CN103509095B - Plant stress tolerance associated protein GmNF-YB8 as well as coding gene and application thereof - Google Patents
Plant stress tolerance associated protein GmNF-YB8 as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a plant stress tolerance associated protein GmNF-YB8 as well as a coding gene and an application thereof. The protein disclosed by the invention is the following (a) or (b): (a) a protein consisting of amino acid sequences shown in the sequence 1 in a sequence table; and (b) a protein which is formed through substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequences in the sequence 1, is associated with plant stress tolerance and is derived from the sequence 1. The protein disclosed by the invention can specifically regulate transcriptional expression of genes containing CCAAT-box cis elements and improve the salt tolerance of plants. Experiments show that the survival rates of GmNF-YB8 transgenic plants can reach 90.3% after salt stress treatment and are far higher than 28.8%, the survival rate of control plants. The protein disclosed by the invention provides a basis for artificially controlling expression of stress tolerance associated genes and plays an important role in stress tolerance enhanced plant breeding.
Description
Technical field
The present invention relates to a kind of plant stress tolerance correlative protein GmNF-YB8 and encoding gene thereof and application.
Background technology
The environment stresses such as arid, high salt and low temperature seriously govern growth, the growth of soybean.Therefore, understand soybean to the response of adverse environmental factor and signal transduction mechanism, improve the resistance of soybean varieties, become one of vital task of soybean heredity research and breed improvement.
A series of responsing reaction can be produced in plant materials, along with many Physiology and biochemistries and change developmentally under environment stress.Specify the reaction mechanism of plant to adverse circumstance, science argument will be provided for adversity gene engineering research and application.At present, plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, and exploration biotechnology improves plant growth characteristics, its objective is and improves plant to the adaptive faculty of adverse circumstance.
Under the adverse environmental factor of the environment-stress such as arid, high salt and low temperature, plant can make corresponding adjustment in molecule, cell and integral level, to reduce the injury existence that environment causes to the full extent.Many genes are expressed by stress-inducing, the product of these genes can not only participate in the stress response of plant directly, and the expression of other genes involved can be regulated or participate in signal transduction path, thus plant is avoided or reduces injury, strengthen the resistance to stressful environmental.To coerce relevant gene product and can be divided into two large classes: the product of first kind genes encoding comprises ionophorous protein, aquaporin, osmotic factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participate in the gene product that plant stress is replied directly; The product of Equations of The Second Kind genes encoding comprises participation and coerces relevant signal transmission and the protein factor of Gene expression and regulation, as protein kinase, transcription factor etc.Wherein, play an important role in the gene expression regulation that transcription factor is replied at plant stress.
Transcription factor also referred to as trans-acting factor, be can with the DBP of cis-acting elements generation specific effect in eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress transcribe.The DNA land of transcription factor determines the specificity that it is combined with cis-acting elements, and transcription regulatory region determines it and plays activation or restraining effect to genetic expression.In addition, himself activity is also subject to the impact of the effect such as nuclear location and oligomerization.
At present known in plant to coerce relevant transcription factor and mainly contain: the AP2(APETALA2 with AP2 structural domain)/EREBP(element responsive to ethylene associated proteins, ethylene responsive element bindingprotein) transcription factor family, bZIP(basic region/leucine zippermotif transcription factors containing basic region and leucine zipper) class transcription factor, WRKY transcription factor family containing conservative WRKY aminoacid sequence, CBF(CCAAT binding factor in conjunction with the main nuclear factor of CCAAT-box) class transcription factor, MYC family containing basic helix-loop-helix (bHLH) and leucine zipper and there is the MYB family of tryptophane bunch (Trp cluster).
NF-Y is the transcription factor of a class in conjunction with cis-acting elements CCAAT-box, special identification in conjunction with the cis-acting elements CCAAT-box in the promotor of many eukaryote composing types, inducibility and cell cycle dependant gene or enhanser, and then in the expression of these genes of transcriptional level control.The heterozygosis tripolymer that NF-Y is made up of NF-YA, NF-YB and NF-YC tri-different subunits.NF-YB albumen and NF-YC albumen guard territory by HFM each other, adopt connected head-to-tail mode to form heterodimer and make platform mutually, attract NF-YA protein binding to this dimer platform thus form the activated heterotrimer nuclear factor of tool.NF-Y is attached to the CCAAT box of target gene promoters part by the DNA binding domain on NF-YA subunit, performs transcriptional activation or Transcription inhibition function.The conservative territory of three subunits of NF-Y has different protein structure domains respectively, and wherein NF-YA guards territory and has DNA binding domains (DNAbinding domain) and make structural domain (subunit interaction domain) mutually with NF-YB/C heterodimer.NF-YB and NF-YC albumen is guarded territory and is then made up of histone fold motif (Histone-fold motif).Wherein NF-YB and H2B histone fold motif is similar, and NF-YC and H2A histone fold motif is similar, and histone motif is made up of three α spirals and two rings, is responsible for the dimeric formation of H2A/H2B.
