CN102140134A - Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof - Google Patents
Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof Download PDFInfo
- Publication number
- CN102140134A CN102140134A CN2011100253641A CN201110025364A CN102140134A CN 102140134 A CN102140134 A CN 102140134A CN 2011100253641 A CN2011100253641 A CN 2011100253641A CN 201110025364 A CN201110025364 A CN 201110025364A CN 102140134 A CN102140134 A CN 102140134A
- Authority
- CN
- China
- Prior art keywords
- sequence
- seq
- protein
- gene
- dre
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a dehydration responsive element (DRE)-binding protein and an encoding gene thereof. The DRE-binding protein is a protein having an amino acid residue sequence shown as SEQ ID No:2 in a sequence table or a protein, which is obtained by substituting, deleting or adding one or more amino acid residues on the amino acid residue sequence shown as SEQ ID No:2, has the same activity as that of the amino acid residue sequence shown as SEQ ID No:2 and is derived from the SEQ ID No:2. An experiment proves that the ZmDBP2 disclosed by the invention is expressed under the induction of drought, high salt, low temperature and ABA (Abscisic Acid), and the transcription expression of a gene containing a DRE/CRT (Calreticulin) cis element (core sequence: CCGAC) can be regulated and controlled specifically. By adopting the ZmDBP2, a basis is laid for manual control over the expression of a stress resistance and stress tolerance related gene. The ZmDBP2 is about to play an important role in cultivating plant breeds with enhanced stress resistance and stress tolerance.
Description
Technical field
The present invention relates in the plant a kind of with coerce relevant transcription factor and encoding gene thereof, particularly a kind of dehydration response element conjugated protein and encoding gene thereof.
Background technology
Environment stresses such as arid, high salt and high temperature are the obstruction factors that influences corn growth, growth.Therefore, the understanding corn is replied and signal transduction mechanism adverse environmental factor, improves the resistance of corn variety, becomes one of vital task of maize genetic research and corn variety improvement.
Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Clear and definite plant is to the reaction mechanism of adverse circumstance, will provide the science argument for adversity gene engineering research and application.At present, the plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, explores and improves plant growth characteristics with biotechnology, its objective is and improves the adaptive faculty of plant to adverse circumstance.
Under the adverse environmental factor of environment-stress such as arid, high salt and high temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.Many genes are expressed by stress-inducing, the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.With coerce relevant gene product and can be divided into two big classes: the product of first kind genes encoding comprises that ionophorous protein, aquaporin, the osmoregulation 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 second genoid coding comprises the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, transcription factor plays an important role in the gene expression regulation that plant stress is replied.
Transcription factor is also referred to as trans-acting factor, is can be conjugated protein with the DNA of cis-acting elements generation specific effect in the eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress and transcribe.The DNA land of transcription factor has determined it and cis-acting elements bonded specificity, and transcription regulatory region has determined it that genetic expression is risen to activate or restraining effect.In addition, himself activity also is subjected to appraising and deciding the influence of effects such as position and oligomerization.
At present known in plant with coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (element responsive to ethylene is conjugated protein, the ethylene responsive element binding protein) transcription factor family with AP2 structural domain, bZIP (basic region/leucine zipper motif transcription factors) the class transcription factor that contains alkalescence zone and leucine zipper, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, the MYC family and MYB family of containing alkaline helix-loop-helix (bHLH) and leucine zipper with tryptophane bunch (Trp cluster).These five transcription factor families, except that WRKY family not the water of involved in plant coerce the reaction, other four families all participate in regulating the environment stress reaction of plant to arid, high salt etc.Wherein, AP2/EREBP class transcription factor extensively exists in higher plant, it is the peculiar class transcription factor of plant, in recent years, report is all arranged in Arabidopis thaliana, tobacco, corn, paddy rice, soybean and rape, and this shows AP2/EREBP class transcription factor ubiquity and have vital role in higher plant.
