CN103509094A - Plant stress tolerance-related protein GmNF-YC9 as well as encoding gene and application thereof - Google Patents
Plant stress tolerance-related protein GmNF-YC9 as well as encoding gene and application thereof Download PDFInfo
- Publication number
- CN103509094A CN103509094A CN201210214142.9A CN201210214142A CN103509094A CN 103509094 A CN103509094 A CN 103509094A CN 201210214142 A CN201210214142 A CN 201210214142A CN 103509094 A CN103509094 A CN 103509094A
- Authority
- CN
- China
- Prior art keywords
- sequence
- gene
- plant
- gmnf
- protein
- 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.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a plant stress tolerance-related protein GmNF-YC9 as well as an encoding gene and an application thereof. As shown in experiments, in a drought tolerance test, the survival rate of T3 generation of homozygous transgenic plants obtained by transforming a recombinant expression vector pBI121-GmNF-YC9 of a DAN molecule expressed by 34th to 897th nucleotide sequences in a sequence 2 in a sequence table into arabidopsis is 90.2%, and the survival rates of wild plants and transgenic empty vector plants are respectively 27.2% and 28.4%; and in a salt-tolerant germination rate test, the germination rate of the T3 generation of homozygous transgenic plants is 90.4%, and the germination rates of the wild plants and the transgenic empty vector plants are respectively 65.8% and 66.2%. The GmNF-YC9 protein and the encoding gene thereof provided by the invention have great significance for improving the stress resistance of plants.
Description
Technical field
The present invention relates to relevant albumen and encoding gene and the application of a kind of resistance of reverse in biological technical field, particularly a kind of plant stress tolerance correlative protein GmNF-YC9 and encoding gene and application, this protein G mNF-YC9 derives from soybean, has the ability that improves drought resistance in plants and salt tolerance.
Background technology
The environment stresses such as arid, high salt and low temperature are seriously restricting growth, the growth of soybean.Therefore, understanding soybean is replied and signal transduction mechanism adverse environmental factor, improves the resistance of soybean varieties, becomes one of vital task of soybean heredity research and breed improvement.
Under environment stress, in plant materials, can produce a series of responsing reactions, the variation that is accompanied by many Physiology and biochemistries and grows.Define the reaction mechanism of plant to adverse circumstance, will provide science argument 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, explores and improves plant growth characteristics by biotechnology, its objective is and improves the adaptive faculty of plant to adverse circumstance.
Under the adverse environmental factor of the environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment in molecule, cell and integral level, the injury the existence that to reduce to the full extent environment, cause.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 the expression of other genes involved or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.To coerce relevant gene product and can be divided into two large classes: the product of first kind genes encoding comprises that 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 the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, in the gene expression regulation that transcription factor is replied at plant stress, play an important role.
Transcription factor is 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 and transcribe.The DNA land of transcription factor has determined that specificity ，Er transcription regulatory region that it is combined with cis-acting elements has determined that it rises and activate or restraining effect genetic expression.In addition, himself activity is also subject to appraising and deciding the impact of the effects such as position and oligomerization.
At present known in plant to coerce relevant transcription factor and mainly contain: have AP2 structural domain AP2 (APETALA2)/(element responsive to ethylene is in conjunction with albumen for EREBP, ethylene responsive element binding protein) transcription factor family, the bZIP(basic region/leucine zipper motif transcription factors that contains alkaline region and leucine zipper) class transcription factor, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, CBF(CCAAT binding factor in conjunction with the main nuclear factor of CCAAT-box) class transcription factor, the MYC family of containing alkaline helix-loop-helix (bHLH) and leucine zipper and the MYB family with tryptophane bunch (Trp cluster).
NF-Y is that a class is in conjunction with the transcription factor of cis-acting elements CCAAT-box, special identification in conjunction with the promotor of many eukaryote composing types, inducibility and cell cycle dependent gene or the cis-acting elements CCAAT-box in enhanser, and then in the expression of these genes of transcriptional level control.The heterozygosis tripolymer that NF-Y is comprised of NF-YA, NF-YB and tri-different subunits of NF-YC.NF-YB albumen and NF-YC albumen, by the conservative territory of HFM each other, adopt connected head-to-tail mode to form heterodimer and make mutually platform, thereby attract NF-YA protein binding to form the activated heterotrimer nuclear factor of tool to this dimer platform.NF-Y is attached to the CCAAT box of target gene promoters part in conjunction with territory by the DNA on NF-YA subunit, carry out transcriptional activation or transcribe inhibit feature.The conservative territory of three subunits of NF-Y has respectively different protein structure domains, and wherein the conservative territory of NF-YA has DNA binding domains (DNA binding domain) and makes mutually structural domain (subunit interaction domain) with NF-YB/C heterodimer.The conservative territory of NF-YB and NF-YC albumen consists of the folding motif (Histone-fold motif) of histone.Wherein NF-YB is similar to the folding motif of H2B histone, and NF-YC is similar to the folding motif of H2A histone, and histone motif is comprised of three α spirals and two rings, is responsible for the dimeric formation of H2A/H2B.
Because the stress tolerance of plant is the complex character being regulated and controled by polygene, relies on and import the comprehensive raising that individual feature protein gene is difficult to realize stress resistance of plant.Therefore, utilize a crucial transcription factor to promote the expression of a plurality of functional genes, 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 plant stress tolerance correlative protein GmNF-YC9 and encoding gene and application.
