
DNA ligase, 2.5 2 × Rapid of μ L connection buffers and 1.5 μ L target DNA fragments composition.Wherein ligase and connection are slow
Fliud flushing is included for pGEM-T Easy carrier.
(2) 5 μ L linked systems are connected at 4 DEG C overnight.
(3) 5 μ L linked systems are all added into 50 μ L bacillus coli DH 5 alpha competent cells, after mixing, ice bath 30
min。
(4) by step, (3) resulting mixed system after heat shock 90s, is placed in 2 min in ice bath in 42 DEG C of water-baths at once.
(5) 400 μ L LB liquid mediums are added in step (4) resulting mixed system, with the speed of 160rpm at 37 DEG C
Rate vibrates 1h, the DH5 α bacterium solution after being activated.
(6) DH5 α bacterium solution and 5 μ L IPTG (1M) after taking 200 μ L to activate are mixed with 40uL x-gal (20mg/ml),
Coated plate on LB plating medium containing ampicillin (Amp) (100 μ g/ml) cultivates 12 ~ 16h at 37 DEG C.
7. the identification and sequencing of positive colony:
(1) with the anti-ampicillin positive bacterium colony of white grown on toothpick picking LB plating medium, in it is another containing
Amp(100 μ g/ml) LB plating medium on scribing line culture 8 ~ 14h, obtain single colonie.
(2), with the single colonie of toothpick picking setting-out culture, the bottom of PCR pipe is applied to.
(3) PCR reaction system:
PCR reaction system is by 1.6 μ L dNTP, 2 μ L 10 × PCR Buffer, 0.4 μ L P1 (10 μM), 0.4 μ L P2
(10μM)、0.1μL Taq™Enzyme (Takara product) composition, and use ddH2It is 20 μ L that O, which adds to final volume,.
Wherein: 10 × PCR Buffer is Taq™The mating buffer of archaeal dna polymerase.
DNTP is made of dATP, dTTP, dGTP and dCTP；DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is carried out by following procedure:
After reaction, it is detected with 0.7% Ago-Gel, determines that target gene fragment has been inserted into pGEM-T
In Easy carrier.
(4) (3) identify that the single bacterium of acquisition is fallen on containing Amp(100 μ g/ml with toothpick picking step) LB liquid medium
In, it is stayed overnight at 37 DEG C with the rate shake culture of 200rpm.
(5) 700 μ L bacterium solutions are drawn in the 1.5mL centrifuge tube of sterilizing, 300 μ L, 50% glycerol is added, are mixed, and send raw work raw
The sequencing of object engineering (Shanghai) Co., Ltd..
It is obtained after sequencingMrDHN3The nucleotide total order as shown in Fig. 2 and sequence table SEQ ID NO.1 of gene 890bp
Column, include the 5 ' areas UTR of the opening code-reading frame of 666bp, the 3 ' areas UTR of 51bp and 173bp.
Two, Medicago ruthenica dehydrated proteinMrDHN3The prokaryotic expression of gene.
1, prokaryotic expression design of primers:
According to sequencing result design primer P3 and P4, to expandMrDHN3The code area DNA sequence dna of gene, and exist respectively
DNA sequence dna 5 '-end and 3 '-ends introduce I two restriction enzyme sites (underscore) of BamH I and Sac.
P3:5 '-GCGGATCCATGGCTGATCAGGAGAATCAGAAC -3’
P4:5 '-CGCGAGCTCTCAATGAGTAGTAGTCTCATCCTT -3’
2, recombinant plasmid and expression vector pET-30a (+) plasmid are extracted with the small extraction reagent kit of plasmid (the raw chemical product in Shanghai)
(Novagen product).
3、MrDHN3Coding sequence PCR amplification and clone:
PCR reaction system is by 12.5 μ 2 × LA of L Buffer I, 2 μ L dNTP, 0.5 μ L P3 (10 μM), 0.5 μ L P4
(10μM)、0.05μL Pyrobest™Archaeal dna polymerase (Takara product) (5U/ μ L), 1 μ L containMrDHN3The pGEM- of gene
T Easy recombinant plasmid (50 times of dilution) composition, and use ddH2It is 25 μ L that O, which adds to final volume,.
Wherein dNTP is made of dATP, dTTP, dGTP and dCTP；DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is carried out by following procedure:
After reaction, it obtainsMrDHN3The PCR product of gene coding region.
4, it is recycled with UNIQ-10 pillar DNA plastic recovery kitMrDHN3Code area PCR product.
