CN103319580A - Plant stress tolerance-associated protein TaNHSF1, coding genes thereof and applications - Google Patents
Plant stress tolerance-associated protein TaNHSF1, coding genes thereof and applications Download PDFInfo
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Abstract
The invention discloses a plant stress tolerance-associated protein TaNHSF1, coding genes thereof and applications. The protein is characterized in that: (1) the protein is composed of an amino acid sequence shown as a sequence 1 in a sequence table; and (b) the protein is a at least one plant stress tolerance- associated protein, which is derived from the protein in (a), and of which the amino acid sequence of the sequence 1 in the sequence table is substituted and/or deleted and/or added by one or more amino acid residues, wherein the plant stress tolerance comprises drought tolerance and heat tolerance. Proved in an experiment, compared a T3 transgenic plant obtained by converting Arabidopis thaliana by a recombination expression vector pBI121-TaHSF1 of a DNA molecule shown in a sequence 2 of a sequence table, with a wild type and trans-empty carrier plant under same conditions, a survival rate in drought tolerance experiments can be raised from 21.3% to 75%; and a survival rate in heat tolerance experiments can be raised from 20% to 90%. The TaNHSF1 protein and coding genes thereof provided by the invention has important meanings for raising stress tolerance.
Description
Technical field
The present invention relates to relevant albumen and encoding gene and the application of a kind of contrary property in the biological technical field, particularly a kind of plant stress tolerance correlative protein TaHSF1 and encoding gene and application, this protein TaHSF1 derives from wheat, has the ability that raising plant drought and high temperature resistance are coerced.
Background technology
The environment stresses such as high temperature are the obstruction factors that affects Growth of Wheat.Therefore, the understanding wheat is replied and signal transduction mechanism adverse environmental factor, improves the resistance of wheat breed, becomes one of vital task of wheat genetic research and wheat breed 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, to improve plant to the adaptive faculty of adverse circumstance.
Plant is being coerced lower can the various defence of generation the response, and wherein, the synthetic obvious increase of the heat shock protein that heat shock factor (Heat-shock factor, HSF) causes (Heat shock protein, HSP) has caused people's concern.Heat shock response (Heat shock response; HSR) be a kind of aversion response that vegetable cell or organ produce when running into extraneous thermal stimulus; it is movable to be that normal protein synthesis produces a kind of cell physiological of HSP when being obstructed, and its expression is regulated and control by HSF.Discovered in recent years HSF plays an important role in the expression regulation of Heat Resistance of Plant gene, and it is positioned at the upstream of coding HSP gene, has certain conservative dna sequence dna and specific transcription factor binding site point (Heat-shock element, HSE).When plant is subject to extraneous thermal stimulus, HSF can with the HSE specific binding, activate the expression of HSP gene, cause the in vivo run-up of HSP as molecular chaperones, thereby help other associated protein again folding, stable, assembling and intracellular distribution degraded, improve plant materials to the tolerance of high temperature.
HSF is called again Features of The Heat Shock Transcription Factor, to be combined under heat stress, oxidative stress and the virus infection on the heat shock element of HSP gene, attract other transcription factors to be combined on the HSP gene and form transcription complex, cause the accumulation with HSP of transcribing of HSP gene.HSF is a kind of protein in essence, has widely homology, all has HSF in the eukaryotes such as fungi, fruit bat, chicken, the mankind.The research of HSF at first from animal, has been widely used in human and animal's disease research.In the organic evolution process, poor because of the motility of plant, in order to conform, evolve out and the different and complicated and diversified HSF type of animal body.According to the comparison of DNA in conjunction with territory research and the zone of convergency, the middle HSF family of plant exists A, B, C three class members.In the plant that all have been studied, all there is polytype HSF, it at most, is respectively 55 and 54 kind in soybean and corn, have 25 kinds in Arabidopis thaliana, has 18 kinds in tobacco, is no less than 14 kinds in wheat.
HSF be a kind of high conservative and be prevalent in prokaryotic organism and eukaryotic cells in heat shock factor, for biology reply poor environment is coerced the nucleotide sequence of avoiding huge damage with various infringements.As a kind of heat shock factor, HSF is non-stress situation to be that abundant kind is arranged in the cell, and the diversity of the function of In The Research of Heat Stress Transcription Factors In Plants Hsfs family group composition and species specificity are to adapt to quick and various and extreme environment.
HSF mainly comprises following important structural domain: conservative N end DNA is in conjunction with territory (DNA-binding domain, DBD), oligomerization territory (Oligomerization domain, OD), nuclear localization signal (Nuclear localization signal, NLS), seven peptide duplicate domains (the C-terminal heptad repeats of C end, HR-C) and activation domain (Activator domain, CTAD).The oligomerization territory is comprised of two 7 hydrophobic peptide repeat region A and B (HR-A/B), and the insertion sequence between these two zones is alterable height, can be used as a foundation of HSF subfamily classification.Thus, Features of The Heat Shock Transcription Factor can be divided into A, B, C three classes.There is not category-A HSF mainly to be responsible for the regulation and control that heat shock gene is expressed between HsfB class HR-A and the HR-B; The transcriptional activation activity that does not have heat-inducible although category-B HSF has dna binding activity may play a role jointly with category-A HSF.Different types of HSF member can exist in cell simultaneously, but its immunogenicity is different, shows that they may there are differences on function.
