CN103524607A - Wheat heat stress associated protein TaGCN5 as well as coding gene and application thereof - Google Patents
Wheat heat stress associated protein TaGCN5 as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat heat stress associated protein TaGCN5 as well as a coding gene and an application thereof. The protein is named as protein TaGCN5 and is the following (a) or (b), namely, (a) a protein formed by amino acid sequences shown in a sequence 1 in a sequence table; and (b) a protein obtained through substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequences in the sequence 1, associated with high temperature stress resistance of plants and is derived from the sequence 1. An over-expression vector of a gene of TaGCN5 is constructed and the high temperature stress resistance of plants is obviously improved after transforming arabidopsis thaliana. The protein TaGCN5 has a great value in culturing high temperature resistant plants.
Description
Technical field
The present invention relates to a kind of wheat heat and coerce associated protein TaGCN5 and encoding gene and application.
Background technology
In plant growth, growth course, except being subject to, the invasion and attack of the biotic factors such as disease worm, being also usually subject to the impact of bad climate and soil factor, and yield and quality is affected, this detrimentally affect is called environment-stress or abiotic stress.Along with Global warming, shortage of water resources, soil desertification, salinization, the abiotic stress that crop is stood can be more and more frequent, more and more serious.
Wheat is the important food crop of China, and its yield and quality is directly connected to people's life.Grow the very important impact of high temperature on wheat, high temperature not only can affect ovary and ovule capsule and pollen growth, bloom and be fertilized, also can affect the synthetic of grouting, albumen and starch, cause that grain yield declines, product qualitative change is bad.Carry out the molecular mechanism research of Heat Tolerance of Wheat Cultivars, identify heat-resisting genes involved, and apply it in the molecular breeding of wheat and go, contribute to the cultivation of wheat heat resistant variety.
Summary of the invention
The object of this invention is to provide a kind of wheat heat and coerce associated protein TaGCN5 and encoding gene and application.
The invention provides a kind of protein, called after TaGCN5 albumen is following (a) or (b):
(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to the high temperature resistant ability of coercing of plant through one or several amino-acid residue by the aminoacid sequence of sequence 1.
In order to make the protein in (a) be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 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 |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Protein 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 protein in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.
The gene of described TaGCN5 albumen of encoding also belongs to protection scope of the present invention.
Described gene specifically can be the DNA molecular of (1) or (2) or (3) as follows:
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular of the albumen that the DNA sequence dna hybridization limiting to (1) under stringent condition and coding are relevant with the high temperature resistant ability of coercing of plant;
(3) DNA sequence dna limiting to (1) has the DNA molecular of 90% above homology and the albumen relevant with the high temperature resistant ability of coercing of plant.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, under 65oC, hybridizes, and then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively to wash film once.
The recombinant expression vector that contains described gene, expression cassette, transgenic cell line or recombinant bacterium all 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.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.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other plant promoter; In addition, while using described gene constructed recombinant expression vector, also enhanser be can use, translational enhancer or transcriptional enhancer comprised, 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 of luminophor, have the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Described recombinant expression vector specifically can be the recombinant plasmid that the multiple clone site (between KpnI and SacI restriction enzyme site) of described gene insertion pCAMBIA-1300 carrier is obtained.
The total length of described gene that increases or the primer pair of its any fragment also belong to protection scope of the present invention.
The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in object plant, obtains the high temperature resistant ability of coercing higher than the transgenic plant of described object plant.Described gene can import in described object plant by described recombinant expression vector.Described recombinant expression vector can by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.Described object plant is monocotyledons or dicotyledons.
Described dicotyledons is preferably Arabidopis thaliana, as gcn5 mutant.The described high temperature resistant ability of coercing height can be presented as that relative conductivity reduces.
The recombinant bacterium that the present invention also protects described TaGCN5 albumen, described gene, the recombinant expression vector that contains described gene, the expression cassette that contains described gene, the transgenic cell line that contains described gene or contains described gene has the application in the transgenic plant of the high temperature resistant ability of coercing in cultivation.Described plant is monocotyledons or dicotyledons.Described dicotyledons is preferably Arabidopis thaliana, as gcn5 mutant.