5、MrDHN3Code area PCR product rTaq® After the processing of archaeal dna polymerase (Takara product) tailing, it is connected to
Sequencing confirmation is carried out after pGEM-T Easy carrier.
6, the double digestion of target gene fragment and expression vector pET-30a:
It will containMrDHN3The pGEM-T Easy carrier and prokaryotic expression carrier pET-30a of code area DNA sequence dna
(Novagen product) is with BamH I (NEB product) and Sac I(NEB product) carry out double digestion, digestion products with 0.7% fine jade
Sepharose separation, is separately recovered target gene fragment and carrier segments with UNIQ-10 pillar DNA plastic recovery kit.
7, connection and identification:
The target gene double enzyme digestion product of segment and the double enzyme digestion product of pET-30a (+) will be inserted by molal quantity 10:1
Ratio mixing, be added T4 DNA ligase (Promega product), at 4 DEG C connect overnight.
It takes 5 μ L connection products to be added into 50 μ L bacillus coli DH 5 alpha competent cells to be converted, using kanamycins
(Kan) resistance screening, the bacterium colony grown are directly identified with PCR, and the positive colony identified is shaken bacterium, extracts plasmid, then use
BamH I and Sac I carries out mono-/bis-digestion identification (Fig. 9).
8, conversion and protein expression:
By PET-30a plasmid conversion e. coli bl21 (DE3) (Novagen product) impression containing target DNA fragment
State cell, with kanamycins (Kan+) carry out resistance screening.Picking positive colony is inoculated in containing 50 mg/L Kan+LB liquid
In culture medium.After rate at 37 DEG C with 250rpm shakes bacterium overnight, by 1:100(v/v) it is transferred in fresh LB culture medium
After 2 ~ 3 h of culture reach 0.6 ~ 0.8 to OD600,1mL bacterium solution is taken out as preceding sample is induced, isopropyl sulphur is added in remaining bacterium solution
For galactoside (IPTG) to final concentration of 0.5 mmol/L, with the rate Fiber differentiation 3h of 250rpm at 37 DEG C.
9, protein extraction and Analysis of Heat Tolerance are expressed:
After Fiber differentiation, the bacterium solution after drawing 1mL induction is in 1.5mL centrifuge tube, together with the 1mL bacterium solution before induction
It is centrifuged 30s with 2000rpm, abandons supernatant, 1mL ddH is added2O, sufficient vortex.It is collected by centrifugation remaining thallus, 100 μ L 2 ×
SDS-PAGE sample-loading buffer (2 × SDS-PAGE sample-loading buffer: 100mmol/L Tris pH6.8,4% (w/v) SDS,
0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 2% (v/v) beta -mercaptoethanol) thallus is resuspended, it is cold after 100 DEG C of water-bath 10min
But to room temperature, 10min is centrifuged with the rate of 10000rpm, supernatant is taken to carry out 12% SDS-PAGE electrophoresis.After inducing simultaneously
Thallus PBS buffer solution (PBS buffer solution (pH8.0): 0.2mol/L NaH2PO4,0.2mol/L Na2HPO4) thallus is resuspended,
5 min of ultrasound are crushed thallus, and 12 000 r/min are centrifuged 15 min, collect supernatant, and dividing supernatant is 2 parts, a to boil in 100 °C
10 min are handled in water-bath, take supernatant for electrophoresis after centrifugation；Another supernatant is directly used in electrophoretic analysis.Such as Figure 10 institute
Show after IPTG is induced, occur one and protein bars similar in expected molecular weight in culture of Escherichia coli total protein
Band still has target protein to there is (Figure 10) in supernatant after boiling water bath is handled, showsMrDHN3Gene can be in Escherichia coli
Middle high efficient expression simultaneously has thermal stability.
Three, Medicago ruthenica dehydrin geneMrDHN3Expression characterization analysis.
1, Medicago ruthenica seedling stress is handled with ABA:
By Medicago ruthenica seed press embodiment 1(mono-) the method by dense sulfuric acid treatment, sprouting, transplanting growth 3 weeks with
Afterwards, stress and ABA processing are carried out, is rounded strain seedling with different disposal time point, after liquid nitrogen flash freezer, -80 DEG C of preservations, in case extracting
Total serum IgE.
(2) low-temperature treatment: carrying out in -4 DEG C of illumination cultivation incubators, respectively in 0h, 8h, for 24 hours, 3d, 7d and 14d, be rounded strain
Seedling.
