CN102492030B - Gene comprising dehydrin functional domain and application thereof in anti-drought alkali-resistant gene engineering - Google Patents

Abstract

The invention provides a gene comprising a dehydrin functional domain and an application thereof in anti-drought alkali-resistant gene engineering, and belongs to the technical field of plant gene engineering. The invention provides a plant stress-resistant associated protein AcDHN (Atriplex canescens dehydrin), which comprises a protein composed of amino acid sequences in SEQ ID NO: 2 and a protein which is derived from SEQ ID NO: 2 and is obtained by substituting or deleting or adding one or more amino acid residues to the amino acid sequences in SEQ ID NO: 2; and the encoding sequence for encoding the gene of AcDHN is as follows: a. nucleotide sequence shown in SEQ ID NO: 1; b. nucleotide molecule encoding the protein sequences in SEQ ID NO: 2; c. nucleotide molecule having more than 90% homology with the nucleotide sequence in a and encoding protein; and d. nucleotide molecule hybridizing with the nucleotide sequence shown in a and encoding protein. AcDHN is introduced to Saccharomyces cerevisiae to obtain transgenic Saccharomyces cerevisiae, thereby improving drought, salt and alkali resistance of yeast.

Description

The gene and the drought resisting alkali resistant genetically engineered thereof that comprise the plain functional domain that dewaters are used

Technical field

The invention belongs to the plant gene engineering technology field, be specifically related to use Saccharomyces Cerevisiae in S accharomyces cerevisiae INVSc1 bacterial strain to express a kind of albumin A cDHN relevant with plant stress-resistance.

Background technology

Occurring in nature exists multiple as multiple biology and abiotic stress such as salt, alkali, high temperature, low temperature, arid, disease and pests, limited growing of crop, thereby influence crop yield and directly threaten grain security, therefore, the gene that degeneration-resistant border is coerced changes over to and carries out the interest that molecular breeding improvement crop character has caused researcher in the crop.And the research of clone's adverse circumstance genes involved and function thereof can provide valuable information and material for carrying out the plant stress-resistance breeding by genetically engineered.But current a lot of researchs mainly concentrate on herbaceous plant, seldom to xylophyta particularly the living xylophyta of salt carry out the research of molecular genetics.Xylophyta and herbaceous plant in reply during biological and abiotic stress in aspect property of there are differences such as structure, growth, growth and physiology, therefore can provide more candidate gene to the tolerance of biology and abiotic stress for improve crop by genetically engineered with degeneration-resistant relevant gene in clone and the research xylophyta.

Atyiplex canescen (Atriplex canescens[Pursh] Nute) be Chenopodiaceae, the atriplex xylophyta, it is the typical plant of arid, semiarid zone, more than annual rainfall 180mm, about 5 ℃ of average annual temperature, arid, semiarid desert, the saltings well-grown of extreme minimum temperature-40 ℃, provenance place of production height above sea level 2377m, the manual inseminating district has reached 3150m.It is reported that Atyiplex canescen is called " biological demineralizer " by some country, plant 6.07 mu of Atyiplex canescens, energy absorbed the salinity more than the 1t from soil in 1 year, it is the extremely valuable good feed shrub in desert, nonirrigated farmland, half-desert mountain, high yield and high quality, be rich in nutrition, branches and leaves contain the crude protein more than 12%, and biomass reaches 15t/hm ², the ability that accumulates selenium is arranged simultaneously, be widely used in the fixing and soil conservation in slope, road in the U.S., but main still for range improvement.In India, from states such as the U.S. 7,000,000 hm that introduced 12 kinds of saltbush breed improvements ²The saltings, some countries of Tunisia and north African are existing 20,000 hm of exploitation saltings resource ²Having planted to desert saltbush, is one of topmost feed shrub in arid and semi-arid area Australia, South Africa, north African, Israel and north America region saltbush, also is the fine tree species of greening and soil conservation.Successively carry out regional introduction and cultivation on China Gansu, Qinghai, Xinjiang, Ningxia and other places in recent years, shown extremely strong drought-resistant, low temperature, barren, anti-good characteristic such as saline and alkaline.