Provided by the present invention and plant stress tolerance correlative protein, derives from soybean (Glycine max L.), and the nuclear factor protein name that is a kind of combination CCAAT-box is GmNF-YC9, this protein be following a) or b) protein:
A) protein being formed by the aminoacid sequence shown in sequence table sequence 1;
B) by 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 to following at least one plant stress tolerance by (a) derivative protein: drought tolerance and salt tolerance.
Aminoacid sequence shown in sequence table sequence 1 is comprised of 287 amino-acid residues, and the aminoacid sequence of the 8th to the 71st is the conservative folding motif of histone.
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 being comprised of the aminoacid sequence shown in sequence table sequence 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Albumen 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 albumen in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in 34-897 position in sequence table sequence 2, and/or carry out the missense mutation of one or several base pair, and/or hold the encoding sequence that connects the label shown in table 1 to obtain at its 5 ＇ end and/or 3 ＇.
The gene of code for said proteins also belongs to protection scope of the present invention.
The encoding gene of described protein is following 1) or 2) or 3) or 4) gene:
1) its nucleotide sequence is the DNA molecular shown in the 34th to the 897th nucleotide sequence in sequence table sequence 2;
2) its nucleotide sequence is the DNA molecular shown in sequence table sequence 2;
3) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a DNA molecular of 99% homology and code for said proteins;
4) under stringent condition with 1) or 2) or 3) 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 in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 2 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.5 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 65 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.
The recombinant vectors that contains described gene, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus also belong to protection scope of the present invention.
The recombinant expression vector that available existing plant expression vector construction contains described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.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 region of foreign gene, 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 transcribing as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean stores protein gene) 3 ' end all has similar functions.While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor (as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (as the promotor of seed specific expression), they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, 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 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 region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene (as is given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to weedicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene of giving methatrexate resistance, give the EPSPS gene to glyphosate resistance) or anti-chemical reagent marker gene etc. (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 YEP-GAP-GmNF-YC9 or pBI121-GmNF-YC9;
Described YEP-GAP-GmNF-YC9 can be the recombinant expression vector that described gene insertion vector YEP-GAP is obtained expressing described albumen; Specifically can be the recombinant expression vector obtaining between the BamHI of the DNA molecular insertion vector YEP-GAP shown in the 34th to the 894th nucleotide sequence in sequence table sequence 2 and XhoI restriction enzyme site;
Described pBI121-GmNF-YC9 can be the recombinant expression vector that described gene insertion vector pBI121 is obtained expressing described albumen; Specifically can be the recombinant expression vector obtaining between the Sma I of the DNA molecular insertion vector pBI121 shown in the 34th to the 897th nucleotide sequence in sequence table sequence 2 and SacI restriction enzyme site.
Another object of the present invention is to provide 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 object plant, obtains resistance of reverse higher than the transgenic plant of described object plant.
In aforesaid method, described object 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/or salt tolerance.
The present invention protects described albumen in the application as in transcription factor.
Experiment showed, the T that in sequence table sequence 2, shown in the 34th to the 897th nucleotide sequence, the recombinant expression vector pBI121-GmNF-YC9 arabidopsis thaliana transformation of DNA molecular obtains
3for isozygotying transfer-gen plant, with wild-type under the same terms with turn empty carrier plant and compare, in drought tolerance experiment, (seedling that normal growth is sprouted 15 days is not watered, until wild-type plant is when withered, rehydration is one week again), wild-type plant is respectively 27.2% and 28.4% with the survival rate that turns empty carrier plant, and the survival rate of transfer-gen plant is 90.2%; In the experiment of salt tolerant germination rate, (after cultivating 10 days on the MS substratum that seed is being contained to 100mM NaCl, add up germination rate, cotyledon is opened completely and is counted sprouting for green seedling), wild-type plant is respectively 65.8% and 66.2% with the germination rate that turns empty carrier plant, and the germination rate of transfer-gen plant is 90.4%.
GmNF-YC9 albumen provided by the present invention and encoding gene thereof are significant aspect raising stress resistance of plant, for the expression of manual control anti contravariance related gene provides the foundation, will play a significant role in as strong drought tolerance and strong salt tolerance plant variety cultivating high resistance to cold and diseases.
Accompanying drawing explanation
Fig. 1 is that real-time fluorescence quantitative PCR is analyzed the expression map that Different stress is processed lower GmNF-YC9 gene.Wherein, A-F respectively is the Stress treatment of dormin, arid, low temperature, high temperature, salt marsh and ethene, and X-coordinate is the time of coercing, and ordinate zou is the relative expression quantity of GmNF-YC9 gene.
Fig. 2 is yeast-one-hybrid system proof transcription factor Binding in vivo specificity and the principle schematic that activates characteristic.
Fig. 3 is the structural representation of recombinant vectors pBI121-GmNF-YC9.
Fig. 4 is that the PCR of transgenic arabidopsis plant cDNA level identifies electrophorogram.Wherein, swimming lane M is molecular weight standard, is followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, and swimming lane C is the environmental Arabidopis thaliana Col-0 of Colombia, swimming lane 1-8 is plant to be identified, and what have expection band is transfer-gen plant.