Stress tolerance due to plant is the complex character regulated and controled by polygene, relies on importing individual feature protein gene to be difficult to the comprehensive raising realizing stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of multiple functional gene, strengthen the resistance of plant, become the engineered study hotspot of plant stress-resistance.
Summary of the invention
The object of this invention is to provide a kind of protein relevant to plant stress tolerance.
Protein provided by the present invention is a kind of nuclear factor albumen in conjunction with CCAAT-box, and name is called GmNF-YB8, derives from Glycine soybean (Glycine max L.), is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
(b) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and the protein that by sequence 1 derived relevant to plant stress tolerance.
The protein of sequence 1 is made up of 188 amino-acid residues.
In order to make the GmNF-YB8 in (a) be convenient to purifying, the N-terminal of the protein that the aminoacid sequence shown in sequence 1 forms or C-terminal label as shown in the table can be connected in by sequence table.
The sequence of table label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
GmNF-YB8 in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the GmNF-YB8 in above-mentioned (b) is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or to hold and/or 3 ＇ end connects the encoding sequence showing shown label and obtains at its 5 ＇.
The nucleic acid molecule of encoding said proteins also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially the gene (called after GmNF-YB8) of described GmNF-YB8 albumen of encoding; Described GmNF-YB8 gene is following 1)-4) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under strict conditions with 1) or 2) DNA molecule hybridize that limits, and the DNA molecular of coding stress tolerance correlative protein;
4) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coding stress tolerance correlative protein.
Described stringent condition is at 0.1 × SSPE(or 0.1 × SSC), in the solution of 0.1%SDS, hybridize under 65 DEG C of conditions and wash film.
Be made up of 567 Nucleotide in sequence 2, open reading frame is whole sequence 2, the protein (GmNF-YB8) in polynucleotide shown in sequence 1.
Recombinant vectors containing described nucleic acid molecule, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Available existing plant expression vector construction contains the recombinant expression vector of described nucleic acid molecule.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as wheat storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.When using described gene constructed described recombinant expression vector, any one enhancement type promotor or constitutive promoter can be added before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can be used alone or are combined with other plant promoter; In addition, when using described nucleic acid molecule to build recombinant expression vector, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process described recombinant expression vector, the coding can expressed in plant as added can produce enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. of colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.
In one embodiment of the invention, the promotor starting described genetic transcription in described recombinant expression vector is specially following a1) or a2):
A1) 35S promoter, as CaMV35S promotor;
A2) Pmin promotor.
More specifically, described recombinant expression vector is following b1) or b2) described recombinant plasmid:
B1) PAHC-PSK carrier (reference: high eastern Yao .2010. wheat ear germinating resistance is correlated with the clone of Vp-1B and AIP2 gene and functional analysis. [Ph.D. Dissertation]. Jilin University) multiple clone site (Spe I) place's forward insert the recombinant plasmid that described GmNF-YB8 gene obtains.
B2) at YEP-GAP carrier (reference Liu Q; Kasuga M; SakumaY; Abe H; Miura S; Yamaguchi-Shinozaki K; Shinozaki K.Two transcription factors; DREB1and DREB2; withan EREBP/AP2DNA binding domain separate two cellular signal transduction pathways indrought-and low-temperature-responsive gene expression; respectively, in Arabidopsis, Plant Cell1998Aug; 10 (8): 1391-1406) recombinant plasmid that described GmNF-YB8 gene obtains is inserted between multiple clone site (BamH I and Xho I).
Described GmNF-YB8 albumen or the application of described nucleic acid molecule in regulating plant resistance of reverse also belong to protection scope of the present invention.
In the present invention, described regulating plant resistance of reverse is embodied as the resistance of reverse improving plant.
Another object of the present invention is to provide a kind of method of cultivating resistance of reverse transgenic plant.
The method comprises the steps: the encoding gene of described GmNF-YB8 albumen to import in object plant, obtains the transgenic plant that resistance of reverse is higher compared with described object plant.
In the present invention, all described resistances of reverse specifically can be salt tolerance.The raising of described salt tolerance specifically may be embodied in the raising of plant survival rate under salt stress.