S. cervisiae Saccharomyces cerevisiae not only has growth and cultivates and be easy to advantages such as genetic manipulation soon, easily, and has a characteristic of typical eukaryotic system, have processes such as translation post-treatment such as glycosylation, disulfide linkage formation and protein folding, its expression pattern and expression of plants pattern are more approaching, so have the irreplaceable superiority of prokaryotic expression system with yeast screening assay plant stress-resistance gene.The method that adopts the different cDNA library of yeast mutants screening plant to identify and excavate new gene has been widely used in the research of plant function gene.With 2 kinds of different aminoacids transhipment defective screening mutant Arabidopis thaliana cDNA libraries of yeast, (1993) such as Frommer etc. and Hsu successfully are cloned into the NAT2/AAP1 that corresponding coding same amino acid sees through enzyme.Yeast expression system also is widely applied in the plant salt tolerance gene functional research, Yamada etc. (2002) utilize yeast expression system to verify that propadiene cyclisation oxydase (AOC) gene strengthens the salt tolerance of transformant greatly, thereby show that the AOC gene strengthens the function of salt stress-resistant in eukaryotic expression system.Tang Yulin etc. (2007) utilize yeast expression system research soybean SAL I3-2 protein function, soybean SAL I3-2 albumen cDNA and deletion fragment thereof are building up on the Yeast expression carrier pYES2, and transformed yeast bacterium INVSc1, detect the upgrowth situation of yeast recon under stress conditions, the result shows that the SALI3-2 gene can obviously improve the salt resistance ability of yeast transformant.Wine brewing yeast strain INVSc1 (genotype is MATa his3D1leu2 trp1-289 ura3-52) is a kind of diploid, and growth is desirable yeast expression bacterial strain rapidly, this bacterial strain is engineered uracil auxotrophy bacterial strain, use SC-U substratum screening transformant, false positive rate is low, can guarantee transformation efficiency.

Dehydration plain (Dehydrin) belongs to LEA family, and molecular mass is 9-200KDa, is a kind of drought-induced albumen that extensively is present in the higher plant, takes place to produce late period at plant embryos.Plant is in the influence of desiccation stresses such as low temperature, Exogenous ABA, arid, the saline and alkaline and outer crystallization of born of the same parents, can great expression and this proteinoid of accumulation.The plain molecule that dewaters has very high wetting ability and thermostability, moisture can be captured in the cell, avoids arid and saline and alkaline damage with the protection cell.Can be that it has the very conservative zone of three classes at plant embryos development later stage and the plain important structure feature of dehydration: K, S and Y fragment, wherein the K fragment be that all dehydration elements all have.The K fragment is rich in Methionin, is usually located at the C end, can have 1 to 12 repetition, generally forms (EKKGIMDKIKEKLPG) by 15 amino-acid residues.Many dehydrations are plain also to contain the S fragment of being made up of serine residue, evidence suggests that the phosphorylation of S fragment can make the dehydration element enter nucleus under the guiding of signal peptide.The plain N end of a lot of dehydrations also has another kind of conservative region: Y fragment, its sequence are (V/T) DEYGNP, with plant and bacteria molecule companion's nucleic acid binding site homology are arranged.

Wherein, the yeast INVSc1 bacterial strain that transforms the pYES-AcDHN recombinant vectors shows strong resistance in drought-resistant and salt alkaline stress, in numerous researchs, also there is not the relevant report aspect the degeneration-resistant physiological function of and plant degeneration-resistant at yeast about the Atyiplex canescen gene that contains the Dehydrin functional domain.

Summary of the invention

The present invention adopts saltbush plant Atyiplex canescen (Atriplex canescens) as research material, coerces down the total length utilization Invitrogen company of Atyiplex canescen by making up low temperature (10 ℃) and salt (400mM NaCl)

Full-Length cDNA Library Construction Kit makes up the full-length cDNA library, and recombinate among the Yeast expression carrier pYES-DEST52 by Gateway technology LR, mix plasmid transformed yeast bacterial strain INVSc1, (saline and alkaline by multiple simulation environment stress, freeze injury, high temperature, SOS coerces, drought stress) filters out the gene relevant with environment stress, for utilizing salt tolerant alkali from now on, arid, freeze injury, transgenic crop and forest that the SOS stress gene obtains anti-corresponding environment stress lay the foundation, and also provide foundation for Atyiplex canescen salt tolerant alkali and low temperature resistant Study on Molecular Mechanism simultaneously.