Fig. 5 wild-type and the comparison of transgenic arabidopsis drought tolerance.Wherein, figure A is wild-type (WT) and transgenic arabidopsis plant (TL) before arid is processed; Figure B is that arid is processed 14 days, wild-type (WT) and the transfer-gen plant (TL) of rehydration after 3 days.
Fig. 6 wild-type and the comparison of transgenic arabidopsis salt tolerance.Wherein, figure A is wild-type (WT) and transgenic arabidopsis (TL) seed germination and growth situation of 10 days on MS substratum; Figure B is that wild-type (WT) and transgenic arabidopsis plant (TL) are containing the germination and growth situation of 10 days on the MS substratum of 100mM NaCl.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
% in following embodiment, if no special instructions, is quality percentage composition.
Quantitative test in following embodiment, all arranges and repeats experiment, results averaged for three times.
Rich No. 8 of soybean iron (Glycine max L.): Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public; Reference: Sun Xiao, Dong Jianhui, Chen Ming, Xu Zhaoshi, the leaf ，Li Liancheng of making the country prosperous, Qu Yanying, clone and the control region piecewise analysis of the strong-willed .2008. soybean of horse adversity gene GmDREB3 promotor. Acta Agronomica Sinica, 34 (8): 1475-1479
Carrier YEP-GAP: Chinese Academy of Agricultural Sciences's crop science research guarantees to provide to the public; Reference: Liu Q; Kasuga M; Sakuma Y; Abe H; Miura S; Yamaguchi-Shinozaki K; Shinozaki K.Two transcription factors; DREB1 and DREB2; with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression; respectively, in Arabidopsis, Plant Cell1998Aug; 10 (8): 1391-1406.
The clone of embodiment 1, GmNF-YC9 gene
One, the separation of mRNA
Rich No. 8 (Glycine max L.) four leaf phase seedling arids of the hydroponics growing soybean iron of 10 days are processed 2 hours, and with liquid nitrogen flash freezer ,-80 ℃ save backup.Employing Quikprep Micro mRNA PurificationKit(Pharmacia) carry out the separation of mRNA.The synthetic ThermoScript II XL(AMV that uses of the first chain cDNA).Adopt the synthetic ds cDNA of SMART method, PCR product carries out 1.0% agarose gel electrophoresis detection.
Two, the acquisition of GmNF-YC9 full length gene sequence
By the method for 5 ' RACE and 3 ' RACE, obtain the full length gene cDNA of the nuclear factor C family sequence of soybean CCAAT-box, as shown in sequence table sequence 2, name is called GmNF-YC9 gene, its open reading frame is for from the 34th to the 897th Nucleotide of 5 of sequence table sequence 2 ' hold, the aminoacid sequence of the Protein G mNF-YC9 of its translation is as shown in sequence table sequence 1, by 287 amino-acid residues, formed, the 8th of this albumen is that conservative histone folds motif to the 71st amino acids sequence, the aminoacid sequence of this albumen is compared on Genabnk, with the albumin A tNF-YC11(At3g12480 in Arabidopis thaliana) there is higher homology, and in soybean, do not find homologous protein, proof GmNF-YC9 albumen is a new albumen.
One, Stress treatment
By potted plant seedling age, be rich No. 8 seedling of soybean iron of 10 days, carry out following processing:
(1) dormin is processed (Figure 1A): be placed in dormin (ABA) solution of 100 μ M, illumination cultivation takes out respectively and use liquid nitrogen flash freezer after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, and-80 ℃ save backup.
(2) arid is processed (Figure 1B): take out and blot the moisture on root, be placed on dry filter paper, arid is cultivated after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours and taken out material, and with liquid nitrogen flash freezer ,-80 ℃ save backup.
(3) subzero treatment (Fig. 1 C): be placed in 4 ℃ of incubators, illumination cultivation takes out and use liquid nitrogen flash freezer after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, and-80 ℃ save backup.
(4) pyroprocessing (Fig. 1 D): be placed at 42 ℃, illumination cultivation takes out respectively and use liquid nitrogen flash freezer after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, and-80 ℃ save backup.
(5) salt marsh is processed (Fig. 1 E): be placed in the NaCl solution of 200mM, illumination cultivation is taken out respectively material after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, and with liquid nitrogen flash freezer ,-80 ℃ save backup.
(6) ethene (EH) is processed (Fig. 1 F): be placed in the EH solution of 50 μ M, illumination cultivation takes out respectively and use liquid nitrogen flash freezer after 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, and-80 ℃ save backup.The processing of contrast: directly get soybean seedling-80 ℃ frozen (0 hour) in contrast without any processing.
Two, the separation of mRNA
Adopt Quikprep Micro mRNA Purification Kit(Pharmacia) material of step 1 is carried out respectively to the separation and purification of mRNA.
Three, reverse transcription is cDNA
Adopt R103-Quant_Reverse_Transcriptase(TIANGEN Biotech (Beijing) Co., Ltd.) by the mRNA reverse transcription of step 2 purifying, be cDNA.
Four, real-time fluorescence quantitative PCR
50 times of templates that are used as afterwards real-time fluorescence quantitative PCR of cDNA dilution that step 3 is obtained.According to the sequence of GmNF-YC9 gene, at its 3 ′Duan non-coding region design special primer, F and R are carried out to real-time fluorescence quantitative PCR amplification to sample, analyze the situation of replying of the various processing of this gene pairs, take actin as reference gene, primer is actin-F and actin-R.