The encoding gene of described GmNF-YB8 albumen is following 1)-4) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of stress tolerance correlative protein of encoding;
4) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coding stress tolerance correlative protein.
Described stringent condition is at 0.1 × SSPE(or 0.1 × SSC), in the solution of 0.1%SDS, hybridize under 65 DEG C of conditions and wash film.
Described GmNF-YB8 gene specifically imports in described object plant by described recombinant expression vector.The described recombinant expression vector carrying described GmNF-YB8 gene transforms object plant by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, the conventional biology methods such as agriculture bacillus mediated, as vegetable cell or tissue, and the plant tissue of conversion is cultivated into plant.
Described object plant both can be dicotyledons also can be monocotyledons.In another embodiment of the present invention, described plant is specially dicotyledons Arabidopis thaliana, as Arabidopis thaliana kind Columbia ecotype Col-0.
Described GmNF-YB8 protein is also belonging to protection scope of the present invention as the application in transcription factor.Described transcription factor can be combined with CCAAT cis-acting elements, and has mobilizing function.
GmNF-YB8 provided by the present invention can express under high Salt treatment, special regulation and control can contain the transcriptional expression of the gene of CCAAT-box cis element (core parts CCAAT); The salt tolerance of plant can be improved.GmNF-YB8 of the present invention is that manual control expression that the is degeneration-resistant and gene of resistance to retrocorrelation provides the foundation, and plays an important role in the plant breeding strengthened in resistance and resistance of reverse.
Accompanying drawing explanation
Fig. 1 is fluorescence real-time quantitative (Real-time) the PCR collection of illustrative plates that GmNF-YB8 is expressed by Salt Stress-induced.
Fig. 2 is the plasmid map of recombinant expression vector PAHCPSK-GmNF-YB8.。
Fig. 3 is T
3for the cDNA level detection result turning the GmNF-YB8 gene in GmNF-YB8 gene Arabidopis thaliana.Wherein, swimming lane M is that Marker(band is followed successively by 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp); Swimming lane Col-0 is Columbia ecotype arabidopsis thaliana; Swimming lane L1-L5 is respectively 5 and to be identifiedly turns GmNF-YB8 gene Arabidopsis plant.
Fig. 4 is T
3in generation, turns GmNF-YB8 gene Arabidopis thaliana and wildtype Arabidopsis thaliana salt tolerance compares.Wherein, A is before high Ficus caricaL; B is that high Ficus caricaL rehydration is after 3 days.TL-1, TL-2 and TL-3 represent 3 T
3for GmNF-YB8 transgenic arabidopsis strain.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The clone of embodiment 1, GmNF-YB8
One, the separation of mRNA
By the rich No. 8 (reference: Sun Xiao of the growth soybean of about 10 days (Glycine max L.) kind iron of water planting, Dong Jianhui, Chen Ming, Xu Zhaoshi, leaf is made the country prosperous, Li Liancheng, the clone of Qu Yanying, horse strong-willed .2008. soybean adversity gene GmDREB3 promotor and control region piecewise analysis. Acta Agronomica Sinica, 34 (8): 14751479) the seedling 200m NaCl aqueous solution process 2 hours of four leaf phases, with liquid nitrogen flash freezer ,-80 DEG C save backup.Adopt Quikprep Micro mRNA PurificationKit(Pharmacia) carry out the separation of mRNA.ThermoScript II XL(AMV is used in first chain cDNA synthesis).Adopt SMART method synthesis ds cDNA, PCR primer carries out 1.0% agarose gel electrophoresis detection.
Two, the acquisition of GmNF-YB8 full length gene sequence
The nuclear factor B race full length gene sequence of soybean CCAAT-box is obtained by the method for 5 ' RACE and 3 ' RACE.
This unnamed gene, as shown in sequence in sequence table 2, is GmNF-YB8 gene by the nucleotide sequence of this gene, and its open reading frame is the whole sequence of sequence 2.Protein (called after GmNF-YB8 albumen) in GmNF-YB8 gene coded sequence table shown in sequence 1, GmNF-YB8 albumen is made up of 188 amino-acid residues, has conservative histone fold motif.
Embodiment 2, real-time fluorescence quantitative PCR analyze the expression characterization of GmNF-YB8
One, Stress treatment
Seedling age is rich No. 8 seedling of the soybean varieties iron of 10 days, carries out following process:
(1) salt marsh process: soybean seedling is placed in 200mM by NaCl solution, illumination cultivation takes out material after 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours respectively, and with liquid nitrogen flash freezer ,-80 DEG C save backup.
(2) process contrasted: directly get without the soybean seedling-80 DEG C of any process frozen in contrast (0 hour).