The molecular weight of plant HSF usually between 31.2 to 57.5kD at present, the functional study of plant HSF is mainly concentrated on HsfA1a, A2 and this three class of B1.HSF molecular species and the size separated in the different organisms are had nothing in common with each other, and such as the HSF that separates in from yeast, fruit bat, human body, its molecular weight is respectively: 150,110,80 * 103; But its structure is very similar, jointly has a very conservative core area domain dna calmodulin binding domain CaM (DNAbinding domain) and trimerization zone (Trimerization domain).Under stress situation, HSF exists with the monomeric form of non-activity.When cell was in stress situation, intracellular environment changed, and the activity of having removed HSF suppresses, and promoted that HSF is changed to tripolymer by monomer.By trimerization zone combination, concrete site is 3 seven amino acid tumor-necrosis factor glycoproteinss in trimerization zone between the HSF.Stress not the time, these 3 sequences form stable coiled coil structure HSF is inner, to keep the monomer whose state; Stress the time under not clear machining function, the coiled coil structure is opened, the seven amino acid tumor-necrosis factor glycoproteins interactions of adjacent HSF form intermolecular coiled coil, under this structure helped, per 3 HSF monomers were combined together to form a tripolymer.
HSF is prokaryotic organism and the eukaryote camber is conservative and ubiquity, the research discovery, and HSF is positioned in nucleus and the part vacuole.
HSF plays vital effect in the growth course of cell, comprise Growth of Cells, apoptosis, canceration, stress reaction, plant immunization, growth even evolution.Recently, research on Arabidopis thaliana, tobacco and paddy rice finds, HSF plant stress-resistance, to hot environment reply and disease resistance in play an important role.The plant that Mishra obtains with the reticent Arabidopis thaliana HSF of HsfA iRNA, thermotolerance obviously descends.Chen Xiaojun etc. (2006) adopt information biology and comparative genomics method combination technology to be cloned into the GmHSFA1 factor gene from the soybean gene group, can obviously improve the thermotolerance of soybean.Above-mentioned studies show that, plant HSF plays an important role in plant adaptation adverse circumstance environmental process.Therefore, the high thermal resistance that the resistance that the further investigation function of plant HSF under adverse environmental factor, the regulation and control that align the conduction of confirming to know the induction heat stress signal, anti contravariance related gene promote the expression of anti contravariance related gene and improve crop has important directive significance and actual application value and improves crop has important directive significance and actual application value.
Summary of the invention
The purpose of this invention is to provide a kind of plant stress tolerance correlative protein TaHSF1 and encoding gene and application.
Provided by the present invention and plant stress tolerance correlative protein derives from wheat, and name is called TaHSF1, this protein be following a) or b) protein:
A) protein that is formed by the aminoacid sequence shown in the sequence table sequence 1;
B) with the aminoacid sequence of sequence table sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with following at least a plant stress tolerance by (a) derivative protein: drought tolerance and thermotolerance.
Aminoacid sequence shown in the sequence table sequence 1 is comprised of 236 amino-acid residues.
Albumen in above-mentioned in order to make (a) is convenient to purifying, can connect label as shown in table 1 at N-terminal or the C-terminal of the protein that is comprised of the aminoacid sequence shown in the sequence table sequence 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 |
| |
8 | DYKDDDDK |
| Strep- |
8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the albumen synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence table sequence 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of code for said proteins also belongs to protection scope of the present invention.
The gene of code for said proteins can be following 1) or 2) or 3) gene:
1) its nucleotide sequence is the dna molecular shown in the sequence table sequence 2;
2) with 1) dna sequence dna that limits has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or the dna molecular that has at least 99% homology and code for said proteins;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of code for said proteins.
Described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 2 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.5 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 65 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of lower hybridization, then use 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The recombinant vectors, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain described gene also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (such as kermes synthetic enzyme Nos gene), plant gene (storing protein gene such as soybean) 3 ' end to transcribe such as the Agrobacterium crown-gall nodule all has similar functions.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor (such as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (such as the promotor of seed specific expression) before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene (gus gene of luminophor as adding the coding that in plant, to express, luciferase genes etc.), antibiotic marker gene (as is given nptII gene to kantlex and associated antibiotic resistance, give the bar gene to weedicide phosphinothricin resistance, give the hph gene to the microbiotic hygromycin resistance, with the dhfr gene of giving the methatrexate resistance, give the EPSPS gene to the glyphosate resistance) or anti-chemical reagent marker gene etc. (such as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.