Real-time fluorescence quantitative PCR is at ABI
on 7000 real-time fluorescence quantitative PCR instrument, carry out, 3 repetitions are established in a parallel test.Utilize the method for Livak KJ and Schmittgen TD (2001) report, 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.
The 481-499 position of the corresponding sequence table sequence 2 of primers F: 5'-CGTGGCCGAGGGCGACCAC-3'()
The 671-690 position of the corresponding sequence table sequence 2 of primer R:5'-ATTCAGATCGATGTTCCGGA-3'()
Primer act in-F:5'-CGGTGGTTCTATCTTGGCATC-3';
Primer act in-R:5'-GTCTTTCGCTTCAATAACCCTA-3'
Result is as shown in the A-F in Fig. 1, and above-mentioned 6 kinds of arid (Figure 1B), low temperature (Fig. 1 C), salt marsh (Fig. 1 E) and the ethene (Fig. 1 F) in coercing of GmNF-YC9 gene pairs are coerced and shown response.
The activation characteristic of embodiment 3, GmNF-YC9
By the cardinal principle of the activation characteristic of yeast-one-hybrid system proof transcription factor as shown in Figure 2, CCAAT cis-acting elements and mutant CCAAT cis-acting elements are building up to respectively to the basic promotor Pmin(minimal promoter of pHISi-1 carrier and pLacZi carrier) upstream, Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).When being connected with the expression vector YEP-GAP(of the goal gene of the encoding transcription factor, do not contain mobilizing function) be transformed into respectively after the yeast cell that is connected with CCAAT cis-acting elements and mutant CCAAT cis-acting elements, if the reporter gene being connected with in the yeast cell of mutant CCAAT cis-acting elements can not be expressed, and the reporter gene being 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, activated Pmin promotor, impel reporter gene to express.Thereby Binding in vivo specificity and the mobilizing function of object transcription factor have been proved.
YPD liquid nutrient medium: yeast extract for microbial culture (Bacto-Yeast Extract) 10g/L, tryptone for microbial culture (Bacto-Peptone) 20g/L, regulate pH to 5.8,121 ℃/15min sterilizing, be down to 60 ℃ and add 40% Glucose later, making its final concentration is 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 ℃/15min sterilizing, after being down to 60 ℃, add 40%Glucose, making its final concentration is 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-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.
1×PEG/LiAc：50％PEG3350 8ml，10×TE buffer 1ml，10×LiAc 1ml。
10 * TE Buffer:100mM Tris-Hcl, 10mM EDTA, pH=7.5,121 ℃ of autoclavings, room temperature preservation.
1×TE/LiAc：10×TE buffer 1ml，10×LiAc 1ml，ddH
2O 8ml。
Z Buffer:Na
2hPO
47H
2o 16.1g/L, NaH
2pO
4h
2o 5.5g/L, KCl 0.75g/L, MgSO
47H
2o 0.246g/L, regulates pH to 7.0,121 ℃/15min sterilizing, 4 ℃ of preservations.
X-gal storage liquid (X-gal Stock Solution): with N, N-dimethyl-formamide(DMF) dissolve X-gal, making its final concentration is 20mg/ml ,-20 ℃ of storages.
The Z buffer damping fluid 100ml(Z buffer with X-gal that contains X-gal), matching while using: Z buffer 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 ℃ of autoclavings, room temperature preservation.
One, the structure of recombinant expression vector
1, the acquisition of GmNF-YC9 gene
According to primers GmNF-YC9-BHI and the GmNF-YC9-XI of GmNF-YC9 gene, primer end is introduced respectively BamHI and XhoI restriction enzyme site, the cDNA of rich No. 8 of soybean varieties iron of take is template, pcr amplification GmNF-YC9 gene, carries out 1.2% agarose gel electrophoresis detection by pcr amplification product.
GmNF-YC9-BHI：5'-TTT
GGATCCATGAGGAAGAAGCTCGATAC-3'；
GmNF-YC9-XI：5'-GGT
CTCGAGCCCCTCCTCTTCATCATAGTC-3'。
Pcr amplification product carries out 1.2% agarose gel electrophoresis detection.
Adopt Agarose Gel DNA PurificationKit Ver.2.0 (TaKaRa company, Code No.DV807A) to reclaim the PCR product of purifying 882bp.
2, the structure of recombinant expression vector
1. with restriction enzyme BamHI and XhoI enzyme, cut the PCR product that step 1 reclaims purifying, reclaim enzyme and cut product;
2. with restriction enzyme BamHI and XhoI enzyme, cut expression vector YEP-GAP, reclaim carrier framework;
3. step enzyme 1. being cut to product is connected with step carrier framework 2.;
4. step connection product electric shock is 3. transformed to JM109 bacterial strain (purchased from Clontech company), 37 ℃ of incubated overnight, picking positive colony checks order; Sequencing result shows, obtained recombinant vectors YEP-GAP-GmNF-YC9(and between the BamHI of YEP-GAP and XhoI restriction enzyme site, inserted sequence table sequence 2 and hold the DNA fragmentation shown in the Nucleotide of 34-894 position from 5').