Two, the separation of mRNA
Adopting Quikprep Micro mRNA Purification Kit(Pharmacia) each sample that obtains step one carries out the separation of mRNA.
Three, reverse transcription is cDNA
After purifying step 2 obtained, mRNA reverse transcription is cDNA.
Four, real-time fluorescence quantitative PCR
CDNA is diluted the template that 50 times are used as Q-RT-PCR afterwards.Hold the special primer of non-coding region to carry out Q-RT-PCR amplification to sample with gene 3 ', analyzing gene, to the response situation of process various in step one, does internal reference with Actin.Q-RT-PCR is at ABI
7000 real-time fluorescence quantitative PCR instrument carry out, and 3 repetitions are established in a parallel test.Utilize the method that Livak KJ and Schmittgen TD (2001) reports, namely 2
-Δ Δ CTcalculate relative expression quantity.
ΔΔC
T=（C
T.GmNF-YB8—C
T.Actin）
Timex—（C
T.GmNF-YB8－C
T.Actin）
Time0
Time x represents random time point, Time
0represent the time point corresponding to target gene (GmNF-YB8) expression of 1 times amount after Actin corrects.
The results are shown in Figure 1.
The activation characteristic of embodiment 3, GmNF-YB8
YEP-GAP carrier: Chinese Academy of Agricultural Sciences's crop science research ensures to provide to the public; Reference Liu Q; Kasuga M; Sakuma Y; Abe H; Miura S; Yamaguchi-Shinozaki K; Shinozaki K.Twotranscription factors; DREB1and DREB2; with an EREBP/AP2DNA binding domainseparate two cellular signal transduction pathways in drought-andlow-temperature-responsive gene expression; respectively, in Arabidopsis, Plant Cell1998Aug; 10 (8): 1391-1406.
YPD liquid nutrient medium: microbial culture yeast extract (Bacto-Yeast Extract) 10g/L, microbial culture tryptone (Bacto-Peptone) 20g/L, regulate pH to 5.8,121 DEG C/15min sterilizing, be down to the Glucose that 60 DEG C add 40% later, make its final concentration be 20g/L.
SD/His-/Ura-/Trp-selective medium: not containing amino acid whose yeast nitrogen (Yeast nitrogen base) 6.7g/L, auxotroph mixture (drop-out media without His/Ura/Trp) 100ml, agar powder (Bacteriological agar) 20g/L, regulate pH to 5.8,121 DEG C/15min sterilizing, add 40%Glucose after being down to 60 DEG C, make its final concentration be 20g/L.
Auxotroph mixture (drop-out mix): (10 ×) L-Isoleucine(Isoleucine) 300mg/L, L-Valine(α-amino-isovaleric acid) 1500mg/L, L-Adenine(VITAMIN B4) 200mg/L, L-Arginine(arginine) 200mg/L, L-Leucine(leucine) 1000mg/L, L-Lysine HCl(Methionin) 300mg/L, L-Methionine(methionine(Met)) 200mg/L, L-Phenylalanine(phenylalanine) 500mg/L, L-Threonine(Threonine) 2000mg/L, L-Tyrosine(tyrosine) 300mg/L.
1×PEG/LiAc：50％（w/v）PEG33508ml，10×TE buffer1ml，10×LiAc1ml。
10 × TE Buffer:100mM Tris-HCl, 10mM EDTA, pH=7.5,121 DEG C of autoclavings, room temperature preservation.
1×TE/LiAc：10×TE buffer1ml，10×LiAc1ml，ddH
2O8ml。
Z solution: Na
2hPO
47H
2o16.1g/L, NaH
2pO
4h
2o5.5g/L, KCl0.75g/L, MgSO
47H
2o0.246g/L, regulates pH to 7.0,121 DEG C/15min sterilizing, 4 DEG C of preservations.
X-gal storage liquid (X-gal Stock Solution): with N, N-dimethyl-formamide, (DMF) dissolves X-gal, makes its final concentration be 20mg/ml ,-20 DEG C of storages.
Z damping fluid 100ml(Z buffer with X-gal containing X-gal), matching while using: Z solution 98ml, beta-mercaptoethanol (β-mercaptoethanol) 0.27ml, X-gal storage liquid (X-gal stock solution) 1.67ml.
10×LiAc：100Mm Tris-HCl，100mM EDTA，pH=7.5。121 DEG C of autoclavings, room temperature preservation.