The described recombinant vectors that contains described gene specifically can be pGBKT7-TaHSF1,16318hGFP-TaHSF1 or pBI121-TaHSF1;
Described pGBKT7-TaHSF1 is for obtaining described gene insertion vector pGBKT7 to express the recombinant expression vector of described albumen; Specifically can be EcoR I and the BamH I enzyme of the dna molecular insertion vector pGBKT7 shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site;
Described 16318hGFP-TaHSF1 is for obtaining described gene insertion vector hGFP to express the recombinant expression vector of described albumen; Specifically can be Sal I and the BamH I enzyme of the dna molecular insertion vector hGFP shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site;
Described pBI121-TaHSF1 is for obtaining described gene insertion vector pBI121 to express the recombinant expression vector of described albumen; Specifically can be BamH I and the Sac I enzyme of the dna molecular insertion vector pBI121 shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site.
Another object of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant of the present invention is that described gene is imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.
In aforesaid method, described purpose plant can be monocotyledons or dicotyledons.
In aforesaid method, described dicotyledons specifically can be Arabidopis thaliana.
In aforesaid method, described resistance of reverse is drought tolerance and thermotolerance.
In aforesaid method, described thermotolerance is anti-42 ℃ high temperature.
The present invention protects described albumen as the application in the transcription factor.
Experiment showed, the T that the recombinant expression vector pBI121-TaHSF1 arabidopsis thaliana transformation with dna molecular shown in the sequence table sequence 2 obtains
3For transfer-gen plant, with wild-type under the same terms with turn the empty carrier plant and compare, (be that the seedling that normal growth was sprouted 15 days is not watered, until the wild-type plant is when withered, one week of rehydration again) survival rate brings up to 75% from 21.3% in drought tolerance experiment; In the thermotolerance experiment, (seedling in 4 weeks of normal growth is carried out 42 ℃ of pyroprocessing 3h, cultivated for 1 week through normal afterwards) survival rate and bring up to 90% from 20%.TaHSF1 albumen provided by the present invention and encoding gene thereof are significant aspect the raising stress resistance of plant, for the artificial expression of controlling anti contravariance related gene provides the foundation, will in cultivating high resistance to cold and diseases such as strong drought tolerance and strong thermotolerance plant variety, play a significant role.
Description of drawings
Fig. 1 is the structural models of TaHSF1 albumen.Wherein, DBD is that N holds DNA in conjunction with the territory, and HR-A/B is the oligomerization territory that two 7 hydrophobic peptide repeat region A and B form, and NLS is nuclear localization sequence, and AHA is the activation structure territory of acid C end.
Fig. 2 is the space-filling model of TaHSF1 albumen.
Fig. 3 is that real-time fluorescence quantitative PCR is analyzed the expression map that Different stress is processed lower TaHSF1 gene.Wherein, A-F respectively is arid, salt marsh, damages to plants caused by sudden drop in temperature, the Stress treatment of dormin (ABA), Plant hormones regulators,gibberellins (GA) and high temperature, and X-coordinate is the time of coercing, and ordinate zou is the relative expression quantity of TaHSF1 gene.
Fig. 4 is the structural representation of recombinant vectors 16318hGFP-TaHSF1.
Fig. 5 is TaHSF1 Subcellular Localization result.Wherein, A is the contrast of hGFP empty carrier, and B is that TaHSF1 is positioned in the nucleus.
Fig. 6 is the structural representation of recombinant vectors pBI121-TaHSF1.
Fig. 7 is that the PCR of transgenic arabidopsis plant identifies electrophorogram.Wherein, A identifies electrophorogram for turning pBI121-TaHSF1 Arabidopis thaliana plant PCR, swimming lane M is DL2000DNA Marker, be followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, swimming lane 1 is plasmid pBI121-TaHSF1 positive control, and swimming lane 2-6 is the plant that turns pBI121-TaHSF1 Arabidopis thaliana strain 1; Swimming lane 7-11 is the plant that turns pBI121-TaHSF1 Arabidopis thaliana strain 2; Swimming lane 12-15 is the plant that turns pBI121-TaHSF1 Arabidopis thaliana strain 3; Swimming lane 16 is Arabidopis thaliana Col-0 negative control; Swimming lane 17 is H
2The O blank; B is that the PCR that turns the empty carrier adjoining tree identifies electrophorogram, swimming lane M is DL2000DNA Marker, be followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, swimming lane 1 is plasmid pBI121 positive control, and swimming lane 2-15 is for turning empty carrier Arabidopis thaliana plant; Swimming lane 16 is Arabidopis thaliana Col-0 negative control; Swimming lane 17 is H
2The O blank.
Fig. 8 is Arabidopis thaliana plant and the phenotype of wild-type Arabidopis thaliana Col-0 under drought condition that turns pBI121-TaHSF1.Wherein, 1 is the wild-type Arabidopis thaliana, and 2-4 is the Arabidopis thaliana plant that turns pBI121-TaHSF1.