Two, the checking of the Binding in vivo specificity of GmNF-YC9 and 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.
DNA fragmentation A is building up to the Pmin of pHis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company)
hIS3promotor upstream, obtains recombinant vectors pHis-1-CCAAT, with Xho I and Nco I restriction endonuclease, pHis-1-CCAAT carrier is cut into wire.
DNA fragmentation A is building up to pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P
cYCIpromotor upstream, obtains recombinant vectors pLacZi-CCAAT, with Xho I and Nco I restriction endonuclease, pLacZi-CCAAT carrier is cut into wire.
First wire pHis-1-CCAAT carrier is transformed in yeast cell (YM4271 strain, MATCHMAKER One-Hybrid System, Clontech company), acquisition can be at SD/His
-the yeast transformant of normal growth on substratum (Yeast transformant).Then take this yeast transformant as host cell, continue to transform the pLacZi-CCAAT carriers that contain 4 repetition CCAAT elements.The SD/His that lacks so at the same time Histidine and uridylic
-/ Ura
-on substratum, select to obtain the normal dual yeast reporter that contains pHis-1-CCAAT and pLacZi-CCAAT.
(2) structure of the dual yeast reporter of mutant
DNA fragmentation B (containing 4 mCCAAT elements): 5 '-GAATTC-mCCAAT-mCCAAT-mCCAAT-mCCAAT-GTCGAC-3'(MDRE: 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.
With DNA fragmentation B, replace DNA fragmentation A, the same step of method (1), obtains the dual yeast reporter of mutant.
2, PEG/LiAc method transformed yeast and interpretation of result
(1) inoculation yeast bacterial strain (YM4271 strain) is in 1ml YPD liquid nutrient medium, concuss 2 minutes, disperse after 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 approximately 4 * 10
7individual/mL);
(2) get 30ml step (1) overnight culture and receive in the YPD substratum that 300ml is fresh, 30 ℃/250rpm cultivates, approximately 3 hours (to OD600=0.5 ± 0.1), the centrifugal 5min of room temperature 1000g, collects thalline, abandons supernatant, with 1/2 volume 1 * TE, suspend, 1000g/5min is centrifugal;
(3) inhale and abandon supernatant, with the freshly prepared 1 * TE/LiAc solution of 1.5ml, suspend, vibration mixes standby;
(4) take out 0.1ml yeast competence and transform, add successively following solution: 0.1 μ g YEP-GAP-GmNF-YC9,0.1mg ssDNA(salmon sperm dna, SiTaa), 0.6mlPEG/LiAc vibrates at a high speed 1 minute, 30 ℃/200rpm shaking culture 30 minutes;
(5) add 70ul DMSO(siTaa#D8779), be inverted gently and mix, 42 ℃ of heat shocks 30 minutes, vibration gently therebetween, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g;
(6) inhale and abandon supernatant, add 0.5ml 1 * TE buffer suspension cell;
(7) with transfering loop, dip suspension, respectively containing 0, the SD/His of 15mmol/L3-AT
-/ Ura
-/ Trp
-on selective medium, setting-out is cultivated.
(8) dull and stereotyped half cultivated normal dual yeast reporter, and second half cultivates the dual yeast reporter of mutant, to do check analysis.
(9) be placed upside down in incubator, 30oC cultivates 3-4 days.
(10) found that the SD/His at 0mmol/L 3-AT
-/ Ura
-/ Trp
-culture medium flat plate on the yeast reporter of normal yeast reporter and sudden change have growth, but the diameter of the 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 energy normal growth, but the yeast reporter of sudden change is not restrained 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 bacterium colony of the normal yeast reporter of picking and sudden change respectively.Go in YPD liquid nutrient medium, in 30 ℃ of shaking culture, in the logarithmic growth later stage to be grown to, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm;
(2) abandon supernatant, liquid in control main, is placed in liquid nitrogen quick-frozen 10min by 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 does not change in 12h, is still white.Illustrate that transcription factor GmNF-YC9 can be combined with CCAAT cis-acting elements, and there is mobilizing function, activated Pmin promotor, impel reporter gene to express.Thereby Binding in vivo specificity and the mobilizing function of GmNF-YC9 have been proved.
One, the structure of recombinant expression vector
1, the clone of GmNF-YC9 gene
According to the primers of GmNF-YC9 gene to (GmNF-YC9-121F and GmNF-YC9-121R), primer end introduces respectively Sma I and SacI enzyme is cut recognition site, rich No. 8 (Glycine max L.) cDNA of the soybean iron of take are template pcr amplification GmNF-YC9 gene, pcr amplification product is carried out to 1.2% agarose gel electrophoresis, adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim the band of purifying 900bp left and right.
GmNF-YC9-121F：5'-TCC
CCCGGGATGAGGAAGAAGCTCGATAC-3'；
GmNF-YC9-121R：5'-C
GAGCTCTTACCCCTCCTCTTCATCATAGTC-3'。
2, the structure of recombinant expression vector
1. with restriction endonuclease sma I and SacI enzyme, cut the PCR product that step 1 reclaims purifying, reclaim enzyme and cut product;
2. use restriction endonuclease sma I and SacI Mei Qie pBI121(Clontech company), reclaim carrier framework;
3. step enzyme 1. being cut to product is connected with step carrier framework 2.;
4. step connection product electric shock is 3. transformed to TOP10 bacterial strain (purchased from Beijing Tian Gen company), 37 ℃ of incubated overnight, picking positive colony checks order; Sequencing result shows, obtained recombinant vectors pBI121-GmNF-YC9(and between the Sma of pBI121 I and SacI restriction enzyme site, inserted sequence table sequence 2 and hold the DNA fragmentation shown in the Nucleotide of 34-897 position from 5', and its structural representation as shown in Figure 3).