The cardinal principle of the activation characteristic of transcription factor is proved: basic promotor Pmin(minimal promoter CCAAT cis-acting elements and mutant CCAAT cis-acting elements being building up to respectively pHISi-1 carrier and pLacZi carrier with yeast-one-hybrid system) upstream, Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).When being connected with the expression vector YEP-GAP(of goal gene of encoding transcription factors not containing mobilizing function) be transformed into the yeast cell being connected with CCAAT cis-acting elements and mutant CCAAT cis-acting elements respectively after, if the reporter gene be connected with in the yeast cell of mutant CCAAT cis-acting elements can not be expressed, and the reporter gene be connected with in the yeast cell of specific CCAAT cis-acting elements can be expressed, illustrate that this transcription factor can be combined with CCAAT cis-acting elements, and there is mobilizing function, have activated Pmin promotor, reporter gene is impelled to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of object transcription factor.
Below detailed description is detected GmNF-YB8 albumen and whether there is activation characteristic.Concrete operations are as follows:
One, the structure of recombinant expression vector YEP-GAP-GmNF-YB8
1, the acquisition of GmNF-YB8 gene
According to primers GmNF-YB8 and GmNF-YB8-XI of GmNF-YB8 gene, prime end introduces BamH I and Xho I restriction enzyme site respectively, with the cDNA of soybean varieties iron rich No. 8 (Institute of Crop Science, Chinese Academy of Agricultural Science) for template, pcr amplification obtains GmNF-YB8 gene.
GmNF-YB8:5'-TTT
gGATCCthe sequence of AATGGCGGACTCGGACAACGAC-3'(underscore part is the recognition sequence of BamH I restriction enzyme site, the 1-21 position of the corresponding sequence 2 in 11-31 position)
GmNF-YB8-XI:5'-GGT
cTCGAGthe sequence of CTACCTGGGCCTACCGGCA-3'(underscore part is the recognition sequence of Xho I restriction enzyme site, and sequence pair thereafter answers the reverse complementary sequence of latter 19 of sequence 2).
Pcr amplification product carries out 1.2% agarose gel electrophoresis detection.
Adopt Agarose Gel DNAPurifcation Kit Ver.2.0(TaKaRa company, Code No.:DV807A) reclaim the PCR primer of purifying about 585bp.
2, the structure of recombinant expression vector YEP-GAP-GmNF-YB8
1. cut with restriction enzyme BamH I and Xho I enzyme the PCR primer that step 1 reclaims purifying, reclaim digestion products;
2. cut expression vector YEP-GAP with restriction enzyme BamH I and Xho I enzyme, reclaim carrier framework;
3. step digestion products is 1. connected with step carrier framework 2.;
4. by the electroporated JM109 bacterial strain of step connection product 3. (Clontech company), 37 DEG C of incubated overnight, picking positive colony checks order; Sequencing result shows, the positive recombinant plasmid obtained inserts the DNA fragmentation shown in sequence 2 of sequence table between BamHI and the XhoI restriction enzyme site of YEP-GAP, by this recombinant plasmid called after YEP-GAP-GmNF-YB8.The promotor starting GmNF-YB8 genetic transcription in recombinant expression vector YEP-GAP-GmNF-YB8 is Pmin promotor.
Two, the Binding in vivo specificity of GmNF-YB8 and the checking of activation characteristic
1, the structure of yeast reporter
(1) structure of normal dual yeast reporter
DNA fragmentation A(is containing 4 CCAAT elements; TTTAA
cCAATcAGAAA):
The core sequence of 5 '-GAATTC-CCAAT-CCAAT-CCAAT-CCAAT-GTCGAC-3'(CCAAT: CCAAT).The nucleotide sequence of DNA fragmentation A is shown in the sequence 3 of sequence table.
With above-mentioned DNA fragmentation A for template, pcr amplification is carried out with the primer pair of A1 and A1-XI composition, by the PCR primer restriction enzyme Xho I that obtains and Nco I double digestion, with pHis-1 carrier (the MATCHMAKER One-Hybrid System through same double digestion, Clontech company) large fragment be connected, by the recombinant plasmid order-checking obtained, order-checking is shown the recombinant vectors called after pHis-1-CCAAT containing the DNA sequence dna shown in sequence 3 in ordered list.In recombinant vectors pHis-1-CCAAT, described DNA fragmentation A is positioned at Pmin promotor upstream.With Xho I and Nco I restriction endonuclease, recombinant vectors pHis-1-CCAAT is cut into wire.