The present invention also protects the application of above arbitrary described method in plant breeding.Described plant is monocotyledons or dicotyledons.Described dicotyledons is preferably Arabidopis thaliana, as gcn5 mutant.
The present invention also protects the application of described TaGCN5 albumen in the high temperature resistant ability of coercing of regulating plant.Described plant is monocotyledons or dicotyledons.Described dicotyledons is preferably Arabidopis thaliana, as gcn5 mutant.
Arbitrary described high temperature specifically can be more than 38 ℃ above.
This abiotic stress of high temperature is the critical limitation factor that affects plant biomass.The present invention be take Arabidopis thaliana as basis, by the method for homologous clone, obtain the wheat cdna with Arabidopis thaliana GCN5 homology, by transgenic method, its function is analysed in depth and identified, the further clear and definite effect of TaGCN5 gene in wheat response high temperature stress process.In the present invention, build the overexpression vector of TaGCN5 gene, after arabidopsis thaliana transformation, significantly increased the high temperature resistant ability of coercing of plant.The present invention has great value for cultivating high temperature resistant plant.
Accompanying drawing explanation
Fig. 1 is the phenotype photo in embodiment 2.
Fig. 2 is the specific conductivity result in embodiment 2.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.PCAMBIA-1300 carrier: Cambia company.Agrobacterium GV3101:Biovector company.The difference of the environmental Arabidopis thaliana of gcn5 mutant and gas Lay has only been AtGCN5 transgenation inactivation.
The environmental Arabidopis thaliana of gas Lay (representing with " Ws "): reference: Bertrand, C., Bergounioux, C., Domenichini, S., Delarue, M., and Zhou, D.X. (2003) .Arabidopsis histone acetyltransferase AtGCN5regulates the floral meristem activity through the WUSCHEL/AGAMOUS pathway.J.Biol.Chem.278,28246 – 28251.
Gcn5 mutant (is the AtGCN5mutant in document, with " gcn5 ", represent): reference: Bertrand, C., Bergounioux, C., Domenichini, S., Delarue, M., and Zhou, D.X. (2003) .Arabidopsis histone acetyltransferase AtGCN5regulates the floral meristem activity through the WUSCHEL/AGAMOUS pathway.J.Biol.Chem.278,28246 – 28251.
Embodiment 1, wheat heat are coerced the discovery of associated protein TaGCN5 and encoding gene thereof
On the basis of ncbi database, to deriving from gene and the fragment of the existing correlation function of other plant, compare, according to the result of comparison and sequential analysis, associated clip to wheat is spliced, extension and Function Identification, finally obtain an open reading frame completing, protein shown in the sequence 1 of this open reading frame code sequence list, by this protein called after TaGCN5 albumen.By the unnamed gene of coding TaGCN5 albumen, be TaGCN5 gene, as shown in the sequence 2 of sequence table.
The acquisition of embodiment 2, transgenic plant and evaluation
One, the structure of recombinant plasmid
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, take the synthetic double chain DNA molecule of step 1 is template, with the primer pair that F1 and R1 form, carries out pcr amplification, obtains pcr amplification product.
F1：5’-CACCCGG
GGTACCATGGACGGCCTCGCGGCGCC-3’；
R1：5’-CTGGGTC
GAGCTCCTAGTTCTTCGTTGACGCAG-3’。
3, with the pcr amplification product of restriction enzyme KpnI and SacI double digestion step 2, reclaim enzyme and cut product.
4, with restriction enzyme KpnI and SacI double digestion pCAMBIA-1300 carrier, reclaim carrier framework.
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid.According to sequencing result, recombinant plasmid is carried out to structrual description as follows: between the KpnI of pCAMBIA-1300 carrier and SacI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 2 of sequence table.
Two, the acquisition of transgenic plant
1, recombinant plasmid transformed Agrobacterium GV3101 step 1 being built, obtains the Agrobacterium of recombinating.