Fig. 9 is Arabidopis thaliana plant and the phenotype of wild-type Arabidopis thaliana Col-0 under hot conditions that turns pBI121-TaHSF1.Wherein, 1 is the wild-type Arabidopis thaliana, and 2-4 is the Arabidopis thaliana plant that turns pBI121-TaHSF1.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The information of used Xiao Bai wheat and carrier hGFP is as follows among the following embodiment:
Xiao Bai wheat (Triticum aestivum cv.Xiaobaimai) and carrier hGFP: Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public; Reference Zhao-Shi Xu, Lan-Qin Xia, Ming Chen, et al.Isolation and molecular characterization of the Triticum aestivum L.ethylene-responsive factor1 (TaERF1) that increases multiple stress tolerance.Plant Mol.Biol.2007,65:719-732.
One, total RNA extracts and the cDNA library structure
The sowing of Xiao Bai wheat on the seedbed, about 20～24 ℃ of growth 10d, is taken out from soil, clean with distilled water flushing, be placed on arid processing 2h on the filter paper, get blade and put into immediately liquid nitrogen ,-80 ℃ save backup.
Adopt Trizol method (TIANGEN Biotech (Beijing) Co., Ltd.) to extract the total RNA of wheat leaf blade, the first chain cDNA is (TaKaRa) synthetic with ThermoScript II XL (AMV).Adopt the synthetic ds cDNA of SMART method.The PCR product carries out 1.0% agarose gel electrophoresis and detects.
Two, the acquisition of TaHSF1 albumen and encoding gene thereof
Take paddy rice heat shock factor AK066316 as known array, at wheat database comparison wheat similar sequences.Through DNAMAN software analysis and design primer 5 '-ATGGGAAGCGAGTGCAAG-3 ' and 5 '-TCAGTAGAACACCTGTCCCAGA-3 ', carry out pcr amplification take the cDNA of Xiao Bai wheat as template, reclaim the band about 750bp, be inserted on the pEASY-Blunt behind the purifying, transforming intestinal bacteria TOP10 (TIANGEN Biotech (Beijing) Co., Ltd.) checks order, the result shows that the length of this recovery fragment is 711bp, and its nucleotide sequence is shown in sequence table sequence 2.Be TaHSF1 with the unnamed gene shown in the sequence table sequence 2, proteins encoded TaHSF1, the aminoacid sequence of this albumen are shown in sequence table sequence 1, and structural models as depicted in figs. 1 and 2.
The activation characteristic of embodiment 2, TaHSF1
One, the structure of bait carrier
1, the acquisition of TaHSF1 gene
The Xiao Bai wheat cDNA that obtains take step 1 is as template, and TaHSF1-EI and TaHSF1-BI are primer (the primer end is introduced respectively the recognition sequence of EcoR I and BamH I), carry out pcr amplification, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect.Adopt the PCR product about Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa, DV807A) recovery purifying 750bp.Sequencing result shows, this PCR product contains the 711bp nucleotide sequence shown in the ordered list sequence 2.
TaHSF1-EI：5’-GGAATTC
ATGGGAAGCGAGTGCAAG-3’；
TaHSF1-BI：5’-CGGATCC
TCAGTAGAACACCTGTCCCAGA-3’。
2, the structure of recombinant expression vector
1. cut the PCR product that step 1 reclaims purifying with restriction enzyme EcoR I and BamH I enzyme, reclaim enzyme and cut product;
2. cut expression vector pGBKT7 (Clontech) with restriction enzyme EcoR I and BamH I enzyme, reclaim carrier framework;
3. step enzyme is 1. cut the carrier framework connection that product is connected with step;
4. step connection product electric shock is 3. transformed JM109 bacterial strain (Clontech company), 37 ℃ of incubated overnight, the picking positive colony checks order; Sequencing result shows, has obtained recombinant plasmid pGBKT7-TaHSF1, and this plasmid is for to have inserted the dna fragmentation shown in the sequence table sequence 2 between the EcoR of carrier pGBKT7 I and BamH I restriction enzyme site.
Two, the activation characteristic of TaHSF1
Bait carrier pGBKT7-TaHSF1, empty carrier pGBKT7 and negative control are changed among the yeast strain AH109 (TIANGEN Biotech (Beijing) Co., Ltd.) jointly with pGADT7 (TaKaRa) respectively, be coated onto respectively the upper cultivation of substratum SD/-Trp/-Leu (STL) and substratum SD/-Trp/-Leu/-His/-Ade (STL-HA) 3-5 days.The result shows, negative control (empty AH109) and the AH109 that contains pGBKT7 do not have growth on substratum STL and STL-HA, and the AH109 that contains pGBKT7-TaHSF1 grows at substratum STL and STL-HA, illustrates that pGBKT7-TaHSF1 has the self activation activity.
One, Stress treatment
Be 10 days Xiao Bai wheat seedling with the seedling age of water planting, carry out following processing:
1, arid is processed: Xiao Bai wheat seedling is taken out the moisture that blots on the root, place on the dry filter paper, arid is cultivated after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours and is taken out material, uses liquid nitrogen flash freezer, and-80 ℃ save backup.