A1:5'-CCG
cTCGAGgAATTCTTTAACCAATCAGA-3'(underscore part is the recognition sequence of Xho I, and sequence is thereafter first 20 of sequence 3);
A1-XI:5'-CATG
cCATGGgTCGACTTTCTGATTGGTT-3'(underscore part is the recognition sequence of Nco I, and sequence is thereafter the reverse complementary sequence of latter 19 of sequence 3)
First by wire pHis-1-CCAAT vector in yeast cell (YM4271 strain, MATCHMAKEROne-Hybrid System, Clontech company), acquisition can at SD/His
-the yeast transformant (Yeast transformant) of normal growth on substratum.Then with this yeast transformant for host cell, continue to transform containing wire pLacZi-CCAAT carrier.Lack the SD/His of Histidine and uridylic so at the same time
-/ Ura
-on substratum, select to obtain normal dual yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT.
(2) structure of dual yeast reporter of mutant
DNA fragmentation B(is containing 4 mCCAAT elements):
5 '-GAATTC-mCCAAT-mCCAAT-mCCAAT-mCCAAT-GTCGAC-3'(mCCAAT: the core sequence CCAAT of 4 CCAAT elements is mutated into TTTTA).The nucleotide sequence of DNA fragmentation B is shown in the sequence 4 of sequence table.
Replace DNA fragmentation A with DNA fragmentation B, the same step of method (1), obtain dual yeast reporter of mutant.
2, PEG/LiAc method transformed yeast and interpretation of result
(1) inoculation yeast bacterium YM4271 strain is in 1ml YPD liquid nutrient medium, concuss 2 minutes, and gone to by suspension after dispersion agglomerate in the triangular flask containing 50ml YPD liquid nutrient medium, 30 DEG C/250rpm shakes and spends the night, and surveys OD600=1.7-1.8(about 4 × 10
7individual/mL).
(2) get 30ml step (1) overnight culture to receive in the fresh YPD substratum of 300ml, 30 DEG C/250rpm cultivates, about 3 hours (to OD600=0.5 ± 0.1), the centrifugal 5min of room temperature 1000g, collects thalline, abandons supernatant, suspend with 1/2 volume 1 × TE, 1000g/5min is centrifugal.
(3) supernatant is abandoned in suction, and with the freshly prepared 1 × TE/LiAc solution suspension of 1.5ml, vibration mixing is for subsequent use.
(4) take out 0.1ml competent yeast to transform, add following solutions successively: YEP-GAP-GmNF-YB8,0.1mg ssDNA(salmon sperm dna of 0.1 μ g step one preparation, SiTaa), 0.6ml PEG/LiAc at a high speed vibration 1 minute, 30 DEG C/200rpm shaking culture 30 minutes.
(5) add 70 μ l DMSO, be inverted mixing gently, 42 DEG C of heat shocks 30 minutes, vibrate therebetween gently, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g.
(6) supernatant is abandoned in suction, adds 0.5ml1 × TE buffer suspension cell.
(7) dip suspension with transfering loop, respectively containing 0, the SD/His of 15mmol/L3-AT
-/ Ura
-/ Trp
-on selective medium, setting-out is cultivated.
(8) dull and stereotyped half cultivates normal dual yeast reporter, and second half cultivates dual yeast reporter of mutant, to do check analysis.
(9) be placed upside down in incubator, cultivate 3-4 days for 30 DEG C.
(10) found that the SD/His at 0mmol/L3-AT
-/ Ura
-/ Trp
-culture medium flat plate on yeast reporter of normal yeast reporter and sudden change have growth, but the diameter of yeast reporter of sudden change is obviously little; And at the SD/His of 15mmol/L3-AT
-/ Ura
-/ Trp
-culture medium flat plate on normal yeast reporter can normal growth, but yeast reporter of sudden change is by supression not growth.
3, galactosidase activity detects
(1) from the SD/His of 0mmol/L3-AT
-/ Ura
-/ Trp
-culture medium flat plate on the yeast reporter daughter colony of respectively normal yeast reporter of picking and sudden change.Go in YPD liquid nutrient medium, in 30 DEG C of shaking culture, in the logarithmic growth later stage to be grown to, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm.
(2) supernatant is abandoned, liquid in control main, centrifuge tube is placed in liquid nitrogen quick-frozen 10min, taking-up makes it naturally melt, add 50 μ l Z/X-gal solution (Z buffer with X-gal), 30 DEG C of incubations, found that normal yeast reporter becomes blue in 6-8h, and the not change in 12h of yeast reporter of sudden change, be still white.This illustrates that transcription factor GmNF-YB8 can be combined with CCAAT cis-acting elements, and has mobilizing function, have activated Pmin promotor, impels reporter gene to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of GmNF-YB8.