DREB (dehydration response element conjugated protein, DRE-binding protein) class transcription factor is a member in the EREBP-like subfamily in the AP2 family.They do not have significant homogeny DREB and EREBP class transcription factor on aminoacid sequence, but all contain one section very conservative DNA calmodulin binding domain CaM (EREBP/AP2 structural domain) of being made up of 58 left and right sides amino acid.The protein three dimensional analysis shows that 3 beta sheets are contained in this zone, plays a crucial role to discerning all kinds of cis-acting elements.Wherein be arranged in the difference of two amino-acid residues of the 14th, 19 of second beta sheet, determine the specific combination of this class transcription factor and different cis-acting elements.DREB class transcription factor the 14th amino acids is Xie Ansuan (V14), and the 19th amino acids is L-glutamic acid (E19), and wherein the 19th amino acid is conservative, for example the 19th amino acids of the OsDREB1 transcription factor of paddy rice be exactly Xie Ansuan (
Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, SekiM, Shinozaki K, Yamaguchi-Shinozaki K,2003).Aspect the decision DNA bonded specificity, the effect of V14 is obviously than E19 important (Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K and Yamaguchi-ShinozakiK, 2002) in the DREB associated protein; And ERF class transcription factor the 14th amino acids is a glycine, and the 19th is aspartic acid, thereby DREB specific combination DRE/CRT cis element, ERF specific combination GCC-box.The C-petiolarea of AP2/EREBP structural domain also comprises 1 core sequence of being made up of 18 amino-acid residues, and this sequence forms amphiphatic alpha-helix, and this alpha-helix may participate in the interaction between other transcription factor and DNA.
At present, in many plants, all find the transcription factor of this EREBP/AP2 of containing structural domain, and transmit relevant (Liu Qiang, Zhao Nanming, Yamaguchi-Shinozaki K, Shinozaki K, 2000) with signal such as disease-resistant, degeneration-resistant respectively.Liu Qiang etc. think that a dreb gene can be regulated and control a plurality of and plant arid, high salt and low temperature patience function associated expression of gene (Liu Qiang, Zhao Nanming, Yamaguchi-Shinozaki K, Shinozaki K, 2000).Kasuga etc. studies confirm that, the DREB1A gene that imports to Arabidopis thaliana can promote the expression of gene rd29, rd17, kin1, cor6.6, cor15a and the erd10 relevant with environment stress patience simultaneously, the resistance of transfer-gen plant strengthens (KasugaM greatly, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K., 1999).Equally, the low temperature tolerance ability of the transfer-gen plant of low temperature patience transcription factor CBF1 be significantly increased (Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF., 1998).Because the stress tolerance of plant is the complex character by the polygene regulation and control, rely on to import the comprehensive raising that the individual feature protein gene is difficult to realize stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of a plurality of functional genes, thereby strengthen the resistance of plant, become the engineered research focus of plant stress-resistance.
According to the number that contains the DNA land, the AP2/EREBP transcription factor is divided into AP2 (APETALA2) and the conjugated protein EREBP of element responsive to ethylene (ethylene-responsive element binding protein) and three major types of RAV.AP2 type transcription factor comprises AP2, the ANT of Arabidopis thaliana, the Glossy of corn, idsl etc.Such transcription factor contains two AP2/EREBP structural domains, regulates growing of cell, has found 14 AP2 type transcription factor genes in Arabidopis thaliana; EREBP type transcription factor only contains 1 AP2/EREBP structural domain, regulates plant to ground molecule responsing reactions such as hormone (ethene), cause of disease, low temperature, arid and high salt.In the EREBP type transcription factor, found many members such as tobacco EREBP1-4, tomato Pti4-6, Arabidopis thaliana RAV1-2, AtEBP, AtERF1-5, DREB1A-C (CBF1-3) and DREB2A-B, transmitted relevant with signals such as cell development, hormone, disease-resistant, low temperature and arid, high salt respectively.These EREBP type transcription factors can be further divided into again: EREBP (ethylene-responsive element binding protein, be ERF) subgroup, comprise tobacco EREBP1-4, tomato Pti4-6, Arabidopis thaliana AtEBP, AtERF1-5, this class transcription factor and the GCC-box specific combination that contains core sequence AGCCGCC, therefore, its DNA land is called the GCC-box again in conjunction with territory (GCC-box binding domain, GBD), the 2nd G wherein, the 5th G, the 7th C be proteic identification (the Hao D that plays an important role to ERF, Ohme-Takagi M, Sarai A, 1998).With nucleus magnetic resonance its three-D space structure be studies show that the GBD of AtERF1 combines with the major groove of its target sequence GCC-box by forming 3 reverse β-lamellas; The DREBP subgroup comprises Arabidopis thaliana DREB1A-C (CBF1-3) and DREB2A-B, and this class transcription factor is specific combination arid response element DRE/CRT under arid, high salt, low temperature, finds 124 DREBP type transcription factor genes in the arabidopsis gene group; RAV type transcription factor comprises Arabidopis thaliana RAV1, RAV2, contains two different DNA land-ERF/AP2 and B3, has found 6 RAV type transcription factor genes in Arabidopis thaliana.Also have the special transcription factor AL079349 of a class, it is all different with above transcription factor, constitutes a class by itself.