The present invention uses Yeast expression carrier pYES-DEST52 (available from Invitrogen company), insert AcDHN albumen nucleotide coding sequence of the present invention and obtain recombinant expression vector pYES-AcDHN, it is except carrying AcDHN albumen nucleotide coding sequence, also has the T7 promotor, the GAL1 promotor, CYC1 stops transcribing signal, the URA3 gene, the f1 replicator, the pUC replicator, yeast 2 μ replication sequences, ampicillin resistance gene, attR1 and attR2 are used for the recombination site of LR reorganization, lethal gene ccdB is used for reverse screening transformant, chloramphenicol resistance gene is used for the dual screening to positive transformant, protein expression detected after V5epitope was convenient to recombinate, 6xHis Tag is used for the recombination purifying, and efficiently expresses required various controlling elements for foreign protein.

The AcDHN gene contains a complete open reading frame (Open Reading Frame) 1017bp by 1408 based compositions, and 5 '-UTR and 3 '-UTR sequence also is provided simultaneously, and AcDHN is the SEQ ID NO:1 sequence in the sequence table.The initiator codon of opening code-reading frame is ATG, and terminator codon is TGA.

An object of the present invention is to provide albumen and encoding gene thereof relevant with plant stress-resistance in the Atyiplex canescen that contains the plain functional domain that dewaters, and use.

The anti-Na of a kind of plant anti-adversity associated protein provided by the present invention ₂CO ₃, NaHCO ₃Coerce with arid etc., name is called AcDHN (Atriplexcanescens Dehydrin), derives from Atyiplex canescen.

A kind of plant anti-adversity associated protein AcDHN wherein comprises following two proteinoid:

(a) protein of being formed by the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table;

(b) with the aminoacid sequence of SEQ ID NO:2 in the sequence table through the replacement of one or several amino-acid residue or disappearance or interpolation and relevant with plant stress-resistance protein of being derived by SEQ ID NO:2 in the sequence table.

SEQ ID NO:2 is made up of 338 amino-acid residues in the sequence table, wherein: 91 hydrophobic amino acids, 208 hydrophilic amino acids, wetting ability is extremely strong, 94 basic aminoacidss, 79 acidic amino acids, protein molecular weight is 38.3kD, iso-electric point is 6.47, wherein is rich in Methionin (Lys) and accounts for 18.33% of total protein concentration, and this also is the principal character of Dehydrin family protein.Atyiplex canescen AcDHN albumen does not appear in the newspapers.

The encoding sequence of encoding gene AcDHN is 5 ' the end 99-1113 position deoxynucleotide of SEQ ID NO:1 in the sequence table.

The encoding sequence of encoding gene AcDHN is one of following:

1) nucleotide sequence of encoding sequence is shown in SEQ ID NO:1 in the sequence table;

2) nucleic acid molecule of SEQ ID NO:2 protein sequence in the code sequence tabulation;

3) with 1) described nucleotide sequence has homology more than 90% and the nucleic acid molecule of coded plant resistance relevant protein AcDHN;

4) under the rigorous condition of height with 1) nucleic acid molecule of described nucleotide sequence hybridization and proteins encoded AcDHN.

More than 4) described in high rigorous condition refer to can be at 0.1XSSC, in the solution of 0.1%SDS, under 65 ℃, hybridize and wash the condition of film.

A kind of recombinant expression vector contains the encoding gene AcDHN of plant anti-adversity associated protein AcDHN.

A kind of bacterium of recombinating contains the encoding gene AcDHN (hereinafter to be referred as encoding gene AcDHN) of plant anti-adversity associated protein AcDHN.

In order to make the AcDHN in (a) be convenient to purifying, N-terminal that can the protein that the aminoacid sequence shown in the sequence 2 is formed in by sequence table or the shuttle base is terminal connects label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK

c-myc

10

EQKLISEEDL

The recombinant Saccharomyces cerevisiae expression vector contains encoding gene AcDHN, and the recombinant yeast expression vector transformed host cells is yeast saccharomyces cerevisiae INVSc1.Restructuring yeast strains shows resistance, and described yeast resistance specifically can be the resistance to abiotic stress, particularly to arid and low temperature, Na ₂CO ₃, NaHCO ₃The resistance of reverse of coercing.

AcDHN is imported the transgenosis yeast saccharomyces cerevisiae that yeast saccharomyces cerevisiae obtains, can promote the drought-resistant salt tolerant alkali ability of yeast.