Embodiment 4, GmNF-YB8 improve the detection of the salt tolerance of plant
One, the structure of recombinant expression vector PAHCPSK-GmNF-YB8
1, the clone of GmNF-YB8 gene
According to the primers of GmNF-YB8 gene to (GmNF-YB8-PAHCPSK-F and GmNF-YB8-PAHCPSK-R), prime end is introduced Spe I enzyme respectively and is cut recognition site, is template PCR amplifications GmNF-YB8 gene with the cDNA of Glycine soybean (Glycine max L.) rich No. 8 of kind iron.
GmNF-YB8-PAHCPSK-F：
5'-CTAG
aCTAGTaTGGCGGACTCGGACAACGAC-3'(dashed part is the recognition site sequence of Spe I, and sequence is thereafter first 21 of sequence 2);
GmNF-YB8-PAHCPSK-R：
5'-CTAG
aCTAGTcTACCTGGGCCTACCGGCATTGGATC-3'(dashed part is the recognition site sequence of Spe I, and sequence is thereafter the reverse complementary sequence of latter 26 of sequence 2).
Pcr amplification product carries out 1.2% agarose gel electrophoresis, adopts Agarose Gel DNA Purification KitVer.2.0(TaKaRa company, Code No.:DV807A) reclaim the band of purifying about 587bp.
2, the structure of recombinant expression vector PAHCPSK-GmNF-YB8
1. cut with restriction enzyme Spe I enzyme the PCR primer that step 1 reclaims purifying, reclaim digestion products;
2. with restriction enzyme Spe I enzyme cut PAHC-PSK carrier (reference: high eastern Yao .2010. wheat ear germinating resistance is correlated with the clone of Vp-1B and AIP2 gene and functional analysis. [Ph.D. Dissertation]. Jilin University), reclaim carrier framework;
3. step digestion products is 1. connected with step carrier framework 2.;
4. by the electroporated TOP10 bacterial strain of step connection product 3. (purchased from Beijing Tian Gen company), 37 DEG C of incubated overnight, picking positive colony checks order; Sequencing result is shown, inserts the positive recombinant plasmid called after PAHCPSK-GmNF-YB8(plasmid map of the DNA fragmentation shown in sequence 2 of sequence table at the Spe I restriction enzyme site place forward of PAHC-PSK as shown in Figure 2).The promotor starting GmNF-YB8 genetic transcription in recombinant expression vector PAHCPSK-GmNF-YB8 is CaMV35S promotor.
Two, the acquisition of GmNF-YB8 gene plant is turned
1, with recombinant expression vector PAHCPSK-GmNF-YB8 transformation Agrobacterium C58C1(Beijing Baeyer enlightening biotech company that step one builds), obtain recombinational agrobacterium.Concrete operations are as follows:
Get the competent cell of 20 μ l Agrobacterium C58C1, the recombinant expression vector PAHCPSK-GmNF-YB8 adding 1 μ l step one structure mixes, electroporated, add 1ml YEB liquid nutrient medium 28 DEG C/180rpm renewal cultivation 3h, get 200 μ l and be coated with YEB resistant panel (Kan50mg/L, Rif50mg/L, Gen50mg/L) 28 DEG C cultivate 2 ~ 3d.Afterwards with the primer pair that GmNF-YB8-PAHCPSK-F and GmNF-YB8-PAHCPSK-R forms, carry out bacterium liquid PCR and identify.Show that the Agrobacterium positive colony containing GmNF-YB8 gene (size is about the object band of 587bp) carries out sequencing analysis by through bacterium liquid PCR qualification.The Agrobacterium called after PAHCPSK-GmNF-YB8/C58C1 proceeding to PAHCPSK-GmNF-YB8 is shown by through order-checking.The Agrobacterium proceeding to PAHC-PSK empty carrier is set simultaneously, by its called after PAHC-PSK/C58C1.
2, two kinds of recombinational agrobacterium step 1 obtained are inoculated in LB(containing 50mg/ml Rifampin, 100mg/ml kantlex, 50mg/ml gentamicin) in liquid nutrient medium, 28 DEG C, 3000rpm cultivates about 30 hours.
3, the bacterium liquid of step 2 is gone to new LB(containing 50mg/ml Rifampin, 100mg/ml kantlex, 50mg/ml gentamicin) in, 28 DEG C, 300rpm cultivates about 14 hours (bacterium liquid OD600 reaches 1.5-3.0).
4, collecting thalline, 4 DEG C, the centrifugal 10min of 4000g, being about 0.8-1.0 with being diluted to OD600 containing 10% sucrose MS liquid nutrient medium (silwet77 containing 0.02% (v/v)).