Recent findings EREBPs albumen has participated in the signal conduction and the gene expression regulation of arid, high salt and low temperature stress.People such as Mine have been separated to EREBP transcription factor CIP353 from the potato stem tuber of cryopreservation, be subjected to low temperature stress to induce strong expression (Mine T, Hiyoshi T, KasaokaK, Ohyama A, 2003), illustrate to have the gene expression regulation that EREBP albumen has participated in being subjected to low temperature stress.Park etc. utilize tomato to be material, obtained being subjected to the EREBP transcription factor Tsi gene of high salt, ethene or jasmonic abduction delivering, EMSA (Electrophoretic mobility shift assays) analysis of experiments is found, Tsi albumen can both be in conjunction with (Park JM with GCC-box and DRE/CRT cis element, Park CJ, Lee SB, Ham BK, Shin R, Paek KH, 2001), although the former binding ability greater than the latter, but illustrate that some EREBP albumen can activate the gene that is subjected to the osmotic stress abduction delivering.Under the normal growth condition, the overexpression of Tsi gene improved transfer-gen plant (35S::Tsi1) salt tolerance, strengthened disease resistance (Park JM, Park CJ, Lee SB, Ham BK, Shin R, Paek KH, 2001), illustrated that more than the Tsi gene may participate in biology and coerce and abiotic stress two bars pathways.By a class MAPK of high-salt stress activated signal transfer mode (comprising SIMKK and SIMK), to coerce signal and pass to EIN2 (in the CTR1 downstream of Ethylene Signal pipeline) (Guo HW and EckerJ, 2004), activate some EREBPs transcription factor at last, regulation and control osmotic stress Expression of Related Genes improves the salt tolerance of plant.Contain the GCC-box element for whether existing, and expression product participates in the gene of abiotic stress response directly, be still waiting to do further confirmation.
Comprehensive present result of study, the signal pipeline of plant under the environment stress condition has following six approach at least: the signal pipeline that (1) depends on ABA has three: induced by arid, high salt, activate MYB, MYC class transcription factor gene, regulation and control have the target gene of MYBR or MYCR cis-acting elements; Be subjected to arid, high salt, high temperature induction, activate ABF/AREB class transcription factor gene, regulation and control have the target gene of ABRE cis-acting elements; Induced by arid, high salt, activate CBF4, DREB1 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.(2) the signal pipeline that does not rely on ABA has three: induced by arid, high salt, activate DREB2 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements; Be subjected to low temperature induction, activate CBF1-3/DREB1A-C class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements; Induced by arid, high salt or ethene, activate ERF class transcription factor gene, regulation and control have the target gene of DRE/CRT or GCC cis-acting elements.
With the cardinal principle of the activation characteristic of yeast-one-hybrid system proof transcription factor as shown in Figure 3, DRE cis-acting elements and mutant DRE cis-acting elements are building up to basic promotor Pmin (minimal promoter) upstream of pHISi-1 carrier and pLacZi carrier respectively, and Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).After the expression vector YEP-GAP (not containing mobilizing function) of the goal gene that is connected with the encoding transcription factor is transformed into the yeast cell that is connected with DRE cis-acting elements and mutant DRE cis-acting elements respectively, if the reporter gene that is connected with in the yeast cell of mutant DRE cis-acting elements can not be expressed, and the reporter gene that is connected with in the yeast cell of specific DRE cis-acting elements can be expressed, illustrate that this transcription factor can combine with the DRE cis-acting elements, and has mobilizing function, activate the Pmin promotor, impelled reporter gene to express.Thereby interior binding specificity of the body that has proved the purpose transcription factor and mobilizing function.