Description of drawings

Fig. 1 is that the total RNA of Atyiplex canescen extracts picture

Wherein: NaCl extracts blade and the total RNA electrophorogram of stem for 400mM NaCl after handling Atyiplex canescen; MW is that 1kb Plus Nucleotide Ladder Marker is made of nucleotide fragments 10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 800bp, 500bp and 300bp, wherein the Nucleotide amount of 3000bp is 100ng, and all the other band Nucleotide amounts are about 50ng; Cold extracts blade and the total RNA electrophorogram of stem behind-10 ℃ of processing Atyiplex canescens.

Fig. 2 inserts fragment length PCR for the cDNA library and detects picture

Wherein: 1-8,9-16 drops into capable bacterium colony PCR qualification result for the picking positive bacteria; MW is that 1kb Plus Nucleotide LadderMarker is made of nucleotide fragments 10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 800bp, 500bp and 300bp, wherein the Nucleotide amount of 3000bp is 100ng, and all the other band Nucleotide amounts are about 50ng.

Fig. 3 is reorganization pYES-AcDHN plasmid pcr amplification electrophorogram

Wherein: M:marker size from top to bottom is: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp1,2: the purpose band

Fig. 4 is AcDHN functional domain comparison result in GenBank

Fig. 5 is AcDHN and other species Dehydrin protein system evolutionary tree

Fig. 6 is pYES-AcDHN and pYES-DEST52 yeast conversion figure

Wherein: A is the INVSc1 yeast strain; B transforms the INVSc1 yeast strain for recombinant plasmid pYES-AcDHN; C is the INVSc1 yeast strain; D transforms the INVSc1 yeast strain for the pYES-DEST52 plasmid.

Fig. 7 is that pYES-AcDHN yeast positive transformant PCR identifies figure

Wherein: M:marker size from top to bottom is: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp

1,2,3: positive transformant purpose band

Fig. 8 induces the back growth curve chart for pYES-AcDHN yeast positive transformant adds semi-lactosi

Wherein: X-coordinate represents to add growth time after the semi-lactosi; Ordinate zou is represented absorbancy OD ₆₀₀Value.

Fig. 9 is whole protein expression amount graphic representation in the growth of pYES-AcDHN yeast positive transformant

Wherein: X-coordinate is represented growth time; Ordinate zou is represented pYES-AcDHN yeast whole protein amount.

Figure 10 coerces the photo I for the treatment of effect for pYES-AcDHN yeast and pYES-DEST52 yeast

Wherein: A handles the yeast effect for 4M KCl; B handles the yeast effect for 5M NaCl; C is pyroprocessing yeast effect; D is subzero treatment yeast effect.

Figure 11 coerces the photo II for the treatment of effect for pYES-AcDHN yeast and pYES-DEST52 yeast

Wherein: A is 6%NaHCO ₃Handle the yeast effect; B is 8%NaHCO ₃Handle the yeast effect; C is 6%Na ₂CO ₃Handle the yeast effect; D is 8%Na ₂CO ₃Handle the yeast effect.

Figure 12 coerces the photo III for the treatment of effect for pYES-AcDHN yeast and pYES-DEST52 yeast

Wherein: A is that 40%PEG6000 handles the yeast effect; B handles the yeast effect for the 5M sorbyl alcohol; C handles the yeast effect for the 4mM Paraquat.

Embodiment

Embodiment one: acquisition and the sequential analysis of encoding gene AcDHN full-length cDNA

1. coerce the processing Atyiplex canescen:

The trimestral Atyiplex canescen plant that will grow is transplanted to plant science institute of Jilin University plant culturing chamber from Jilin Province's forest-science institute-10 ℃ wild country, and plantation 10d treats the plant survival, sends young leaves.Handle every strain Atyiplex canescen plant with 500mL 400mM NaCl every day, handles 4d, plucks blade, and stem and root clean up uses liquid nitrogen flash freezer, and-80 ℃ frozen, in order to building the storehouse.

2. construction cDNA library:

To gather under-10 ℃ of conditions and extract total RNA (Fig. 1) simultaneously with blade, stem and root that 400mMNaCl handles the Atyiplex canescen plant, use the cap antibody module of Invitrogen company to carry out enrichment at a chain cDNA who contains complete cap sequence then, then according to Invitrogen company

Full-Length cDNA Library Construction Kit construction cDNA total length uncut library (Fig. 2), and recombinate among the Yeast expression carrier pYES-DEST52 by Gateway technology LR, PCR detects and inserts fragment length, fragment length is surpassed 300 library clone order-checkings of 1000bp, carry out amino acid comparison and bioinformatic analysis, obtain Atyiplex canescen resistance ESTs sequence.