(3) NaCl Stress treatment: being poured with 150 mmol/L NaCl aqueous solutions from flowerpot bottom weekly, until culture substrate is full
With, respectively at processing 0h, 8h, for 24 hours, 3d, 7d and 14d, be rounded strain seedling.
(4) when desiccation stress is handled: seedling being removed from culture substrate, root matrix is cleaned with distilled water, uses blotting paper
After sucking excessive moisture, it is placed in culture dish natural-dehydration at room temperature, is rounded strain seedling respectively at 0h, 2h, 4h, 8h, 12h.
(5) ABA is handled: seedling being removed from culture substrate, cleans root matrix with distilled water, is sucked with blotting paper surplus
It after remaining moisture, is placed in the culture dish for being covered with blotting paper, whole strain uses 100 μm of ol/L containing 0.05% Tween20 (v/v) de-
It falls after acid solution (ABA) spray pours, capping is rounded strain children to prevent plant natural-dehydration, respectively at 0h, 0.5h, 1h, 3h, 6h, 12h
Seedling.
2, Total RNAs extraction withMrDHN3The semi-quantitative RT-PCR analysis of genetic transcription:
(1) Total RNAs extraction synthesizes same embodiment 1(mono- with the first chain of cDNA).
(2) PCR reacts:
PCR reaction system is by 1.6 μ L dNTP, 2 μ L 10 × PCR Buffer, 0.4 μ L P1 (10 μM), 0.4 μ L P2
(10μM)、0.1μL rTaq™Archaeal dna polymerase (Takara product) composition, and use ddH2It is 20 μ l that O, which adds to final volume,.
Wherein dNTP is made of dATP, dTTP, dGTP and dCTP；DATP, dTTP, dGTP and dCTP are 2.5mM.
PCR reaction system is carried out by following procedure:
Carry outMrDHN3When gene magnification, under same reaction system and reaction condition, Medicago ruthenica Actin gene is expanded
Increase, as reference gene, Actin primer sequence used is as follows:
MrActinF:5’-TGCTTCTAACTGAGGCTCCACT-3’
MrActinR:5 '-AAAGGACTTCTGGGCAACG-3 '
Amplified reaction terminates, and after 6 × loading of 4uL Buffer is added in reaction system, draws 5uL sample, uses matter
The Ago-Gel that concentration is 0.7% is measured to detect.
(3) in the Medicago ruthenica seedling handled by Different stress and ABAMrDHN3The Semiquatitative RT-PCR assay of genetic transcription
Detection, discoveryMrDHN3Gene is constitutive expression in Medicago ruthenica seedling, and expression is not influenced by stress and ABA processing
(Fig. 5 ~ 8).
2 Medicago ruthenica dehydrin gene of embodiment, the gene areMrDHN3Encoding gene, it has SEQ in sequence table
Formed in ID NO.1 the 1st to 890 by the missing of one or more bases, insertion or replacement with functional activity
Allele.
Medicago ruthenica dehydrated proteinMrDHN3The clone of gene and Medicago ruthenica dehydrated proteinMrDHN3The prokaryotic expression of gene is same
Embodiment 1.
3 Medicago ruthenica dehydrin gene of embodiment, the gene areMrDHN3Encoding gene, it has shown in Fig. 2 and sequence
The protein of amino acid residue sequence shown in SEQ ID NO.2 the 1st to 221 in table.
Medicago ruthenica dehydrated proteinMrDHN3The clone of gene and Medicago ruthenica dehydrated proteinMrDHN3The prokaryotic expression of gene is same
Embodiment 1.
It is rightMrDHN3Protein product coded by cDNA reading frame is made of 221 amino acid residues, molecular weight (Mw) be
24.84 kD, theoretical isoelectric point (pI) are 5.56, are acidic protein.Overall average hydrophobicity index Grand average of
Hydropathicity (GRAVY) is -1.444, hydrophily with higher.
Knot of Pfam database (http://pfam.janelia.org) database to the MrDHN3 amino acid sequence encoded
Structure domain scans for, and shows that MrDHN3 albumen belongs to LEAII protein family, and there are a S- segment (solid box marks) and 2
K- segment (dotted line frame marks) (Fig. 3) rich in lysine is SK2 type dehydrated protein.BLAST pairs is utilized in ncbi database
The amino acid sequence of MrDHN3 coding protein and other species protein amino acid sequences compare (Fig. 3) discovery and M. truncatula
(Medicago truncatula) and Trifolium repense (Trifolium repens) Amino acid sequence identity (identity) are most
Height, up to 81% or more.Systematic evolution tree (Fig. 4) is constructed to the SKn type dehydrated protein amino acid sequence of 6 species of NCBI downloading
It was found that the affiliation of MrDHN3 and leguminous plant pea SK2 albumen are nearest, it is one group that pulse family different plant species, which are gathered, and grass family is not
Gathering with species is one group, other section plants are another group, shows that DHN3 albumen has phylogenetic feature.