2, salt marsh is processed: with Xiao Bai wheat seedling place 2% by NaCl and Na
2SO
4(NaCl and Na in the sodium salt solution that forms
2SO
4Mass percent be 3: 2) in, illumination cultivation is taken out respectively material after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, use liquid nitrogen flash freezer ,-80 ℃ save backup.
3, damage to plants caused by sudden drop in temperature processing: Xiao Bai wheat seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ save backup.
4, ABA processes: Xiao Bai wheat seedling is placed the ABA solution of 100 μ M, illumination cultivation takes out respectively and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ save backup.
5, GA processes: Xiao Bai wheat seedling is placed the GA solution of 50 μ M, illumination cultivation takes out respectively and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ save backup.
6, pyroprocessing: Xiao Bai wheat seedling is placed under 42 ℃, and illumination cultivation takes out respectively and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ save backup.
The processing of contrast: directly get the Xiao Bai wheat seedling-80 ℃ frozen in contrast (0 hour) without any processing.
Two, the separation of mRNA
Use respectively QuikprepMicro mRNA PurificationKit (Pharmacia) to carry out the separation of mRNA the material that step 1 obtains.
Three, reverse transcription is cDNA
Adopting R103-Quant_Reverse_Transcriptase (TIANGEN Biotech (Beijing) Co., Ltd.) is cDNA with the mRNA reverse transcription of step 2 purifying.
Four, real-time fluorescence quantitative PCR
With after 50 times of the cDNA dilutions as the template of real-time fluorescence quantitative PCR.According to the sequence of TaHSF1 gene, at its variable region design special primer HSF1F and HSF1R.Take actin as reference gene, primer is actin-2F and actin-2R.
Real-time fluorescence quantitative PCR is at ABI
Carry out on the 7000 real-time fluorescence quantitative PCR instrument, 3 repetitions are established in a parallel test.Utilize the method for Livak KJ and Schmittgen TD (2001) report, namely 2
-Δ Δ CTCalculate relative expression quantity.
ΔΔC
T＝(C
T.Target-C
T.Actin)
Timex-(C
T.Target-C
T.Actin)
Time0
Time x represents random time point, Time
0The target gene of expression 1 times of amount after actin proofreaies and correct is expressed.
HSF1F:5 '-ACTTCCTCCTCCCCTCCTACT-3 ' (the 164-184 position of corresponding sequence table sequence 2);
HSF1R:5 '-ATCAGCGGCAGCAGCTT-3 ' (the 298-314 position of corresponding sequence table sequence 2);
actin-2F：5’-CTCCCTCACAACAACCGC-3’；
actin-2R：5’-TACCAGGAACTTCCATACCAAC-3’。
Each is coerced TaHSF1 and hormone shows response, and the result as shown in Figure 3.
One, the structure of recombinant expression vector
1, the acquisition of TaHSF1 gene
Sequence (being the sequence of sequence table sequence 2) design primer TaHSF1-SI and TaHSF1-BI according to the TaHSF1 gene, the primer end is introduced respectively the recognition sequence of Sal I and BamH I, take the cDNA of Xiao Bai wheat as template, pcr amplification TaHSF1 gene carries out 1.2% agarose gel electrophoresis with pcr amplification product and detects.
Adopt the PCR product about Agarose Gel DNA PurificationKit Ver.2.0 (TaKaRa, DV807A) recovery purifying 750bp.
TaHSF1-SI:5 '-GCGTCGAC
ATGGGAAGCGAGTGCAAG-3 ' (in the line part corresponding sequence table sequence 2 the 1st to the 18th);
TaHSF1-BI:5 '-CGGATCC
GTAGAACACCTGTCCCAGA-3 ' (in the line part corresponding sequence table sequence 2 the 708th to the 690th).
2, the structure of recombinant expression vector
1. cut the PCR product that step 1 reclaims purifying with restriction enzyme Sal I and BamH I enzyme, reclaim enzyme and cut product;
2. cut expression vector hGFP with restriction enzyme Sal I and BamH I enzyme, reclaim carrier framework;
3. step enzyme is 1. cut the carrier framework connection that product is connected with step;
4. step connection product electric shock is 3. transformed Top10,37 ℃ of incubated overnight, the picking positive colony checks order; Sequencing result shows, has obtained recombinant plasmid 16318hGFP-TaHSF1, and this plasmid is for to have inserted the dna fragmentation shown in the sequence table sequence 2 between the Sal of carrier hGFP I and BamH I restriction enzyme site, and its structural representation as shown in Figure 4.
Two, material is prepared
It is the bronze suspension 6 μ l (50mg/ml) of 1.0 μ M that the recombinant plasmid 16318hGFP-TaHSF1 that 3 μ g step 1 obtain adds diameter, 0.1M spermidine (spermidine) 4 μ l, 2.5M CaCl
26 μ l, with bronze, DNA, spermidine and the calcium chloride mixing that vibrates respectively first, then mix vibration mixing 3min after, leave standstill 15min on ice.The centrifugal 10s of 12000rpm (rotating speed reach 12000rpm after 10s) abandons supernatant.Add 140 μ l dehydrated alcohols, the centrifugal 10s of (breaing up bronze) 12000rpm after the thick vibration collects the bronze precipitation.20 μ l dehydrated alcohols suspend and precipitate, the some film.