2.1Total the extraction of RNA:

2.11 get the 100mg tissue sample, in the mortar of precooling, use the liquid nitrogen grinding powder, change over to then in the 1.5mL centrifuge tube;

2.1.2 add the Trizol reagent of 1ml, the vibration mixing, room temperature leaves standstill the abundant lysing cell of 10min;

2.1.3 add the chloroform of 200 μ L, concuss 15sec, room temperature leaves standstill 5min;

2.1.4 at 4 ℃, 12,000rpm high speed frozen centrifugation 15min;

2.1.5 get supernatant in new 1.5mL centrifuge tube, add the 0.5mL Virahol, put upside down mixing, room temperature leaves standstill 10min;

2.1.6 at 4 ℃, 12,000rpm high speed frozen centrifugation 10min;

2.1.7 remove supernatant, add the 75% washing with alcohol precipitation of 1mL, 7,500rpm high speed frozen centrifugation 5min;

2.1.8 repeating step 2.1.7 once;

2.1.9 remove supernatant, air drying RNA precipitation is dissolved in the 50 μ L DEPC water then.

3. screen the clone of encoding gene AcDHN fragment:

CDNA sequence according to the encoding gene AcDHN that finds out in the Atyiplex canescen ESTs sequence, LR recombinates and obtains recombinant yeast expression vector pYES-AcDHN among the Yeast expression carrier pYES-DEST52, with recombinant yeast expression vector pYES-AcDHN transformed into escherichia coli competence DH5 α, after the picking list bacterium colony, LB (Amp resistance) shakes the training back and extracts plasmid.According to the sequences Design PCR primer on the carrier, the performing PCR of going forward side by side amplification, checking cDNA sequence and sequencing result.

Upstream (T7): 5 '-TAATACGACTCACTATAGGG-3 '

Downstream (R): 5 '-AGGGTTAGGGATAGGCTTACCTTC-3 '

The PCR reaction system: the clone PCR reaction conditions is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s; 56 ℃ of annealing 30s; 72 ℃ are extended 3min, 30 circulations; 72 ℃ are extended 10min.Amplified production detects through 1% agarose gel electrophoresis, and a specific band (Fig. 3) is arranged in the 1400bp position.

Embodiment two: the sequential analysis of encoding gene AcDHN

Comprise 1017bp through order-checking encoding gene AcDHN cDNA sequence open reading frame, formed by 338 amino-acid residues, wherein, 91 hydrophobic amino acids, 208 hydrophilic amino acids, wetting ability is extremely strong, protein molecular weight is 38.3kD, iso-electric point is 6.47, wherein is rich in Methionin (Lys) and accounts for 18.33% of total protein concentration, and this also is the principal character of Dehydrin family protein.The result shows the cDNA sequence that obtains full-length gene, and its GenBank accession number is JN974246.

Utilize NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) that the amino acid of this genes encoding is carried out conservative region analysis (Fig. 4), the result shows: this albumen belongs to the Dehydrin family protein.Aminoacid sequence to encoding gene AcDHN on the NCBI website carries out BLAST, carry out evolutionary analysis and homology analysis with other species aminoacid sequences of determining function, and draw evolutionary tree with clustalX and MEGA4 software: encoding gene AcDHN and spinach Dehydrin amino acids coding evolutionary distance are nearest.With Dehydrin albumen homology in the Arabidopis thaliana be 57% (Fig. 5).

Analyze AcDHN according to DNAMAN and do not contain signal peptide.Carry out secondary structure prediction with the AcDHN of DNAMAN, find to contain 10.65% helix, 3.84% strand, 84.9% coil.