4 Medicago ruthenica dehydrin gene of embodiment, the gene areMrDHN3Encoding gene, it has SEQ in sequence table
Substitution of the amino acid residue sequence shown in ID NO.2 the 1st to 221 through one or several amino acid residues lacks or adds
Add but contain the S- segment and 2 K- segments rich in lysine residue of a series of 1 serine (Ser) residue
(EKKGIMDKIKEKLPG or derivatives thereof) and have identical as SEQ ID NO.2 amino acid residue sequence active by SEQ
Protein derived from ID NO.2；The sequence of the protein as derived from SEQ ID NO.2 is corresponding with the sequence of allele.
Medicago ruthenica dehydrated proteinMrDHN3The clone of gene and Medicago ruthenica dehydrated proteinMrDHN3The prokaryotic expression of gene is same
Embodiment 1.
Recombinant expression carrier pET30a- MrDHN3 of the embodiment 5 containing Medicago ruthenica dehydrin gene, refers to this is flat
The encoding gene of Mu beans dehydrated proteinMrDHN3Opening code-reading frame sequence is inserted into coli expression carrier pET-30a
Between (Novagen product) BamH I and Sac I restriction enzyme site to get.
Medicago ruthenica dehydrated proteinMrDHN3The clone of gene and Medicago ruthenica dehydrated proteinMrDHN3The prokaryotic expression of gene is same
Embodiment 1.
The recombinant bacterium BL/ of recombinant expression carrier pET30a- MrDHN3 of the embodiment 6 containing Medicago ruthenica dehydrin gene
MrDHN3 refers to that recombinant expression carrier pET30a- MrDHN3 is transformed into E. coli expression strains BL21 (DE3), i.e.,
?.
The recombinant bacterium generates the base after addition IPTG induction by culture in fluid nutrient medium containing kanamycin
Because of the Medicago ruthenica dehydrated protein of coding.
Application of the 7 Medicago ruthenica dehydrin gene of embodiment in improvement plant and microorganism salt resistance and heat-resisting aspect.
(1) bacterium solution contact plate is tested:
Escherichia coli culture and IPTG inductive condition are as described in embodiment 3, when the Escherichia coli bacteria liquid of IPTG induction
When OD600 to 1.0 or so, with the LB liquid medium of the kanamycins containing 50mg/L and the IPTG of 0.5mmol/L by bacterium solution
10 times of dilution, carries out the Stress treatment of next step.Salt stress processing is to drip to each 10 μ L of bacterium solution after original bacteria liquid and dilution
Containing 50mg/L kanamycins, 0.5 mmol/L IPTG and 500mM NaCl or KCl solid LB media on, 37 DEG C culture
The Escherichia coli Growth situation of observation control and processing after 3d；By the bacterium solution after original bacteria liquid and dilution 55 in heat stress test
After DEG C water bath processing 30min, 10 μ L bacterium solutions is therefrom taken to drip to containing on 50mg/L kanamycins solid LB media respectively, 37 DEG C
It is observed after cultivating 16 h.Simultaneously to be control containing pET-30a (+) BL21 (DE3) bacterial strain in test.
(2) bacterium colony analysis of accounts:
By such as the culture (OD600 ≈ 1.0) that (1) IPTG is induced, with containing 50mg/L kanamycins,
The fresh LB liquid medium of 0.5mmol/L IPTG dilutes 10 times, and taking 100 μ L to be spread evenly across the card containing 50mg/L, that is mould
On the solid LB plate of element, bacterium colony counting is carried out after 37 DEG C of cultures, calculates E. coli clones survival rate.As qualitative analysis tries
It tests, 3d is cultivated in salt stress test；Heat stress test, cultivates 16 h.
High-salt stress and high temperature stress treatment conditions are tested with bacterium solution contact plate.
(3) result:
MrDHN3 expression can significantly improve Escherichia coli growth and survival energy under the conditions of above-mentioned with high salt and high temperature stress
Power (Figure 11 ~ 12).Bacterium colony count results are shown, under 0.5mol/L NaCl and KCl high-salt stress, MrDHN3 expresses bacterial strain
Survival rate is respectively 16.71 % and 20.8 %, and contains empty carrier pET-30a(+) Strain survival rate distinguish 3.55 % and
4.29 %；After 55 DEG C of high-temperature process, 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 there is protecting effect therefore to pass through biological skill Escherichia coli under environment stress
The gene can be used to improve the salt resistance and heat resistance of plant and microorganism by art method.