The innovation and creation content
The purpose of this invention is to provide a kind of dehydration response element conjugated protein and encoding gene thereof.
Dehydration response element conjugated protein provided by the present invention, name is called ZmDBP2, derive from Zea corn (Zea mays L.), be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.
The protein that the amino acid residue sequence of sequence 2 is made up of 343 amino-acid residues in the sequence table is conservative AP2/EREBP structural domain from the 166th-223 amino acids residue sequence of aminoterminal.
The dehydration response element conjugated protein encoding gene, name is called ZmDBP2, derives from Zea corn (Zea mays L.), is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.
CDNA sequence in the sequence 1 is by 1501 based compositions, and the open reading frame of this gene is from the 155th-1186 bit base of 5 ' end, 343 amino acid of encoding altogether (sequence 2 in the sequence table).
Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention among the amplification ZmDBP2.
Experimental results show that ZmDBP2 of the present invention expresses under the inducing of arid, high salt, low temperature, ABA, and can special regulation and control contain DRE/CRT cis element (core sequence: gene transcription expression CCGAC).ZmDBP2 of the present invention will play an important role in cultivating resistance and the plant breeding of resistance of reverse enhanced for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.
Description of drawings
Fig. 1 is the homology comparison result of ZmDBP2 and corn ZmDREB aminoacid sequence
Fig. 2 is expressed by stress-inducing for ZmDBP2 real-time fluorescence quantitative PCR collection of illustrative plates
Fig. 3 is the principle schematic with binding specificity in the body of yeast-one-hybrid system proof transcription factor and activation characteristic
Fig. 4 is real-time fluorescence quantitative PCR reaction conditions figure
Fig. 5 is for obtaining the pcr amplification information drawing of ZmDBP2 gene coding amino acid complete sequence partly
Embodiment
The clone of embodiment 1, ZmDBP2
One, the separation of mRNA
About 20 days two leaf phase of corn seedling of growth in the sandy soil is carried out arid handle 5 hours (treatment processs: the taking-up that seedling is careful, note not hindering root, blot with the moisture of clean thieving paper blade and root, seedling is placed on the clean thieving paper again, In Shade 5 hours), use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.
Adopt Trizol method (TianGen) to extract the total RNA of wheat leaf blade, the first chain cDNA is synthetic with ThermoScript II XL (AMV) (TaKaRa).Adopt SMART method (BD) to synthesize ds cDNA: get the LD-PCR amplification ds cDNA that 2 μ l cDNA carry out 100 μ l systems, cycle number is 24, and the extension time is 6min.Get 5 μ lPCR products after amplification finishes and carry out sepharose (1.0%) electrophoresis detection.
Two, the acquisition of ZmDBP2 full length gene sequence
Method by 5 ' RACE and 3 ' RACE obtains a new dehydration response element conjugated protein gene nucleotide series and a corresponding amino acid sequence from corn, the result obtains having the ZmDBP2 of the nucleotide sequence shown in the sequence 1 in the sequence table, its open reading frame is from the 155th-1186 bit base of 5 ' end, 343 amino acid (sequence 2 in the sequence table) of encoding altogether are conservative AP2/EREBP structural domain from the 166th-223 amino acids residue sequence of aminoterminal.The homologous sequence comparison result shows that ZmDBP2 is the highest with corn ZmDBF1 (AAM80486) homology of having reported, has only 34.47% homology (as shown in Figure 1), illustrates that ZmDBP2 is a newfound corn gene.Among Fig. 1, black surround is represented consistent amino acid moiety.
Embodiment 2, real-time fluorescence quantitative PCR are analyzed the expression characterization of ZmDBP2
One, seedling age is 20 days a corn seedling, carries out following processing:
(1) arid is handled: the corn seedling of water planting is taken out the moisture that blots on the root, place on the exsiccant filter paper, arid is cultivated after 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 hours and is taken out material, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.