Embodiment three: the conversion of recombination yeast and detection

Yeast saccharomyces cerevisiae INVSc1 is urinary ammonia acid auxotroph (Ura), is not containing on the SC minimum medium (SC-Ura) of urinary ammonia acid the irreproducible and growth of this yeast.Contain the URA3 gene on the pYES2-DEST52 plasmid, make recombination yeast can be on the SC-Ura substratum normal growth.Therefore, can select substratum to screen with SC-Ura and transform positive yeast and non-positive yeast.Respectively with in plasmid pYES-AcDHN and the pYES-DEST52 transformed yeast bacterium INVSc1 bacterial strain, transformed yeast is coated on the SC-Ura solid and selects to do contrast with unconverted yeast on the substratum, cultivates, except empty yeast, all there is bacterium colony to grow behind the 2d, yeast conversion success (Fig. 6) is described.

1. use Lithium acetate chemical transformation transformed saccharomyces cerevisiae INVSc1, its concrete step of converting:

1.1 add a yeast strain mono-clonal (INVSc1) in the 10mLYPD liquid nutrient medium, 30 ℃ are spent the night and shake training;

1.2 detect the OD that cultivates bacterium liquid ₆₀₀, the yeast liquid of adding incubated overnight is diluted to OD in 50mL YPD substratum ₆₀₀Be 0.4, shake training 2-4h again;

1.32500rpm frozen centrifugation 5min precipitation is collected thalline, uses the resuspended thalline of 40mL1xTE afterwards;

1.42500rpm frozen centrifugation 5min precipitation is collected thalline, uses the resuspended thalline of 2mL1xLiAc/0.5xTE afterwards;

1.5 be distributed into 100 μ L/1.5mLEP pipe;

1.6 yeast cell is placed incubated at room 10min;

1.7 in each transformation system (100 μ L), add 1 μ g plasmid Nucleotide, 100 μ g sex change salmon sperm nucleus thuja acids, mixing;

1.8 add 700 μ L 1x LiAc/40%PEG-3350/1x TE, mixing;

1.930 the mixed solution 30min in ℃ incubation step 8;

1.10 add 88 μ LDMSO, mixing, 42 ℃ of thermal shock 7min;

1.115000rpm frozen centrifugation 1min removes supernatant;

1.12 with the resuspended thalline of 1mL1xTE, 5000rpm frozen centrifugation 1min removes supernatant;

1.13 with the resuspended thalline of 100 μ L1xTE, coat on the screening culture medium.

2. the evaluation of positive transformant

3 INVSc1 of picking (pYES2-AcDHN) mono-clonal at random on the screening culture medium, 30 ℃, the 200rpm shaking culture is spent the night, get overnight culture, boiling water bath 5min places the 5min smudge cells on ice, carries out bacterium liquid PCR as template and carries out pcr amplification with primer T7 and R with centrifugal concentrate of cytoclasis liquid, PCR reaction system (20 μ L) is: PCR Buffer 2 μ L, dNTP 1 μ L, T7 Primer 0.5 μ L, R Primer 0.5 μ L, Template 1 μ L, ddH ₂O 14.5 μ L.Amplified production is electrophoresis on 1% sepharose, the 1400bp length of its amplified fragments and expection match (Fig. 7).Proof recombinant plasmid pYES2-AcDHN has transformed and has entered into yeast.

Embodiment four: transgenic yeast growth and protein expression

Use among the Wine brewing yeast strain INVSc1 in the laboratory, the GAL1 promotor can be suppressed by glucose, and can inducible transcription (Fig. 8) under as the situation of carbon source at semi-lactosi.Wherein albumen is at 12h left and right sides expression amount the highest (Fig. 9).

1. protein induced expression step

Be inoculated in the SC-U substratum that contains 2% glucose 1.1 will contain the pYES-AcDHN of target gene, 30 ℃, spend the night and shake training;

The OD of bacterium liquid 1.2 measuring and calculating is spent the night ₆₀₀, guarantee to make to be inoculated in OD in 50mL SC-U (the containing semi-lactosi) substratum ₆₀₀Equal 0.4;

1.3 the yeast cell volume that calculates in the sucking-off step 2,3500rpm, 5min, 4 ℃ are centrifugal, abandon supernatant;

1.4 the cell in the step 3 is resuspended in the 50mL inducing culture (SC-U, semi-lactosi), and 30 ℃ are shaken training;

1.5 respectively 0,4,8,12,16 and the 24h place take out the inducing cell of equal volume, and measure OD ₆₀₀Value;

1.6 with the cell 3500rpm that takes out, 5min, 4 ℃ are centrifugal, collect thalline;

1.7 abandon supernatant, cell be resuspended in the 500 μ L sterilized waters;

1.8 with cell transfer in the centrifuge tube of 1.5mL of sterilization, 12000rpm, 30s abandons supernatant;

1.9 cell is kept at-80 ℃, standby.