2, get gcn5 mutant seed, 4 ℃ of vernalization are seeded in MS substratum after 3 days, cultivate (22 ℃/18 ℃, 16 hours illumination/8 hour dark, 60%-70% humidity), while growing into two true leaves, be transplanted in the culture pan that is filled with culture medium (culture medium is Nutrition Soil and the vermiculite that equal-volume mixes), after plant blossom, cut off major branch top to promote side shoot development, the plant of 4-6 after beta pruning days is inverted in the bacteria suspension of the restructuring Agrobacterium that step 1 obtains and soaks, then take out plant, with the black plastic bag that is full of gas, encase, keep flat, 22 ℃ of dark cultivations 24 hours, then remove plastics bag culture pan is upright, cultivate (22 ℃/18 ℃, 16 hours illumination/8 hour dark, 60%-70% humidity) to plant to solid, results T
0for seed.
3, get T
0for seed, 4 ℃ of vernalization are laid in the MS substratum containing 60 μ l/100ml Totomycin after 3 days, cultivating (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) selects positive plant after 7 days (positive plant shows as: true leaf health is deep green, root is stretched in substratum), positive plant is transferred to MS substratum and adopts the same terms to cultivate 10 days, and then be transferred to soil and adopt the same terms to cultivate plant to solid, results T
1for seed.
4, get T
1for seed, according to the method for step 3, screen positive plant and gather in the crops T
2for seed.
5, get T
2for seed, according to the method screening positive plant of step 3.
For a certain T
1for plant, if its T
2for plant, be positive plant, this T
1for plant and self progeny thereof, it is a transgenic line isozygotying.
6, by T
2for plant selfing, obtain T
3for seed.
Three, turn the acquisition of empty carrier plant
The recombinant plasmid that replaces step 1 to build with pCAMBIA-1300 carrier, other same step 2, obtains turning empty carrier plant.
Four, phenotype comparison and surviving rate statistics
The T of the transgenic line respectively step 2 being obtained
3the T that turns empty carrier strain obtaining for seed, step 3
3the phenotype of carrying out under heat stress for seed, the environmental seed of Arabidopis thaliana of gas Lay and the seed of gcn5 mutant (50 of each strains) compares and surviving rate detection, method is as follows: after seed disinfection, by sterile water wash 6-7 time, be laid in MS substratum, 4 ℃ of vernalization 3 days, then cultivate (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 8 days, then cultivate (instant heating Stress treatment; 38 ℃, 16h illumination/8h is dark, 60%-70% humidity) 4 days, then cultivate (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 2 days, take pictures and add up surviving rate.Figure 1A is shown in by photo.The environmental Arabidopis thaliana of transgenic line and gas Lay all grows fine, and there is no significant difference between the two.Turn empty carrier strain and the equal growing way of gcn5 mutant is not good, between the two, there is no significant difference.The T of transgenic line
3surviving rate for seed is 92%, and the surviving rate of the seed of the environmental Arabidopis thaliana of gas Lay is 91%, turns the T of empty carrier strain
3the surviving rate that is the seed of 9.6%, gcn5 mutant for the surviving rate of seed is 9.5%.Result shows, imports the high temperature resistant ability of coercing that TaGCN5 gene can significantly strengthen gcn5 mutant.
The T of the transgenic line respectively step 2 being obtained
3the T that turns empty carrier strain obtaining for seed, step 3
3for seed, the environmental seed of Arabidopis thaliana of gas Lay and the seed of gcn5 mutant, carry out the phenotype comparison under heat stress, method is as follows: after seed disinfection, by sterile water wash 6-7 time, be laid in MS substratum, 4 ℃ of vernalization 3 days, then cultivate (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 8 days, then transplant to cultivating in culture medium (culture medium is Nutrition Soil and the vermiculite that equal-volume mixes) (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 10 days, then cultivate (instant heating Stress treatment; 38 ℃, 16h illumination/8h is dark, 60%-70% humidity) 4 days, then cultivate (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 2 days, take pictures.Figure 1B is shown in by photo.The environmental Arabidopis thaliana of transgenic line and gas Lay all grows fine, and there is no significant difference between the two.Turn empty carrier strain and the equal growing way of gcn5 mutant is not good, between the two, there is no significant difference.Result shows, imports the high temperature resistant ability of coercing that TaGCN5 gene can significantly strengthen gcn5 mutant.