Three, particle gun bombardment receptor material
1. select the split film (this experiment 1100psi) of certain pressure, with the bombardment film, soak 1～2h in 70% alcohol, taking-up is dried;
2. metal baffle is sterilized at spirit lamp with alcohol-pickled, the Bechtop ultraviolet sterilization of particle gun;
3. get the above-mentioned bronze for preparing of 20 μ l-plasmid complex body, evenly coat on the mid-way of bombardment film, be not applied on the whole film, size is consistent with the pore diameter range on the carrier fixed ring, dries, and then is fixed on the carrier fixed ring;
4. above-mentioned carrier fixed ring is installed on the launching device;
5. can split film and be installed to gas acceleration tube lower end;
6. the onion epidermis culture dish is put into vacuum chamber, take off the culture dish lid;
7. vacuumize pointer to 26In/Hg;
8. put helium in the gas acceleration tube, until pressure reaches in the time of can splitting the pressure that film can bear in the pipe, can split film and break;
9. gas is flushed on the bombardment film, and carrier moves downward, and blocked by metal baffle, and following bronze-plasmid complex body sees through the mesh directive target cell of metal baffle;
10. will bombard good onion epidermis cell and put into 25 ℃ of incubators, under laser confocal microscope, observe after secretly cultivating 16～24h.
Four, onion epidermis cell microscopy
Particle gun is bombarded, secretly cultivates 16-24h onion epidermis compressing tablet afterwards, then at laser scanning co-focusing microscope (Bio-Rad MicroRadiance) (Laser scanning confocal microscopy, LSMC) observe GFP (green fluorescent protein) fluorescence, the line scanning of going forward side by side is taken pictures (as shown in Figure 5).The result shows that TaHSF1 is positioned in the nucleus.The working parameter of LSCM is: Ex=488nm, Em=525 ± 15nm, Power=10%, Zoom7, medium sweep, Frame512 * 512.Software is TIME-COURSE and PHOTOSHOP5.0.
One, the structure of recombinant expression vector
1, TaHSF1 gene cloning
To (TaHSF-1F and TaHSF1-1R), the primer end introduces respectively BamH I and Sac I enzyme is cut recognition site according to the primers of TaHSF1 gene, take the cDNA of Xiao Bai wheat as template, and pcr amplification TaHSF1.
TaHSF1-1-121F:5 '-CCGGATCC
ATGGGAAGCGAGTGCAAG-3 ' (in the line part corresponding sequence table sequence 2 the 1st to the 18th);
TaHSF1-1-121R:5 '-CGAGC
TCTCAGTAGAACACCTGTCCCAGA-3 ' (the 711st to the 690th sequence reverse complemental in line part and the sequence table sequence 2)
Pcr amplification product carries out 1.2% agarose gel electrophoresis, adopts the band about Agarose Gel DNA PurificationKitVer.2.0 (TaKaRa, DV807A) recovery purifying 750bp.
2, the structure of recombinant expression vector
1. cut the PCR product that step 1 reclaims purifying with restriction enzyme BamH I and Sac I enzyme, reclaim enzyme and cut product;
2. cut pBI121 (Clontech company) with restriction enzyme BamH I and Sac I enzyme, reclaim carrier framework;
3. step enzyme is 1. cut the carrier framework connection that product is connected with step;
4. step connection product electric shock is 3. transformed TOP10 bacterial strain (TIANGEN Biotech (Beijing) Co., Ltd.), 37 ℃ of incubated overnight, the picking positive colony checks order; Sequencing result shows, obtained recombinant plasmid pBI121-TaHSF1 and (inserted sequence table sequence 2 from the dna fragmentation shown in the 1st-711 Nucleotide of 5 ' end between the BamHI of pBI121 and Sac I restriction enzyme site; As shown in Figure 6).
Two, the acquisition of transgenic arabidopsis
1, transforms Agrobacterium C58 (Beijing Baeyer enlightening biotech company) with recombinant plasmid pBI121-TaHSF1, obtain the Agrobacterium of recombinating.
2, the Agrobacterium of will recombinating is inoculated in the YEP liquid nutrient medium, and 28 ℃, 3000rpm were cultivated approximately 30 hours;
3, the bacterium liquid with step 2 goes in the YEP liquid nutrient medium (containing 50 μ g/ml kantlex and 50 μ g/ml Rifampins), and 28 ℃, 300rpm are cultivated approximately 14 hours (bacterium liquid OD600 reaches 1.5-3.0);
4, collect thalline, 4 ℃, the centrifugal 10min of 4000g are diluted to OD600 with 10% sucrose (containing 0.02%silwet) and are about 0.8-1.0;
5, with Arabidopis thaliana (the environmental Col-0 of Colombia, SALK company) whole strain tips upside down in the container of the bacterium liquid that fills step 4 with flowerpot, flower is soaked about 50s, soak complete after, take out flowerpot, be sidelong in pallet, cover black plastic cloth, open plastic cloth after 24 hours, upright placing flowerpot carries out normal illumination cultivation, T1 is for seed for results, kantlex screening (concentration is the 50mg/L kantlex) resistant plant, the yellow of most of non-transgenic plant is die, and transfer-gen plant can normal growth.