(2) salt marsh is handled: with corn seedling place 2% by NaCl and Na
2SO
4(NaCl and Na in the sodium salt solution of forming
2SO
4Mass percent be 3: 2) in, illumination cultivation is taken out material respectively after 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 hours, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.
(3) damage to plants caused by sudden drop in temperature processing: corn seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer after 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 hours, and-80 ℃ of preservations are standby.
(4) ABA handles: corn seedling is placed the ABA solution of 200 μ M, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 hours, and-80 ℃ of preservations are standby.
(5) Dui Zhao processing: the corn seedling of directly getting without any processing-80 is ℃ in contrast frozen.
Two, the separation of mRNA
After the processing of 20 days seedling of growth,, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.Adopt Quikprep Micro mRNA PurificationKit (Pharmacia) to carry out the separation of mRNA.
Three. reverse transcription is cDNA
Adopting R103-Quant_Reverse_Transcriptase (TIANGEN) is cDNA. with the mRNA reverse transcription of purifying
Reaction system:
10×RT?buffer 2μl
dNTP?mix(2.5mM?each) 4μl
Oligo-dT primer (10) 2 μ l
RNase?inhibitor(10U/) 1μl
Quant?reverse?transcriptase 1μl
RNase?free?H2O 5μl
Template ribonucleic acid 5 μ l
Totally be 20
Hatch 60min for 37 ℃.
Four, real-time fluorescence quantitative PCR
According to known ZmDBP2 sequence, at its variable region design special primer.ZmDBP2RTF：5’-GCCCGATGGCATTTTAGACG-3’；ZmDBP2RTR：5’-AACCAGGAGATTAGCACGCA-3’
With actin is internal control gene.
Reaction system:
cDNA 1μl
2.5×RealMasterMIX 8μl
20×SYBR 1μl
ddH
2O 10μl
Reaction conditions: Fig. 4
Each is coerced ZmDBP2 and hormone shows response.Fig. 2
The activation characteristic of embodiment 3, ZmDBP2
One, ZmDBP2 is gene constructed to expression vector YEP-GAP
1, obtains the complete sequence of ZmDBP2 gene coding amino acid part
According to the sequences Design primer of the ZmDBP2 gene of having cloned, the primer end adds EcoRI and XhoI restriction enzyme site respectively, and pcr amplification obtains the complete sequence of coded amino acid part, and program and system are as follows:
Primer sequence:
ZmDBP2-EI：5’-GGGGAATTCGCTCCATCCAAGCTGTAGTCCT-3’
ZmDBP2-XI：5’-GGGCTCGAGGCTCATGACAGGATGGAATCC-3’
Reaction system (50 μ l):
Template (60ng/ul) 0.5 μ l
dNTP(10mM) 1μl
Primer (25 μ M) 1 μ l
10×buffer 5μl
ddh
2O 42.1μl
Taq(5U/μl) 0.4μl
Amplification condition (PTC-200): Fig. 5
Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, the scanning of ultraviolet gel imaging instrument, whether the position of observing about 1.1Kb has a bright band.
Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim the purified pcr product recovery of the probe of cDNA library screening (with three).
2, ZmDBP2 is gene constructed to expression vector YEP-GAP
PCR product and expression vector YEP-GAP (Liu Q, Kasuga M, Sakuma Y, Abe H with purifying in the step 1, Miura S, Yamaguchi-Shinozaki K, Shinozaki K., 1998), with EcoRI (Takara) and XhoI (Takara) respectively enzyme cut 4-6hr, reaction system is as follows:
10 * damping fluid H, 5 μ l
EcoR?I(12U/μl) 2μl
XhoI(12U/μl) 2μl
PCR product/YEP-GAP 20 μ l
ddH
2O 21μl
Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim purifying enzyme and cut the product recovery of the probe of cDNA library screening (with three).