2. recombinant protein is quantitative

With after the cracking Buffer cracking, use the Bradford method to carry out the mensuration of total protein concentration yeast cell, simulation yeast cell growth curve and total protein are expressed curve

2.1 be ready to cell pyrolysis liquid, before preparing experiment, preferably can understand the OD of lysing cell ₆₀₀

2.2 cell is resuspended in the 500 μ l lysis buffers, 3500rpm, 5min, 4 ℃ are centrifugal, collect thalline;

2.3 the taking-up supernatant adds an amount of (calculating the volume that adds lysis buffer) lysis buffer then, makes OD600 to 50-100;

2.4 add the pickling glass pearl of equivalent;

2.5 the treatment solution in the step 4 is placed on vortex 1min on the vortex instrument, place 1min on ice, repeat this step 20 time, lysing cell.Cell can microscope bottom be observed lytic effect by the mechanical shear stress cracking;

2.6 with mixed solution after the cracking in the step 5,12000rpm, 10s, 4 ℃ are centrifugal;

2.7 supernatant is transferred in the new 1.5mLEP pipe, can be carried out quantification of protein with BSA.

Embodiment five: positive transformed yeast environment stress is handled

1. environment stress is handled pre-treatment

Draw preservation bacterium liquid or the picking list bacterium colony of the correct INVSc1 (pYES2-AcDHN) of yeast INVSc1 (pYES-DEST52) and checking, be inoculated in the SC-U liquid nutrient medium (glucose that adds final concentration 2%), 30 ℃ of shaking table shaking culture 24h measure its OD ₆₀₀Be worth, and with SC-U liquid nutrient medium (glucose) bacterial concentration adjusted OD ₆₀₀Be 0.4, volume is 5mL, the centrifugal 1min of 8000rpm, add the resuspended thalline of 2mL inducing culture (SC-U+2% semi-lactosi), be connected in the inducing culture of 5mL 30 ℃ of shaking culture 24h, the OD of measurement INVSc1 (pYES-DEST52) and INVSc1 (pYES2-AcDHN) ₆₀₀Be worth, and OD is adjusted in the concentration unification of two kinds of recombination yeasts ₆₀₀Value is 2, gets the yeast of equivalent, and the centrifugal 1min of 8000g abandons supernatant.This two primary yeast is carried out the different processing of coercing, and relatively the resistance of (pYES-DEST52) and INVSc1 (pYES2-AcDHN) is tested for every kind and is repeated 3 times.

2. the simulation environment stress is handled

Plant is present in occurring in nature will face various biologies and abiotic stress, wherein topmost have following several: salt damage (NaCl, KCl etc.), alkali evil (Na ₂CO ₃, NaHCO ₃Deng), drought stress, freeze injury low temperature stress, high temperature stress and phytopathogen are coerced.

Under indoor, use high density NaCl and KCl simulation salt stress, high density Na ₂CO ₃And NaHCO ₃The simulation alkaline stress, PEG6000 and sorbyl alcohol simulating drought are coerced, low temperature (20 ℃) simulation frozen stress, high temperature (53 ℃) simulation high temperature stress infects the biologies such as reactive oxygen species simulating plant pathogenic bacteria that produce in the plant process and coerces thereby use Paraquat can produce active oxygen simulating plant pathogenic micro-organism.Use above the processing, simulate biology and abiotic stress to the influence of transgenosis yeast saccharomyces cerevisiae, thereby estimate the various response effect of coercing of gene pairs (Figure 10).

2.1NaCl handle: above-mentioned thalline is resuspended in 5mol/L NaCl solution, 24h is placed in 30 ℃ of vibrations behind the mixing, then with undiluted thalline with dilute 10,100,1000,10000 times thalline respectively, get 2 μ L point samples on the SC-U solid medium of (containing 2% glucose), cultivate 48h for 30 ℃, relatively the growth differences of the bacterium colony of two kinds of bacterium.

2.2KCl handle: thalline is resuspended in mixing in the 4mol/L KCL solution, 24h is coerced in 30 ℃ of vibrations, then with undiluted thalline with dilute 10,100,1000,10000 times thalline respectively, get 2 μ L point samples on the SC-U solid medium of (containing 2% glucose), cultivate 48h for 30 ℃, relatively the growth differences of the bacterium colony of two kinds of bacterium.