5, by Arabidopis thaliana (Columbia ecotype Col-0, SALK company) whole strain tips upside down in the container of the bacterium liquid filling step 4 together with flowerpot, make flower soak about 50s, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth, after 24h, open plastic cloth, upright placing flowerpot, carry out normal illumination cultivation, results T
1for seed, kantlex screening (concentration is 50 μ g/L kantlex) positive plant.T
2t is shown in representative
1the seed produced for selfing and the plant grown up to by it, T
3t is shown in representative
2the seed produced for selfing and the plant grown up to by it.By T
3carry out cDNA level qualification (all following F and R of primer pair of cDNA level qualification, expection band is 369bp) for plant, what cDNA level identified part sample the results are shown in Figure 3.Positive plant (DNA and cDNA level detection is positive plant) the called after PAHCPSK-GmNF-YB8/Col-0 containing GmNF-YB8 gene will be shown through qualification.
F:5'-TAGTATGGCGGACTCGGACAACGAC-3'(5-25 position is first 21 of sequence 2);
R:5'-CTAGACTAGTCTACCTGGGCCTACCGGCATTGGATC-3'(11-36 position is the reverse complementary sequence of latter 26 of sequence 2);
Three, the acquisition of empty vector control plant is turned
With recombinational agrobacterium PAHCPSK/C58C1 arabidopsis thaliana transformation Col-0, obtain the adjoining tree proceeding to PAHC-PSK empty carrier, called after PAHC-PSK/Col-0.The same step 2 of concrete method for transformation.
Four, the Salt-Tolerance Identification of GmNF-YB8 gene plant is turned
The T of the positive (DNA level and the qualification of cDNA level are the positive) will be accredited as respectively through step 2
3for transfer-gen plant PAHCPSK-GmNF-YB8/Col-0(TL), T
3in generation, turns empty vector control plant PAHC-PSK/Col-0(CK) and Arabidopis thaliana Col-0(WT) (each 60 strains) carry out salt tolerance detection.Arrange and repeat experiment for three times, results averaged.
By the sprouting seedling of 15 days of normal growth, water the NaCl aqueous solution of 200mM, (about 2 weeks), then rehydration one week until WT lines Col-0 wilts, observe phenotype, take pictures and add up survival rate.
As shown in Figure 4 and Table 1, turn GmNF-YB8 gene plant PAHCPSK-GmNF-YB8/Col-0 has 90.3% survival and energy normal growth to result; And the survival rate of wildtype Arabidopsis thaliana Col-0 is only 28.8%, and growth conditions is not good enough, and the phenotype turning empty vector control plant PAHC-PSK/Col-0 is consistent with Arabidopis thaliana Col-0, and survival rate and Arabidopis thaliana Col-0 do not have significant difference.
Table 1T
3for the survival rate statistics of transgenic Arabidopsis plants Salt-Tolerance Identification
| Repeat 1 | Repeat 2 | Repeat 3 | On average | |
| TL | 88.60% | 90.00% | 92.30% | 90.30% |
| CK | 27.70% | 26.50% | 28.60% | 27.60% |
| WT | 28.90% | 27.60% | 29.90% | 28.80% |
Claims (10)
1. protein, the protein be made up of the aminoacid sequence shown in sequence in sequence table 1.
2. the nucleic acid molecule of protein described in coding claim 1.
3. nucleic acid molecule according to claim 2, is characterized in that: described nucleic acid molecule is the gene of protein described in coding claim 1, and described gene is the DNA molecular shown in sequence in sequence table 2.
4. the recombinant vectors containing nucleic acid molecule described in Claims 2 or 3.
5. the expression cassette containing nucleic acid molecule described in Claims 2 or 3.
6. the recombinant bacterium containing nucleic acid molecule described in Claims 2 or 3.
7. recombinant vectors according to claim 4, is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector; The promotor starting described genetic transcription in described recombinant expression vector is following a1) or a2):
A1) 35S promoter;
A2) Pmin promotor.
8. protein described in claim 1, or the application of nucleic acid molecule described in Claims 2 or 3 in regulation and control arabidopsis thaliana salt-tolerance.
9. cultivate the method for salt tolerant transgenic arabidopsis, comprise the steps:, by the channel genes object Arabidopis thaliana of protein described in coding claim 1, to obtain the transgenic arabidopsis that salt tolerance is higher compared with described object Arabidopis thaliana.
10. protein according to claim 1 is as the application in transcription factor.
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| CN102234318A (en) * | 2010-04-27 | 2011-11-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein TaTPRPK1, encoding gene thereof, and application thereof |
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| CN101805401A (en) * | 2010-04-27 | 2010-08-18 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein TaHSP90-1 and coding gene and application thereof |
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