The enzyme of purifying is cut the PCR product cut carrier YEP-GAP with enzyme and be connected 4-8hr, reaction system is as follows:
10×Ligase?buffer 1μl
Enzyme is cut PCR product 4 μ l
Enzyme is cut carrier YEP-GAP 4 μ l
T4DNA?Ligase 1μl
Get 0.5 μ l and connect product, electric shock transforms the JM109 bacterial strain, 37 ℃ of incubated overnight, and the picking positive colony, whether the sequencing analysis sequence is correct, obtains containing the recombinant expression vector YEP-GAP-ZmDBP2 of ZmDBP2 gene.
Two, the checking of binding specificity and activation characteristic in the body of ZmDBP2
1, the structure of yeast reporter
Fragment 5 '-GAATTC-DRE-DRE-DRE-DRE-GTCGAC-3 ' (core sequence of DRE: TACCGACAT) be building up to the Pmin of pHis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company) respectively that will contain 4 DRE elements
HIS3Promotor and pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P
CYCIThe promotor upstream obtains recombinant vectors pHis-1-DRE and pLacZi-DRE respectively, respectively pHis-1-DRE and pLacZi-DRE carrier is cut into wire with Xho I and Nco I restriction endonuclease.Earlier wire pHis-1-DRE carrier is transformed in the yeast cell (YM4271 strain system, MATCHMAKER One-Hybrid System, Clontech company), acquisition can be on the SD/His substratum yeast transformant (Yeast transformant) of normal growth.Be host cell then, continue to transform the pLacZi-DRE carriers that contain 4 repetition DRE elements with this yeast transformant.The SD/His that lacks Histidine and uridylic so at the same time
-/ Ura
-On the substratum, select to obtain to contain the normal dual yeast reporter of pHis-1-DRE and pLacZi-DRE; The core sequence CCGAC of 4 DRE elements is mutated into TTTTT (MDRE), i.e. 5 '-GAATTC-MDRE-MDRE-MDRE-MDRE-GTCGAC-3 ', by normal dual yeast reporter construction process, make up a dual yeast reporter of mutant that contains 4 MDRE boxes again.
2, PEG/LiAc method transformed yeast and interpretation of result
(1) inoculation yeast bacterial strain (YM4271 strain system, MATCHMAKER One-Hybrid System, Clontech company) in 1ml YPD liquid nutrient medium, concuss 2 minutes, disperse behind the agglomerate suspension to be gone in the triangular flask that contains 50ml YPD liquid nutrient medium, 30 ℃/250rpm shakes and spends the night, and surveys OD600=1.7-1.8 and (counts about 4 * 10
7Individual/mL);
(2) get 30ml step (1) overnight culture and receive in the fresh YPD substratum of 300ml, 30 ℃/250rpm cultivates, and 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, suspends with the freshly prepared 1 * TE/LiAc solution of 1.5ml, and the vibration mixing is standby;
(4) taking out 0.1ml yeast competence transforms, add following solution successively: 0.1 μ g expression vector YEP-GAP-ZmDBP2,0.1mg ssDNA (salmon sperm dna, Sigma), 0.6mlPEG/LiAc vibrated 30 ℃/200rpm shaking culture 30 minutes at a high speed 1 minute;
(5) add 70ul DMSO (sigma#D8779), be inverted mixing gently, 42 ℃ of heat shocks 30 minutes, vibration gently therebetween, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g;
(6) supernatant is abandoned in suction, adds 0.5ml 1 * TE buffer suspension cell;
(7) dip in transfering loop and get suspension, containing 0 respectively, the SD/His/Ura of 15mmol/L 3-AT
-/ Trp
-Setting-out is cultivated on the selective medium.
(8) the normal dual yeast reporter of half dull and stereotyped culturing step 1 structure, the dual yeast reporter of mutant that second half culturing step 1 makes up is so that do check analysis.
(9) be placed upside down in incubator, cultivated 3-4 days for 30 ℃.
(10) SD/His that found that at 0mmol/L3-AT
-/ Ura
-/ Trp
-Culture medium flat plate on the yeast reporter of normal yeast reporter and sudden change growth is all arranged, but the diameter of the yeast reporter of sudden change is obviously little; And normal yeast reporter can normal growth on the culture medium flat plate of the SD/His/Ura/Trp of 15mmol/L 3-AT, but the yeast reporter of sudden change is not restrained not growth.