The recombinant expression carrier pET30a- MrDHN3 of 8 Medicago ruthenica dehydrin gene of embodiment is in improvement plant and micro-
The application of biological salt resistance and heat-resisting aspect.
The recombinant bacterium of the recombinant expression carrier pET30a- MrDHN3 of 9 Medicago ruthenica dehydrin gene of embodiment is being improved
The application of plant and microorganism salt resistance and heat-resisting aspect.
SEQ ID NO.1
1 CGTGTCATTA TGTGTAGTAG TGAAGTAGTA TAAAGTGCTA CCCCATCTCT
TTGATTTTCA
61 TCATCGAAAC AACATAATCT CATACTTTAC CTTTAGCATT TCCAGCTACA
TAAGAATTAA
121 TATTATTCCA ATTGATTAAT CTTGTTAATT TGCTTAGTGA TCATCAATTC
ATCATGGCTG
181 ATCAGGAGAA TCAGAACAAG TACGAGGAAA CCACCGCAAC CAACTCTGAG
ACAGAGATCA
241 AAGACAGGGG TGTTTTTGAT TTTCTAGGTG GTAAGAAAAA GGATGAAGAA
CATAAGCCTC
301 AAGAGGAAGC TATTGCAACT GATTTTAATC ACAAGGTGAC TTTGTATGAA
GCTCCATCAG
361 AGACCAAAGT AGAAGAAGAA GAAAAAGGTG AAAAGAAACA CACCAGCCTT
TTGGAGAAAC
421 TTCACCGATC TGATAGCTCT TCAAGCTCTT CGAGCGAGGA GGAAGTTGAT
GGAGAGAAGA
481 GGAAAAAGAA GAAGAAGGAA AAGAAGGAGA AGAAAGAGGA CACATCAGTG
CCAGTAGAGA
541 AAGTTGATGT TGTTGATGGA ACAACAGCAA GTACTGAGGA GAAGAAAGGT
TTCCTAGACA
601 AAATTAAGGA GAAGCTTCCA GGACACAAGA AAACTGAGGA TGTAACAACT
CAACCGCCTG
661 TTGCTGCTGT TGCTGTGCCA TCTGCTGAGA CAACAACAAC AACAACAACT
AGTCATGATC
721 AAGGAGAGAA GAAAGGTATT TTGGAAAAGA TCAAAGAGAA GATTCCTGGT
TATCACCCTA
781 AGACTGCTAC TGATCATGAA GACAAAGATC ATCACAAGGA TGAGACTACT
ACTCATTGAT
841 TATGATCGAT CCATTGTGTT GTTTTGAGGC TTTGGAGGGT GCTTTGTTTA
SEQ ID NO.2
1 MADQENQNKY EETTATNSET EIKDRGVFDF LGGKKKDEEH KPQEEAIATD
FNHKVTLYEA
61 PSETKVEEEE KGEKKHTSLL EKLHRSDSSS SSSSEEEVDG EKRKKKKKEK
KEKKEDTSVP
121 VEKVDVVDGT TASTEEKKGF LDKIKEKLPG HKKTEDVTTQ PPVAAVAVPS
AETTTTTTTS
181 HDQGEKKGIL EKIKEKIPGY HPKTATDHED KDHHKDETTT H
Claims (3)
1. Medicago ruthenica dehydrin gene, it is characterised in that: the gene isMrDHN3Encoding gene, it is SEQ ID in sequence table
Nucleotide sequence shown in NO.1 the 1st to 890.
2. Medicago ruthenica dehydrin gene as described in claim 1, it is characterised in that: the gene is encoded in sequence table
The protein of amino acid residue sequence shown in SEQ ID NO.2 the 1st to 221.
3. Medicago ruthenica dehydrin gene as claimed in claim 1 or 2 is in improvement microorganism salt resistance and the application of heat-resisting aspect.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Medicago truncatula dehydrin mRNA;Tang H.等;《Genbank 登录号：XM_003603939.2》;20150825 * |
| 扁蓿豆Ｍｒ Ｌ ＥＡ２基因的克隆和原核表达;马超等;《西北植物学报》;20141031;第34卷(第10期);1944-1950 * |
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