2.3Na ₂CO ₃Handle: thalline is resuspended in 6% and 8% Na respectively ₂CO ₃Solution, 2h is coerced in 30 ℃ of vibrations of mixing, with undiluted thalline with dilute 10,100,1000,10000 times thalline respectively, gets the direct point sample of 2 μ L with then on the SC-U solid medium of (containing 2% glucose), 30 ℃ of constant temperature culture 48h, the relatively growth differences of the bacterium colony of two kinds of bacterium.

2.4NaHCO ₃Handle: thalline is resuspended in 6% and 8% NaHCO ₃Solution, behind the mixing, 2h is coerced in 30 ℃ of vibrations, with then with undiluted thalline with dilute 10,100,1000,10000 times thalline respectively, get the direct point sample of 2 μ L on the SC-U solid medium of (containing 2% glucose), cultivate 48h for 30 ℃, relatively the growth differences of the bacterium colony of two kinds of bacterium.

2.540%PEG6000 handles: thalline is resuspended in the sorbyl alcohol of 40% PEG6000 and 5mol/L, 24h is coerced in 30 ℃ of vibrations, to not dilute and dilute respectively 10,100,1000,10000 times thalline then, get 2 μ L point samples on the SC-U solid medium of (containing 2% glucose), 30 ℃ of constant temperature culture 48h, the relatively growth differences of the bacterium colony of two kinds of bacterium.

2.65mol/L sorbyl alcohol (Sorbitol) is handled: thalline is resuspended in the sorbyl alcohol of 5mol/L, 24h is coerced in 30 ℃ of vibrations, to not dilute and dilute respectively 10,100,1000,10000 times thalline then, get 2 μ L point samples on the SC-U solid medium of (containing 2% glucose), 30 ℃ of constant temperature culture 48h, the relatively growth differences of the bacterium colony of two kinds of bacterium.

2.7 freezing treatment: thalline is resuspended in the SC-U liquid nutrient medium, and freezing (20 ℃) coerce 48h, therebetween every 6h the thalline freeze thawing once.To not dilute and dilute 10,100,1000,10000 times thalline respectively, get 2 μ L point samples 30 ℃ of cultivation 48h, relatively growth differences of the bacterium colony of two kinds of bacterium on the SC-U solid medium of (containing 2% glucose).

2.8 pyroprocessing: thalline is resuspended in the SC-U liquid nutrient medium, coerces 1h for 53 ℃.To not dilute and dilute 100,1000,10000 times thalline respectively, get 2 μ L point samples 30 ℃ of cultivation 48h, relatively growth differences of the bacterium colony of two kinds of bacterium on the SC-U solid medium of (containing 2% glucose).

2.9 Paraquat (Paraquat): thalline is resuspended in the 4mM Paraquat solution, coerces 2h for 30 ℃.To not dilute and dilute 10,100,1000,10000 times thalline respectively, get 2 μ L point samples 30 ℃ of cultivation 48h, relatively growth differences of the bacterium colony of two kinds of bacterium on the SC-U solid medium of (containing 2% glucose).

Wherein, the pYES-AcDHN transformed yeast is for 4M KCl, 5M NaCl, 6%NaHCO ₃, 8%NaHCO ₃, 6%Na ₂CO ₃, 8%Na ₂CO ₃, adverse circumstance effect such as 40%PEG6000,5M sorbyl alcohol obviously is better than blank plasmid pYES-DEST52 transformed yeast.

Claims (4)

1. plant anti-adversity associated protein AcDHN is characterized in that: the protein of being made up of the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table.

2. by the encoding gene AcDHN of the described plant anti-adversity associated protein AcDHN of claim 1, it is characterized in that the encoding sequence of described encoding gene AcDHN is:

5 ' of SEQ ID NO:1 end 99-1112 position deoxynucleotide in the nucleotide sequence of encoding sequence such as the sequence table.

3. recombinant expression vector is characterized in that containing the encoding gene AcDHN of the described plant anti-adversity associated protein AcDHN of claim 2.

4. a reorganization bacterium is characterized in that containing the encoding gene AcDHN of the described plant anti-adversity associated protein AcDHN of claim 2.

Images