Five, conductance measurement
The T of the transgenic line respectively step 2 being obtained
3the T that turns empty carrier strain obtaining for seed, step 3
3for seed, the environmental seed of Arabidopis thaliana of gas Lay and the seed of gcn5 mutant (50 of each strains), carry out conductance measurement detection, method is as follows:
1,4 ℃ of vernalization of seed are laid in to MS substratum after 3 days, then cultivate (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 8 days, then transplant to cultivating in soil (22 ℃/18 ℃, 16h illumination/8h are dark, 60%-70% humidity) 14 days, select growth consistent, well-developed plant.
2, with punch tool, from the 5th, the 6th of plant and the 7th leaf, beat and get (three leaves are taken off in every strain) respectively, with distilled water flushing 3 times, be placed in 20ml test tube, add 13ml distilled water (whole blade is all immersed in water).
3, test tube is placed in 42 ℃ of water-baths and places 1h, then room temperature is placed 24h, and (writing down the now reading of specific conductivity is T then with conductivitimeter, to measure the specific conductivity of solution in each test tube
1), then centrifuge tube packs tightly with masking foil, and sterilizing (15Psi, 121 ℃, 15min) is measured the now specific conductivity of solution with conductivitimeter again after placement 24h and (is designated as T
2).
4, according to formula, calculate CMS (Cell membrane thermostability) value, the i.e. stability of cytolemma.
CMS=T
1/T
2。
The results are shown in Figure 2.
Claims (10)
1. a protein is following (a) or (b):
(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to the high temperature resistant ability of coercing of plant through one or several amino-acid residue by the aminoacid sequence of sequence 1.
2. the gene of protein described in the claim 1 of encoding.
3. gene as claimed in claim 2, is characterized in that: described gene is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular of the albumen that the DNA sequence dna hybridization limiting to (1) under stringent condition and coding are relevant with the high temperature resistant ability of coercing of plant;
(3) DNA sequence dna limiting to (1) has the DNA molecular of 90% above homology and the albumen relevant with the high temperature resistant ability of coercing of plant.
4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain gene described in claim 2 or 3.
5. recombinant expression vector as claimed in claim 4, is characterized in that: the recombinant plasmid that described recombinant expression vector obtains for gene described in claim 2 or 3 being inserted to the multiple clone site of pCAMBIA-1300 carrier.
6. the total length of gene or the primer pair of its any fragment described in the claim 2 or 3 that increases.
7. cultivating a method for transgenic plant, is that gene described in claim 2 or 3 is imported in object plant, obtains the high temperature resistant ability of coercing higher than the transgenic plant of described object plant.
8. method as claimed in claim 7, is characterized in that: described in claim 2 or 3 gene by right, want 4 or 5 described in recombinant expression vector import in described object plant.
Described in claim 1 described in protein, claim 2 or 3 in gene, claim 4 or 5 arbitrary described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium in cultivation, there is the application in the transgenic plant of the high temperature resistant ability of coercing.
10. the application of method in plant breeding described in claim 7 or 8.
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| CN107641153A (en) * | 2017-11-16 | 2018-01-30 | 河南农业大学 | Biological characters of wheat under drought conditions low temperature stress response protein TaCDCP and its encoding gene and application |
| CN113527451A (en) * | 2020-04-21 | 2021-10-22 | 中国农业科学院作物科学研究所 | Wheat heat stress related protein TaANK and coding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107641153A (en) * | 2017-11-16 | 2018-01-30 | 河南农业大学 | Biological characters of wheat under drought conditions low temperature stress response protein TaCDCP and its encoding gene and application |
| CN107641153B (en) * | 2017-11-16 | 2020-10-30 | 河南农业大学 | Wheat low-temperature stress response protein TaCDCP under drought condition and coding gene and application thereof |
| CN113527451A (en) * | 2020-04-21 | 2021-10-22 | 中国农业科学院作物科学研究所 | Wheat heat stress related protein TaANK and coding gene and application thereof |
| CN113527451B (en) * | 2020-04-21 | 2023-07-14 | 中国农业科学院作物科学研究所 | Wheat heat stress related protein TaANK, and coding gene and application thereof |
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