To T
1Carry out the PCR evaluation of dna level for resistant plant, the qualification result of part sample as shown in Figure 7.Screening obtains positive transfer-gen plant (turning the Arabidopis thaliana plant of TaHSF1 gene) from resistant plant.
The primer that PCR identifies is as follows:
HSF1-PF:5 '-CCAGCTCAACACCTACGGT-3 ' (the 216-234 position of corresponding sequence table sequence 2);
HSF1-PR:5 '-CCTCCTTCTTGGCGACCA-3 ' (complementary with the 553-536 position of sequence table sequence 2);
The size of its predicted segment is 338bp.
T
2T is shown in representative
1The seed that produces for selfing reaches the plant that is grown up to by it, T
3T is shown in representative
2The seed that produces for selfing reaches the plant that is grown up to by it.
Three, turn the acquisition of empty carrier control plant
Transform Agrobacterium with plasmid pBI121, obtain the Agrobacterium of recombinating, with this restructuring Agrobacterium-mediated Transformation Arabidopis thaliana, obtain turning the empty carrier adjoining tree, the same step 2 of method.
The PCR primers designed that turns the empty carrier adjoining tree is primers F: 5 '-TTCAGAAAGAATGCTAACCC-3 ' and primer R:5 '-GAGGCATCTTCAACGATGGCCTT-3 ', and the prediction product is 600bp, and qualification result is shown in Fig. 7 B.
Four, the drought tolerance of transgenic plant is identified
To identify the T that is positive through PCR respectively
3Identify the T that is positive for transgenic arabidopsis plant (Transgenic Line, TL), through PCR
3In generation, turn empty carrier adjoining tree (CK) and Arabidopis thaliana Col-0 (WT) (each 60 strain) carries out the drought tolerance evaluation.Repeated experiments is set three times, results averaged.
15 days seedling of normal growth is carried out arid process, do not water continuously until Arabidopis thaliana Col-0 is withered (approximately 20 days), then rehydration is 7 days, observes phenotype, takes pictures and adds up survival rate, result such as table 2 and shown in Figure 8.
The survival rate statistics that table 2. transgenic arabidopsis plant drought tolerance is identified
| |
|
|
On average | |
| TL | 74％ | 78％ | 73％ | 75％ |
| CK | 23％ | 24％ | 22％ | 23 |
| WT | ||||
| 20％ | 23％ | 21％ | 21.3％ |
The result shows: Arabidopis thaliana Col-0 is most of dead, and surviving rate only is 21.3%, but the survival of 75% transfer-gen plant and energy normal growth.The phenotype that turns the empty carrier adjoining tree is consistent with Arabidopis thaliana Col-0, and survival rate and Arabidopis thaliana Col-0 do not have significant difference.
Five, the Heat tolerance identification of transgenic plant
To identify the T that is positive through PCR respectively
3Identify the T that is positive for transgenic arabidopsis plant (Transgenic Line, TL), through PCR
3In generation, turn empty carrier adjoining tree (CK) and Arabidopis thaliana Col-0 (WT) (each 60 strain) carries out the high thermal resistance evaluation.Repeated experiments is set three times, results averaged.
The seedling in 4 weeks of normal growth is carried out 42 ℃ of pyroprocessing 3h, cultivated for 1 week through normal afterwards, observe phenotype, take pictures and add up survival rate, result such as table 3 and shown in Figure 9.
The survival rate statistics that table 3. transgenic arabidopsis plant high thermal resistance is identified
| |
|
|
On average | |
| TL | 89％ | 93％ | 88％ | 90％ |
| CK | 24％ | 22％ | 20％ | 22％ |
| WT | 23％ | 19％ | 18％ | 20％ |
The result shows: Arabidopis thaliana Col-0 is most of dead, and surviving rate only is 20%, and the transfer-gen plant surviving rate is up to 90%, and the energy normal growth.The phenotype that turns the empty carrier adjoining tree is consistent with Arabidopis thaliana Col-0, and survival rate and Arabidopis thaliana Col-0 do not have significant difference.
Claims (10)
1. protein, be following a) or b) protein:
A) protein that is formed by the aminoacid sequence shown in the sequence table sequence 1;
B) with the aminoacid sequence of sequence table sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with following at least a plant stress tolerance by (a) derivative protein: drought tolerance and thermotolerance.
2. the encoding gene of the described protein of claim 1.
3. gene according to claim 2, it is characterized in that: the encoding gene of described protein is following 1) or 2) or 3) gene:
1) its nucleotide sequence is the dna molecular shown in the sequence table sequence 2;
2) with 1) dna sequence dna that limits has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least 99% homology and a described protein DNA molecule of coding claim 1;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization and the described protein DNA molecule of coding claim 1 that limit.