3, galactosidase activity detects
(1) from the SD/His of 0mmol/L 3-AT
-/ Ura
-/ Trp
-Culture medium flat plate on the yeast reporter bacterium colony of the normal yeast reporter of picking and sudden change respectively.Go in the YPD liquid nutrient medium,, wait to grow to the logarithmic growth later stage, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm in 30 ℃ of shaking culture;
(2) abandon supernatant, liquid in the control main places liquid nitrogen quick-frozen 10min with centrifuge tube, taking-up is melted it naturally, adds 50ul Z/X-gal solution, 30 ℃ of incubations, found that normal yeast reporter becomes blue in 6-8h, and the yeast reporter of sudden change changes not in 12h, still is white.Illustrate that transcription factor ZmDBP2 can combine with the DRE cis-acting elements, and have mobilizing function, activated Pmin promotor (or Pmin
HIS3Promotor or P
CYCIPromotor?), impel reporter gene to express.Thereby interior binding specificity of the body that has proved ZmDBP2 and mobilizing function.
Three, medicament preparation:
(1) YPD liquid nutrient medium
Microbial culture yeast extract (Bacto-Yeast Extract) 10g/L
Microbial culture tryptone (Bacto-Peptone) 20g/L
Regulate pH to 5.8,60 ℃ of Glucose of adding 40% are later on reduced in 121 ℃/15min sterilization, and making its final concentration is 20g/L.
(2) SD/His/Ura/Trp selective medium
Do not contain 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 ℃/15min sterilization adds 40%Glucose after reducing to 60 ℃, and making its final concentration is 20g/L.
(3) auxotroph mixture (Drop-out mix): (10X)
L-Isoleucine (Isoleucine) 300mg/L
L-Valine (Xie Ansuan) 1500mg/L
L-Adenine (VITAMIN B4) 200mg/L
L-Arginine (arginine) 200mg/L
L-Histidine Hcl monohydrate (Histidine) 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
(4)1×PEG/LiAc：
50％(w/v)PEG3350 8ml
10×TE?buffer 1ml
10×LiAc 1ml
(5)10×TE?Buffer：
100mM?Tris-Hcl
10mM?EDTA，pH＝7.5
121 ℃ of autoclavings, room temperature preservation.
(6)1×TE/LiAc：
10×TE?buffer 1ml
10×LiAc 1ml
ddH
2O 8ml
(7)Z?Buffer：
Na
2HPO
4·7H
2O 16.1g/L
NaH
2PO
4·H
2O 5.5g/L
KCl 0.75g/L
MgSO
4·7H
2O 0.246g/L
Regulate pH to 7.0,121 ℃/15min sterilization, 4 ℃ of preservations.
(8) X-gal storage liquid (X-gal Stock Solution):
Use N, N-dimethyl-formamide (DMF) dissolves X-gal, and making its final concentration is 20mg/ml ,-20 ℃ of storages.
(9) contain the Z buffer damping fluid 100ml (Z buffer with X-gal) of X-gal, note matching while using:
Z?buffer 98ml
Beta-mercaptoethanol (the 0.27ml of β-mercaptoethanol)
X-gal storage liquid (X-gal stock solution) 1.67ml
Claims (9)
1. dehydration response element conjugated protein, be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.
2. protein according to claim 1 is characterized in that: described protein has SEQ ID № in the sequence table: 2 amino acid residue sequence.
3. protein according to claim 1 and 2 is characterized in that: described SEQ ID №: 2 be conservative AP2/EREBP structural domain from the 166th-2235 amino acids residue sequence of aminoterminal.
4. dehydration response element conjugated protein encoding gene is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.
5. gene according to claim 4 is characterized in that: described gene has the dna sequence dna of sequence 1 in the sequence table.
6. gene according to claim 5 is characterized in that: the open reading frame of described gene is from the 115th-1186 bit base of 5 ' end.
7. contain claim 4,5 or 6 described expression carrier.
8. the clone that contains claim 4,5 or 6 described genes.