Two, the acquisition of transgenic plant
1, recombinant plasmid pBI121-GmNF-YC9 being transformed to Agrobacterium C58C1(Beijing Baeyer enlightening biotech company buys), obtain the Agrobacterium of recombinating.
2, restructuring Agrobacterium step 1 being obtained is inoculated in LB (containing 50mg/L Rifampin, 100mg/L kantlex, 50mg/L gentamicin) liquid nutrient medium, and 28 ℃, 3000rpm are cultivated approximately 30 hours;
3, the bacterium liquid of step 2 is gone in LB (containing 50mg/L Rifampin, 100mg/L kantlex, 50mg/L gentamicin), 28 ℃, 300rpm are cultivated approximately 14 hours (bacterium liquid OD600 reaches 1.5-3.0);
4, collect the thalline of step 3,4 ℃, the centrifugal 10min of 4000g, be about 0.8-1.0 with being diluted to OD600 containing 10% sucrose MS liquid nutrient medium (containing 0.02%silwet);
5, by Arabidopis thaliana (the environmental Col-0 of Colombia, the purchase of SALK company) whole strain tips upside down in the container of the bacterium liquid that fills step 4 together with flowerpot, make flower soak 50s left and right, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth, after 24hr, open plastic cloth, upright placing flowerpot, carry out normal illumination cultivation, individual plant results T
1for seed.
6, by T
1after seed disinfection, be laid on MS substratum (containing 50 μ g/mL kantlex), 23 ℃, 16h illumination/8h dark culturing, after 7 days, are selected the transgenic arabidopsis plant (showing as four true leaves for green) of anti-kantlex, by T
1for resistant transgenic Arabidopis thaliana plant, go to the upper continuation cultivation of MS solid medium (not containing kantlex) and move in soil after 7 days, press individual plant results T
2for seed.T is screened in plantation after the same method
2for seed, transplant kalamycin resistance separated than being the T of 3:1
2for 30 of strains, and individual plant results T
2for the T that ties on each individual plant in strain
3for seed, get at random 10 T
3for strain seed, carry out after the same method kalamycin resistance screening, obtain 3 T
3in generation, no longer produces the Transgenic wheat line of kalamycin resistance separation.Plant and the seed of breeding Transgenic wheat line, carry out following PCR and identify and further resistance of reverse evaluation.
Meanwhile, the identical method of usining transforms empty carrier pBI121 and contrasts as empty carrier, obtains 3 T
3the empty carrier that turns that generation isozygotys contrasts strain.
T
2t is shown in representative
1the seed producing for selfing and the plant being grown up to by it, T
3t is shown in representative
2the seed producing for selfing and the plant being grown up to by it.
Three, the PCR of transfer-gen plant identifies
Get the T that step 2 obtains
3for Transgenic wheat line plant, in cDNA level, carry out PCR evaluation, primer pair is 5 '-ATGAGGAAGAAGCTCGATAC-3' and 5'-TTACCCCTCCTCTTCATCATAGTC-3'; Expection band is 864bp; Partial results as shown in Figure 4, contains and expects the positive transfer-gen plant of band.
Get the T that step 2 obtains
3in generation, isozygotys and turns empty carrier contrast strain plant, through the PCR of DNA level, identifies all positive.
Four, the drought tolerance of transgenic plant is identified and Salt-Tolerance Identification
1, drought tolerance is identified
The PCR respectively step 3 being obtained identifies the T that isozygotys being positive
3the PCR obtaining for transgenic line (TL), step 3 identifies the T that isozygotys being positive
3in generation, turns empty carrier contrast strain (CK) and each 60 strains of not genetically modified wild-type Arabidopis thaliana Col-0 (WT), and method is in accordance with the following steps carried out drought tolerance evaluation (arrange and repeat for three times to test, results averaged) respectively:
The sprouting of the normal growth seedling of 15 days is not watered, until WT plant withered (about 2 weeks), then rehydration is one week, observes phenotype, takes pictures and add up survival results as shown in table 2 and Fig. 5.
The survival rate statistics that table 2. transgenic arabidopsis plant drought tolerance is identified
| | Repeat | 1 | |
|
On average |
| TL | 89.1% | 91.2% | 90.3% | 90.2% | |
| CK | 28.4% | 29.6% | 27.2% | 28.4% | |
| WT | 27.3% | 28.0% | 26.1% | 27.2% |
Result shows: the survival rate of not genetically modified wild-type Arabidopis thaliana Col-0 (WT) is 27.2%, and transgenic line survival rate is 90.2% and can normal growth, turn empty carrier contrast strain and be 28.4% and with WT without significant difference; This explanation GmNF-YC9 albumen and encoding gene thereof are crossed and are expressed the drought tolerance that can improve plant in Arabidopis thaliana.