4. the recombinant vectors, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain claim 2 or 3 described genes.
5. recombinant vectors according to claim 4, it is characterized in that: described recombinant vectors is pGBKT7-TaHSF1,16318hGFP-TaHSF1 or pBI121-TaHSF1;
Described pGBKT7-TaHSF1 is for obtaining claim 2 or 3 described gene insertion vector pGBKT7 to express the recombinant expression vector of the described albumen of claim 1; Be specially EcoR I and the BamH I enzyme of the dna molecular insertion vector pGBKT7 shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site;
Described 16318hGFP-TaHSF1 is for obtaining claim 2 or 3 described gene insertion vector hGFP to express the recombinant expression vector of the described albumen of claim 1; Be specially Sal I and the BamH I enzyme of the dna molecular insertion vector 163hGFP shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site;
Described pBI121-TaHSF1 is for obtaining claim 2 or 3 described gene insertion vector pBI121 to express the recombinant expression vector of the described albumen of claim 1; Be specially BamH I and the Sac I enzyme of the dna molecular insertion vector pBI121 shown in the sequence table sequence 2 are cut the recombinant expression vector that obtains between the recognition site.
6. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.
7. method according to claim 6, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.
8. method according to claim 7, it is characterized in that: described dicotyledons is Arabidopis thaliana.
9. arbitrary described method according to claim 6-8 is characterized in that: resistance of reverse is drought tolerance and thermotolerance.
10. the described albumen of claim 1 is as the application in the transcription factor.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928317A (en) * | 2015-06-09 | 2015-09-23 | 昆明理工大学 | Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector |
| CN106754974A (en) * | 2017-03-08 | 2017-05-31 | 南京农业大学 | Festuca Arundinacea " Regenerate " FaHsfC1b genes and its application |
| CN110746498A (en) * | 2018-07-04 | 2020-02-04 | 中国农业科学院作物科学研究所 | Application of plant stress tolerance-related protein TaANTL7A.2 in regulation and control of plant stress tolerance |
| CN111620934A (en) * | 2019-02-26 | 2020-09-04 | 中国科学院遗传与发育生物学研究所 | Application of protein GmHSFB2b in regulation and control of accumulation of plant flavonoids |
| CN112251462A (en) * | 2020-10-26 | 2021-01-22 | 南京农业大学 | Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period |
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-
2012
- 2012-03-19 CN CN2012100727182A patent/CN103319580A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION: "《Risk Assessment and Risk Management Plan for DIR 100》", 30 June 2010 * |
| DANDAN QIN ET AL.: "Heat stress-responsive transcriptome analysis in heat susceptible and tolerant wheat (Triticum aestivum L.) by using Wheat Genome Array", 《BMC GENOMICS》 * |
| TAKASHI MATSUMOTO ET AL.: "Comprehensive Sequence Analysis of 24,783 Barley Full-Length cDNAs Derived from 12 Clone Libraries", 《PLANT PHYSIOL》 * |
| 秦丹丹等: "植物热激转录因子及其与耐热性关系的研究进展", 《麦类作物学》 * |
| 陈晓军等: "GmHSFA1基因克隆及其过量表达提高转基因大豆的耐热性", 《遗传》 * |
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| CN104928317B (en) * | 2015-06-09 | 2017-12-22 | 昆明理工大学 | The plant expression vector of tobacco gibberellin synthesis transcription regulatory factor gene and its application |
| CN106754974A (en) * | 2017-03-08 | 2017-05-31 | 南京农业大学 | Festuca Arundinacea " Regenerate " FaHsfC1b genes and its application |
| CN110746498A (en) * | 2018-07-04 | 2020-02-04 | 中国农业科学院作物科学研究所 | Application of plant stress tolerance-related protein TaANTL7A.2 in regulation and control of plant stress tolerance |
| CN111620934A (en) * | 2019-02-26 | 2020-09-04 | 中国科学院遗传与发育生物学研究所 | Application of protein GmHSFB2b in regulation and control of accumulation of plant flavonoids |
| CN111620934B (en) * | 2019-02-26 | 2022-03-08 | 中国科学院遗传与发育生物学研究所 | Application of protein GmHSFB2b in regulation and control of accumulation of plant flavonoids |
| CN112410347A (en) * | 2019-08-21 | 2021-02-26 | 中国农业大学 | Corn ZmHsf21 gene and application thereof |
| CN112251462A (en) * | 2020-10-26 | 2021-01-22 | 南京农业大学 | Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period |
| CN113281521A (en) * | 2021-05-19 | 2021-08-20 | 河南大学 | Gateway binary plasmid vector for rapidly identifying plant stress particle-associated protein, and construction method and application thereof |
| CN113281521B (en) * | 2021-05-19 | 2022-07-22 | 河南大学 | Gateway binary plasmid vector for rapidly identifying plant stress particle associated protein, and construction method and application thereof |
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