9. the arbitrary segmental primer in amplification claim 4, the 5 or 6 described genes is right.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100253641A CN102140134A (en) | 2011-01-24 | 2011-01-24 | Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100253641A CN102140134A (en) | 2011-01-24 | 2011-01-24 | Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102140134A true CN102140134A (en) | 2011-08-03 |
Family
ID=44407946
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100253641A Pending CN102140134A (en) | 2011-01-24 | 2011-01-24 | Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102140134A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015007241A1 (en) * | 2013-07-18 | 2015-01-22 | Institute Of Botany, The Chinese Academy Of Science | Molecular marker |
| CN110129338A (en) * | 2019-06-14 | 2019-08-16 | 吉林省农业科学院 | Maize Transcription Factor ZmEREB160 gene and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009094527A2 (en) * | 2008-01-23 | 2009-07-30 | Pioneer Hi-Bred International, Inc. | Transcriptional activators involved in abiotic stress tolerance |
-
2011
- 2011-01-24 CN CN2011100253641A patent/CN102140134A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009094527A2 (en) * | 2008-01-23 | 2009-07-30 | Pioneer Hi-Bred International, Inc. | Transcriptional activators involved in abiotic stress tolerance |
Non-Patent Citations (2)
| Title |
|---|
| WANG C.T.等: "GenBank：ACO72993.1", 《GENBANK》 * |
| WANG C.T.等: "GenBank：FJ805750.1", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015007241A1 (en) * | 2013-07-18 | 2015-01-22 | Institute Of Botany, The Chinese Academy Of Science | Molecular marker |
| CN110129338A (en) * | 2019-06-14 | 2019-08-16 | 吉林省农业科学院 | Maize Transcription Factor ZmEREB160 gene and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guy | Molecular responses of plants to cold shock and cold acclimation | |
| Li et al. | Soybean DRE-binding transcription factors that are responsive to abiotic stresses | |
| Chen et al. | Over-expression of OsDREB genes lead to enhanced drought tolerance in rice | |
| Cominelli et al. | Transgenic crops coping with water scarcity | |
| CN102234320B (en) | Plant stress-tolerant associated protein TaDREB4B and encoding gene and application thereof | |
| Kurowska et al. | Drought stress and re-watering affect the abundance of TIP aquaporin transcripts in barley | |
| Park et al. | Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas | |
| Huang et al. | Cloning and functional analysis of the novel gene GhDBP3 encoding a DRE-binding transcription factor from Gossypium hirsutum | |
| Mawlong et al. | Isolation and characterization of an AP2/ERF-type drought stress inducible transcription factor encoding gene from rice | |
| CN103509094B (en) | Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application | |
| CN103509093B (en) | Plant stress tolerance-related protein GmNF-YC14 as well as encoding gene and application thereof | |
| CN103319583A (en) | Plant stress tolerance-associated protein TaNF-YB 1, coding genes thereof and applications | |
| Charfeddine et al. | Potato dehydrins present high intrinsic disorder and are differentially expressed under ABA and abiotic stresses | |
| CN102234322B (en) | Protein GmNF-YA1 related with stress tolerance of plants, and coding gene and application thereof | |
| CN102140134A (en) | Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof | |
| CN102140135A (en) | Corn dehydration responsive element bonding protein ZmDBP3 and coding gene thereof | |
| Wang et al. | GmLEA2-1, a late embryogenesis abundant protein gene isolated from soybean (Glycine max (L.) Merr.), confers tolerance to abiotic stress | |
| CN103509096B (en) | Plant stress tolerance associated protein GmNF-YB6 as well as coding gene and application thereof | |
| CN104892738B (en) | Plant stress tolerance correlative protein GmNF-YC17 and its encoding gene and application | |
| CN102234321A (en) | Plant stress-tolerant associated protein GmNF-YB1 and encoding gene and application thereof | |
| CN102140136A (en) | Corn dehydration responsive element (DRE)-binding protein ZmDBP4 and encoding gene thereof | |
| Şahin-Çevik et al. | Two AP2 domain containing genes isolated from the cold-hardy Citrus relative Poncirus trifoliata are induced in response to cold | |
| CN100395338C (en) | Ethylene response component binding protein and its coding gene | |
| CN100395336C (en) | Dehydration response component binding protein and its coding gene | |
| CN100366634C (en) | Binding protein of response component of plant dehydration, and coding genes and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110803 |