2. Salt-Tolerance Identification
The PCR respectively step 3 being obtained identifies the T that isozygotys being positive
3the PCR obtaining for transgenic line (TL), step 3 identifies the T that isozygotys being positive
3in generation, turns each 150, the seed of empty carrier contrast strain (CK) and not genetically modified wild-type Arabidopis thaliana Col-0 (WT), and method is in accordance with the following steps carried out Salt-Tolerance Identification (arrange and repeat for three times to test, results averaged) respectively:
After seed is sterile-processed, with toothpick simple grain, evenly put on the MS substratum that contains different concns NaCl (0mM, 100mM NaCl), with sealed membrane, seal, place after 3 days for 4 ℃, move into 23 ℃, 16h illumination/8h is dark, cultivates after 7 days and add up germination rate in the culturing room of 60% relative humidity, cotyledon is opened completely and is counted sprouting for green seedling, and three repetitions are established in each processing.Result is as shown in table 3 and Fig. 6.
The germination rate statistics of table 3. transgenic arabidopsis plant Salt-Tolerance Identification
Result shows: the germination rate of not genetically modified wild-type Arabidopis thaliana Col-0 (WT) seed is 65.8%, and the germination rate of transgenic line (TL) seed is 90.4%, turns the germination rate of empty carrier contrast (CK) seed and WT without significant difference; This explanation GmNF-YC9 albumen and encoding gene thereof are crossed and are expressed the salt tolerance that can improve plant in Arabidopis thaliana.
Claims (10)
1. a protein, be following a) or b) protein:
A) protein being formed by the aminoacid sequence shown in sequence table sequence 1;
B) by 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 to following at least one plant stress tolerance by (a) derivative protein: drought tolerance and salt tolerance.
2. the encoding gene of protein described in claim 1.
3. gene according to claim 2, is characterized in that: the encoding gene of described protein is following 1) or 2) or 3) or 4) gene:
1) its nucleotide sequence is the DNA molecular shown in the 34th to the 897th nucleotide sequence in sequence table sequence 2;
2) its nucleotide sequence is the DNA molecular shown in sequence table sequence 2;
3) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a protein DNA molecule described in 99% homology and coding claim 1;
4) under stringent condition with 1) or 2) or 3) protein DNA molecule described in the DNA sequence dna hybridization that limits and coding claim 1.
4. the recombinant vectors, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain gene described in claim 2 or 3.
5. recombinant vectors according to claim 4, is characterized in that: described recombinant vectors is YEP-GAP-GmNF-YC9 or pBI121-GmNF-YC9;
Described YEP-GAP-GmNF-YC9 is for obtaining the gene insertion vector YEP-GAP described in claim 2 or 3 to express the recombinant expression vector of albumen described in claim 1; Be specially the recombinant expression vector obtaining between the BamHI of the DNA molecular insertion vector YEP-GAP shown in the 34th to the 894th nucleotide sequence in sequence table sequence 2 and XhoI restriction enzyme site;
Described pBI121-GmNF-YC9 is for obtaining the gene insertion vector pBI121 described in claim 2 or 3 to express the recombinant expression vector of albumen described in claim 1; Be specially the recombinant expression vector obtaining between the Sma I of the DNA molecular insertion vector pBI121 shown in the 34th to the 897th nucleotide sequence in sequence table sequence 2 and SacI restriction enzyme site.
6. cultivating a method for transgenic plant, is that gene described in claim 2 or 3 is imported in object plant, obtains resistance of reverse higher than the transgenic plant of described object plant.
7. method according to claim 6, is characterized in that: described object plant is monocotyledons or dicotyledons.
8. method according to claim 7, is characterized in that: described dicotyledons is Arabidopis thaliana.
9. according to arbitrary described method in claim 6-8, it is characterized in that: resistance of reverse is drought tolerance and/or salt tolerance.
Described in claim 1 albumen in the application as in transcription factor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210214142.9A CN103509094B (en) | 2012-06-25 | 2012-06-25 | Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210214142.9A CN103509094B (en) | 2012-06-25 | 2012-06-25 | Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103509094A true CN103509094A (en) | 2014-01-15 |
| CN103509094B CN103509094B (en) | 2015-09-02 |
Family
ID=49892567
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210214142.9A Active CN103509094B (en) | 2012-06-25 | 2012-06-25 | Plant stress tolerance correlative protein GmNF-YC9 and encoding gene thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103509094B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104892741A (en) * | 2014-03-05 | 2015-09-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein GmNF-YA17, and encoding gene and application thereof |
| CN104892739A (en) * | 2014-03-05 | 2015-09-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein GmNF-YC6, and encoding gene and application thereof |
| CN107532148A (en) * | 2015-02-18 | 2018-01-02 | 衣阿华州立大学研究基金公司 | The sub- binding site of Transcription inhibition of NF YC4 promoters is modified to increase protein content and resistance to stress |
| CN110643733A (en) * | 2019-11-08 | 2020-01-03 | 河南农业大学 | Kit for detecting expression quantity of soybean biological clock gene ELF3 homologous gene |
| CN110684089A (en) * | 2018-07-04 | 2020-01-14 | 中国农业科学院作物科学研究所 | Application of plant stress tolerance related protein GmMYB118 in regulation and control of plant stress tolerance |
| US12037594B2 (en) | 2022-11-21 | 2024-07-16 | Iowa State University Research Foundation, Inc. | Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591383A (en) * | 2008-05-27 | 2009-12-02 | 中国农业科学院作物科学研究所 | A kind of plant stress tolerance correlative protein and encoding gene thereof and application |
| 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 |
-
2012
- 2012-06-25 CN CN201210214142.9A patent/CN103509094B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591383A (en) * | 2008-05-27 | 2009-12-02 | 中国农业科学院作物科学研究所 | A kind of plant stress tolerance correlative protein and encoding gene thereof and application |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 无: "登录号：I1M0K0(I1M0K0_SOYBN)", 《EBI》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104892741A (en) * | 2014-03-05 | 2015-09-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein GmNF-YA17, and encoding gene and application thereof |
| CN104892739A (en) * | 2014-03-05 | 2015-09-09 | 中国农业科学院作物科学研究所 | Plant stress tolerance associated protein GmNF-YC6, and encoding gene and application thereof |
| CN104892741B (en) * | 2014-03-05 | 2017-12-26 | 中国农业科学院作物科学研究所 | Plant stress tolerance correlative protein GmNF YA17 and its encoding gene and application |
| CN104892739B (en) * | 2014-03-05 | 2018-06-19 | 中国农业科学院作物科学研究所 | Plant stress tolerance correlative protein GmNF-YC6 and its encoding gene and application |
| CN107532148A (en) * | 2015-02-18 | 2018-01-02 | 衣阿华州立大学研究基金公司 | The sub- binding site of Transcription inhibition of NF YC4 promoters is modified to increase protein content and resistance to stress |
| CN107532148B (en) * | 2015-02-18 | 2022-06-24 | 衣阿华州立大学研究基金公司 | Modification of the transcriptional repressor binding site of the NF-YC4 promoter to increase protein content and stress resistance |
| US11542520B2 (en) | 2015-02-18 | 2023-01-03 | Iowa State University Research Foundation, Inc. | Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress |
| CN110684089A (en) * | 2018-07-04 | 2020-01-14 | 中国农业科学院作物科学研究所 | Application of plant stress tolerance related protein GmMYB118 in regulation and control of plant stress tolerance |
| CN110684089B (en) * | 2018-07-04 | 2021-07-27 | 中国农业科学院作物科学研究所 | Application of plant stress tolerance related protein GmMYB118 in regulation and control of plant stress tolerance |
| CN110643733A (en) * | 2019-11-08 | 2020-01-03 | 河南农业大学 | Kit for detecting expression quantity of soybean biological clock gene ELF3 homologous gene |
| CN110643733B (en) * | 2019-11-08 | 2021-07-09 | 河南农业大学 | Kit for detecting expression quantity of soybean biological clock gene ELF3 homologous gene |
| US12037594B2 (en) | 2022-11-21 | 2024-07-16 | Iowa State University Research Foundation, Inc. | Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103509094B (en) | 2015-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis | |
| US9809827B2 (en) | Transgenic maize | |
| CN102234320B (en) | Plant stress-tolerant associated protein TaDREB4B and encoding gene and application thereof | |
| 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 | |
| CN103319583B (en) | Plant stress tolerance-associated protein TaNF-YB 1, coding genes thereof and applications | |
| CN102234323A (en) | Plant stress-tolerance-associated protein TaDREB3A and coding gene and application thereof | |
| CN104892741B (en) | Plant stress tolerance correlative protein GmNF YA17 and its encoding gene and application | |
| CN102234322B (en) | Protein GmNF-YA1 related with stress tolerance of plants, and coding gene and application thereof | |
| CN104220596A (en) | Plant body showing improved resistance against environmental stress and method for producing same | |
| CN104892737B (en) | Plant stress tolerance correlative protein GmNF-YA15 and its encoding gene and application | |
| CN102653556B (en) | Plant adverse resistance related transcription factor GmWRKY78 as well as encoding gene and application thereof | |
| CN102234321B (en) | Plant stress-tolerant associated protein GmNF-YB1 and encoding gene and application thereof | |
| 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 | |
| CN102174092B (en) | ABA (abscisic acid) and salt related protein STS1 (steroid sulfatase 1) and encoding genes and application thereof | |
| CN103319582B (en) | Plant stress tolerance-associated protein TaNF-YA 1, coding genes thereof and applications | |
| CN104892740A (en) | Plant stress tolerance associated protein GmEF13, and encoding gene and application thereof | |
| CN103509095B (en) | Plant stress tolerance associated protein GmNF-YB8 as well as coding gene and application thereof | |
| CN104892739B (en) | Plant stress tolerance correlative protein GmNF-YC6 and its encoding gene and application | |
| CN104892736B (en) | Plant stress tolerance correlative protein GmNF-YA20 and its encoding gene and application | |
| CN104892742B (en) | Plant stress tolerance correlative protein GmNF-YA2 and its encoding gene and application | |
| CN102140135A (en) | Corn dehydration responsive element bonding protein ZmDBP3 and coding gene thereof | |
| CN102140134A (en) | Corn dehydration responsive element (DRE)-binding protein ZmDBP2 and encoding gene thereof | |
| CN103725701B (en) | A kind of method of cultivating the drought-resistant plant of transgenosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |