CN103044534B - Related gene of drought resistant medicago sativa as well as encoding protein and application of gene and protein - Google Patents
Related gene of drought resistant medicago sativa as well as encoding protein and application of gene and protein Download PDFInfo
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Abstract
本发明公开了一种由紫花苜蓿抗旱相关基因编码的蛋白，其中，该蛋白具有SEQ ID No：2所示的氨基酸序列，或者该蛋白具有将SEQ ID No：2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加后仍具有抗旱活性的氨基酸序列。本发明还涉及该蛋白的编码基因，以及含有该基因的表达载体和细胞，同时还涉及一种培育抗旱紫花苜蓿的方法和它们的应用。本发明提供的紫花苜蓿抗旱相关基因(bZIP)在紫花苜蓿中的表达可以显著增强紫花苜蓿的抗旱性能。该基因的发现为主要农作物的抗旱研究提供了基因资源，在基因工程改良植物的抗旱性能研究中将发挥重要作用。
The invention discloses a protein encoded by an alfalfa drought-resistance-related gene, wherein the protein has the amino acid sequence shown in SEQ ID No: 2, or the protein has the amino acid sequence shown in SEQ ID No: 2 through one or An amino acid sequence that still has drought resistance activity after substitution, deletion or addition of several amino acid residues. The invention also relates to the coding gene of the protein, the expression vector and the cell containing the gene, and also relates to a method for cultivating drought-resistant alfalfa and their application. The expression of the alfalfa drought-resistance-related gene (bZIP) in the alfalfa can significantly enhance the drought-resistance performance of the alfalfa. The discovery of this gene provides genetic resources for the research on drought resistance of major crops, and will play an important role in the research on the drought resistance performance of genetic engineering improved plants.
Description
Technical field
The present invention relates to a kind of alfalfa gene related to drought tolerance, also relate to the albumen by this genes encoding, and the expression vector that contains described alfalfa gene related to drought tolerance and cell, also relate to a kind of method of cultivating drought resisting alfalfa simultaneously, and the albumen of alfalfa gene related to drought tolerance and coding thereof and the expression vector that contains this gene and the application of cell in the plant of cultivating drought-resistant ability raising.
Background technology
Alfalfa is China and even most important leguminous forage in the world, is described as " King of Pasture ".Alfalfa is more in northwest China plantation, and the Northwest's arid, climatic characteristic short of rain, it is very unfavorable that the normal growth of crop is grown, and seriously restricted the development of husbandry.
Therefore, the drought resistance of further investigation alfalfa, for overcoming the restriction of the natural condition such as this area's arid, wind erosion to clover cultivation, expands its planting range, increases productivity, and has very important meaning.
In the past few decades, the study on drought resistance of alfalfa progressively being transferred to the research that improves alfalfa self drought resistance from the research of the qualification aspect of drought resistance comes up, especially in the research in recent years or in the nearly more than ten years, the research work of the breeding for drought resistance of alfalfa and breed improvement aspect is particularly outstanding.
At present the most effective and conventional method or traditional recurrent selection method, produces offspring by roguing in original population, offspring is identified, then form the average performance of new colony with raising colony with good progeny recombination.Then can utilize pedigree method to select self-mating system to new colony.
Along with molecular biological development, biotechnology has obtained application widely in breeding.But compared with research in other crops (as corn, Chinese sorghum etc.), the research in alfalfa Drought-resistant Breeding is also relatively less.Therefore the Molecular Biology Mechanism that, alfalfa drought resisting is relevant needs further to study.
Summary of the invention
The present invention is based on the research of the Molecular Biology Mechanism relevant to alfalfa drought resisting, the albumen of alfalfa gene related to drought tolerance and this genes encoding is provided on the one hand, the expression vector and the cell that contain described alfalfa gene related to drought tolerance are also provided on the other hand, and the method for cultivation drought resisting alfalfa, their application is also provided.
The invention provides a kind of albumen of alfalfa gene related to drought tolerance coding, wherein, this albumen has the aminoacid sequence shown in SEQ ID No:2, or this albumen has the aminoacid sequence aminoacid sequence shown in SEQ ID No:2 still after replacement, disappearance or the interpolation of one or several amino-acid residue to drought resisting activity.
The present invention also provides a kind of alfalfa gene related to drought tolerance, and wherein, this gene has the nucleotide sequence shown in SEQ IDNo:1, or this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.
The present invention also provides a kind of expression vector, and wherein, this expression vector contains alfalfa gene related to drought tolerance provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains alfalfa gene related to drought tolerance provided by the invention.
The present invention also provides a kind of method of cultivating drought resisting alfalfa, and wherein, the method comprises alfalfa gene related to drought tolerance provided by the invention is imported in alfalfa cell, obtains alfalfa cell and transfer-gen plant that drought-resistant ability improves.
The present invention also provide alfalfa gene related to drought tolerance provided by the invention and coding thereof albumen, the expression vector that contains described gene, contain described gene transgenic cell in the application of cultivating in the plant that drought-resistant ability improves.
Alfalfa gene related to drought tolerance provided by the invention (bZIP) in alfalfa by Drought Stress abduction delivering, and then the present inventor imports to alfalfa gene related to drought tolerance (bZIP) in alfalfa by transgenic technology, result shows that the expression of alfalfa gene related to drought tolerance (bZIP) in alfalfa can significantly strengthen the drought resistance of alfalfa.The anti-drought gene that the present invention clones from alfalfa is that the drought resisting research of staple crops (particularly alfalfa) provides genetic resources, in the drought resistance research of genetically engineered improvement plant, will play a significant role.
Brief description of the drawings
Fig. 1 has shown the analytical results of the real-time quantitative PCR of bZIP gene (SEQ ID NO:1), and result shows that the expression level of bZIP gene can be by raising that drought stress is induced.
Fig. 2 has shown the structure of carrying out the used pCAMBIA1302 carrier that is inserted with bZIP gene of cellular localization in embodiment 2.
Fig. 3 is transferred to the empty expression vector that does not contain object bZIP gene in onion epidermis cell, the Subcellular Localization figure observing.
Fig. 4 is for to be transferred to the expression vector that contains ZIP gene (SEQ ID NO:1) in onion epidermis cell, the Subcellular Localization figure observing.
Fig. 5 has shown the structure of in embodiment 3, Medicago sativa being carried out the used pCAMBIA1302 carrier that is inserted with bZIP gene of agriculture bacillus mediated bZIP gene transformation.
Embodiment
The invention provides a kind of albumen of being encoded by alfalfa gene related to drought tolerance, wherein, this albumen has the aminoacid sequence shown in SEQ ID No:2, or this albumen has the aminoacid sequence aminoacid sequence shown in SEQ ID No:2 still after replacement, disappearance or the interpolation of one or several amino-acid residue to drought resisting activity.Under preferable case, this albumen has the aminoacid sequence shown in SEQ ID No:2.
Correspondingly, the present invention also provides a kind of alfalfa gene related to drought tolerance (bZIP), wherein, this gene has the nucleotide sequence shown in SEQ ID No:1, or this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ IDNo:2.Preferably, this gene has the nucleotide sequence shown in SEQ ID No:1.
Alfalfa gene related to drought tolerance provided by the invention (bZIP) be contriver by building the cDNA library of specifically expressing under alfalfa drought stress, and screening obtains from this cDNA library.
The construction process of described cDNA library comprises: utilize 30% (w/v) PEG8000 to alfalfa (Medicago sativa L.cv. Baoding clover, Zhong Xu east, Beijing grass cultivation science and technology limited Company) carry out simulating drought process 12 hours, results plant, is put in-80 DEG C as experimental group.The plant of not processing through arid is control group.Utilize PCR-Selected cDNA Subtraction Kit (Clontech, Mountain View, CA, USA) to build the cDNA library of specifically expressing under alfalfa drought stress.
In the building process in library, obtain 525 single bacterium colonies and all checked order.Through removing carrier sequence and redundant sequence, 130 est sequences are finally obtained.The size of these sequences is from 250bp to 1000bp.Wherein, the est sequence that length is 567bp has caused experimenter's concern, and to utilizing SMARTer
tMrACE cDNA Amplification Kit (Clontech, USA) has carried out 5 ' RACE and 3 ' RACE clone to this est sequence respectively, has finally obtained this full length gene sequence (SEQ IDNO:2).Wherein, the normal experiment operation that the method for gene clone is biology field, concrete grammar is as follows:
A. prepare basic liquid: 2.0 μ l 5 × the first chain damping fluids, 1.0 μ l DTT (20mM), 1.0 μ ldNTP Mix (10mM);
B. for the preparation of the cDNA:2.75 μ l RNA of 5 ' RACE, 1.0 μ l 5 '-CDS primer A (ACGCGACGGTTTCAACATCCCTCTC);
For the preparation of the cDNA:3.75 μ l RNA of 3 ' RACE, 1.0 μ l 3 '-CDS primer A (GAGCTGATGCTGTGGCTGCTGGTTG);
C. ready liquid is put in 72 DEG C 3 minutes, then be put in 42 DEG C cooling 2 minutes.
D. to the SMARTer IIA oligo that adds 1 μ l in the cDNA of 5 ' RACE in B.
E. prepare the cDNA reaction solution of 5 ' RACE and 3 ' RACE: the damping fluid that the steps A of 4.0 μ l obtains, 0.25 μ l RNA enzyme inhibitors (40U/ μ l), 1.0 μ l SMARTScribe reversed transcriptive enzymes (100U).
F. the liquid in step e is joined in step C, complete the preparation of the cDNA synthesis reaction solution of 3 ' RACE; Liquid in step e is joined in step D, complete the preparation of the cDNA synthesis reaction solution of 5 ' RACE.
G. the reaction solution preparing is put in 42 DEG C to 90 minutes, in last 70 DEG C 10 minutes, completes the synthetic of cDNA.
Wherein, 3 ' RACE reaction system is: 94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 30 circulations.5 ' RACE reaction system is: 94 DEG C 30 seconds, 65 DEG C 30 seconds, 72 DEG C 3 minutes, totally 40 circulations.
Get PCR product and carry out electrophoresis detection on the sepharose of 1.0 (W/V) %, reclaim object band, connect and reclaim on product and pEGM T-Easy carrier, transform bacillus coli DH 5 alpha competent cell, extract plasmid order-checking, sequencing result shows that this gene has the nucleotide sequence shown in SEQ ID:No:1 (1781bp).
The present invention also provides a kind of expression vector, and wherein, this expression vector contains the gene with following nucleotide sequence: the nucleotide sequence shown in SEQ ID No:1, or the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.Gene provided by the invention can be building up in expression vector by existing method, before its transcription initiation Nucleotide, can add any enhancing promotor or inducible promoter.For the ease of transgenic plant cells or plant are identified and are screened, can process used carrier, as add plant selected marker (as BAR gene, gus gene, luciferase gene etc.) or there is the antibiotic marker thing (as Totomycin, kantlex etc.) of resistance.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains the gene with following nucleotide sequence: the nucleotide sequence shown in SEQ ID No:1, or the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.Described cell can be monocotyledonous cell, can be also the cell of dicotyledons, as the cell of paddy rice, wheat, corn, cucumber, tomato or clover etc.Be preferably clover cell, more preferably alfalfa cell.
The present invention also provides a kind of method of cultivating drought resisting alfalfa, and wherein, the method comprises the base with the Nucleotide shown in SEQ ID No:1 is imported in alfalfa cell, obtains alfalfa cell and transfer-gen plant that drought-resistant ability improves.
According to of the present invention be the method for genetically engineered field routine by the method that has gene provided by the invention to import in alfalfa cell, conventional biological method transformed plant cells or the tissue such as for example can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity, agriculture bacillus mediated, afterwards the vegetable cell of conversion be cultivated into plant.
The present invention also provide alfalfa gene related to drought tolerance provided by the invention and coding thereof albumen, the expression vector that contains described gene, contain described gene transgenic cell in the application of cultivating in the plant that drought-resistant ability improves.Preferably, described plant is alfalfa.
The Real-time PCR Analysis of bZIP gene
In order further to study from suppressing the poor expression that subtracts the bZIP gene (gene shown in SEQ ID NO:1) obtaining hybridization library, utilize the method for real-time quantitative PCR to analyze, concrete steps are as follows:
With Medicago sativa (Medicago sativa L.cv. Baoding clover of 50 days sizes, Zhong Xu east, Beijing grass cultivation science and technology limited Company) be experiment material, use respectively 30% (w/v) PEG 8000 to process 0,15,30 minute and 1,6,12,24 hour, extract total RNA.Total RNA of 5 μ g is used to the preparation of the first chain cDNA, concrete steps are as follows: first add 0.5 μ l 50 μ M oligo (dT) (Invitrogen), 1 μ l 10mM dNTP Mix and 5 μ l DEPC-treated water, 65 DEG C of heat shocks 5 minutes, are put in cooled on ice 1 minute; Secondly, add 4 μ l 5 × First Strand Buffer, 2 μ l 0.1M dithiothreitol (DTT), 1 μ l 40U/ μ l RNaseOUT (Invitrogen) and 200USuperScript II Reverse Transcriptase (Invitrogen), be put in 25 DEG C 5 minutes, 50 DEG C 60 minutes and 70 DEG C 15 minutes.Synthetic cDNA is put in-20 DEG C of preservations.
Primer for real-time quantitative PCR is:
Primer 1:5 '-3 ': ACCAAGACTGAAAAGCCTTC
Primer 2: 5 '-3 ': TTCTCCATCAGTGGTCGGTG.
A SYBR green reporter assay kit is used to real-time quantitative PCR reaction.The 18S rRNA of Medicago truncatula is used to interior mark.Do and repeat experiment for 3 times for each gene.Reaction system is as follows: 2 μ l 10 × PCR buffer, 2 μ l 25mM Mg
2+0.5 μ l 25mM dNTP, 0.5 μ l 10 μ M forward primer, 0.5 μ l 10 μ M reverse primer, 1 μ l 20 × SYBR Green Master Mix (Invitrogen), 0.2 μ l 5U/ μ l Taq (Invitrogen, 11304-029), 1 μ l cDNA, finally mends 20 μ l with ultrapure water by cumulative volume.Reaction conditions is as follows: 95 DEG C of dissolving DNAs 2 minutes, the amplified reaction that then carries out 40 circulations, 95 DEG C of 10 second, 60 DEG C of 30 second, 70 DEG C of 45 second.Finally use 2
-Δ Δ Ctstatistical method is analyzed PCR result.Result as shown in Figure 1.
Can find out from the result of Fig. 1, the expression level of bZIP gene (SEQ ID NO:1) can be by raising that drought stress is induced.
The Subcellular Localization of bZIP gene
The structure of plant expression vector: 1) RNA extracts (extraction of Trizol method), 2) reverse transcription (M-MLV, promega company), 3) pcr amplification: taking the cDNA of reverse transcription as template, utilizing Taq enzyme to be PCR, the restriction enzyme site designing (NcoI and SpeI) be connected into PCR product, 4) PCR product and carrier carry out enzyme and cut processing, 5) enzyme is cut product connection, and carrier structure as shown in Figure 2.
Transform intestinal bacteria and detect, utilize particle gun, the empty expression vector that does not contain object bZIP gene is transferred to onion epidermis cell, observe its Subcellular Localization (as shown in Figure 3); Utilize particle gun, the carrier that contains goal gene is imported in onion epidermis cell, observe its Subcellular Localization (as shown in Figure 4).
In Fig. 3, by relatively can finding out of A and B, the empty expression vector that does not contain object bZIP gene all has expression at the each several part of cell; And by relatively can the finding out of A in Fig. 4 and B, the expression vector that contains ZIP gene (SEQ ID NO:1) is only expressed in nucleus.
Embodiment 3
Agriculture bacillus mediated bZIP gene transformation Medicago sativa
One, material and reagent
1, vegetable material
Be alfalfa (Medicago sativa L.cv. Baoding clover, Zhong Xu east, Beijing grass cultivation science and technology limited Company) for examination alfalfa variety.
The acceptor material that the aseptic seedling cotyledon germinateing 7 days is genetic transformation.
2, agrobacterium strains and plasmid vector
Agrobacterium strains used is agrobacterium tumefaciens: GV3103 (sky, Beijing bounties Gene Tech. Company Limited), Agrobacterium substratum:
Plasmid vector: pCAMBIA1302 (purchased from Shanghai Xi Piji Bioisystech Co., Ltd).BZIP gene is inserted in pCAMBIA1302, and concrete steps are as follows:
1) RNA extracts (extraction of Trizol method), 2) reverse transcription (M-MLV, promega company), 3) pcr amplification: taking the cDNA of reverse transcription as template, amplification obtains bZIP gene order, and (primer is: 5 '-3 ': GGGGCTC
cCATGGaTGGGAAATAGTGACGAAGAGAAATC Nco I and 5 '-3 ': CGCGGCTC
tGATCAaCCAGCAGCCACAGCATCAGCTCTAG Spe I), 3) pcr amplification product is to carry out electrophoresis detection on 1% (W/V) sepharose in concentration, and reclaim the pcr amplification product of target sizes, and pCAMBIA1302 carrier and PCR product are carried out to double digestion processing with Nco I and Spe I, 4) after endonuclease reaction finishes, carry out electrophoresis detection with 1.0% (W/V) sepharose, the fragment that recovery contains object band, by T4 DNA ligase, connect, 5) connection product is proceeded in competence bacillus coli DH 5 alpha, obtain being inserted with the pCAMBIA1302 carrier of bZIP gene, it contains CaMV35s promotor, bZIP gene (SEQ ID NO:1) and a moisture resistance mycin screening-gene, the transfer-gen plant that can obtain by hygromycin selection preliminary evaluation when carrying out genetic transformation.Shown in the structure iron 3 of the plasmid vector that contains object fragment.
The pCAMBIA1302 carrier that is inserted with bZIP gene is imported to agrobacterium tumefaciens: in GV3103, concrete steps are as follows:
1) get the agrobacterium tumefaciens of-70 DEG C of preservations: GV3103 in containing 50 μ g/ml Streptomycin sulphate plate streakings, 28 DEG C of cultivations.
2) picking list colony inoculation is in 5ml YM liquid nutrient medium, 28 DEG C of shaking culture 12-16hr of 220rpm.
3) get 2ml bacterium liquid and transfer in 100ml YM liquid nutrient medium, 28 DEG C of 220rpm shaking culture are to OD600=0.5.
4) proceed to aseptic centrifuge tube, the centrifugal 5min of 5000rpm, removes supernatant liquor.
5) add the CaCl2 solution of the 0.1M of 10ml precooling, suspension cell gently, places 20min on ice.4 DEG C of centrifugal 5min of 5000rpm, remove supernatant.
6) add the CaCl2 solution containing the 0.1M of 15% glycerine of 4ml precooling, suspend gently.
7) agrobacterium suspension is sub-packed in aseptic Eppendorf pipe, and every pipe 200 μ l are frozen in-70 DEG C.
8) plasmid DNA of getting about 1 μ g joins in 200ml GV3103 competent cell, and after mixing, ice bath 30min, places 10min for-70 DEG C.
9) again at 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, adds 28 DEG C of 800mlYM liquid nutrient mediums, and 175rpm is coated in containing on the YM flat board of 50 μ g/ml Kanamycin after shaking training 3hr.28 DEG C of cultivations are to forming single bacterium colony.
Extract the plasmid the order-checking that import the Agrobacterium that has bZIP gene, result shows that the nucleotide sequence of quiding gene is consistent with SEQ ID NO:1, shows that Loss does not occur the expression vector that contains goal gene bZIP.
Two, experimental technique
1, the cultivation of Agrobacterium
Importing there is is the Agrobacterium of bZIP gene on the YMB solid medium that contains 50mg/L kantlex and 50mg/L Rifampin, draw flat board, be put in incubator 28 DEG C of cultivations.Two days later, picking list bacterium colony from flat board, is inoculated in the 20ml YMB liquid nutrient medium that contains 50mg/L kantlex and 50mg/L Rifampin 180rpm, 28 DEG C of cultivations.Draw flat board with the bacterium liquid shaking, 28 DEG C of cultivations, after growing single bacterium colony, are put in 4 DEG C of preservations by flat board.
2, the conversion of bZIP gene
Picking list bacterium colony on flat board, is inoculated in the YMB liquid nutrient medium that 20ml contains 50mg/L kantlex and 50mg/L Rifampin, and on constant-temperature table, in 28 DEG C, 180rpm cultivates.Within two days, reclaim bacterial strain later, bacterium liquid is fallen in 10ml centrifuge tube, 4000rpm, centrifugal 10min.Outwell supernatant, with the resuspended thalline of SH liquid nutrient medium that does not contain antibiotic improvement of the bacterium of having gone out, make the OD of bacterium liquid
600value is for 0.6-0.8, stand-by.The preparation of 4 explants: the alfalfa cotyledon of 4-5 days sizes is cut from aseptic seedling with pocket knife, be cut into the long fritter of 3-4mm, the explant scaling off is put in the SH liquid nutrient medium of improvement, prevents from drying up, stand-by.After explant is ready to complete, by the explant being soaked in the SH liquid nutrient medium of improvement, fall in the filter of the bacterium of having gone out in advance, reclaim explant.The explant of recovery is put in ready bacterium liquid, carries out During Agrobacterium.Every for a moment, shake several under, contribute to Agrobacterium to be adsorbed onto on explant.Contaminate after 15 minutes, fall on aseptic filter, reclaim explant.Explant is put on aseptic filter paper, blots the bacterium liquid of explant outside.Explant is put on not containing the filter paper in the common culture medium of cephamycin, culture dish is sealed with Parafilm, be put in dark place, cultivate 4 days for 28 DEG C.
Explant after agrobacterium tumefaciens is infected, is placed on dark cultivation after 4 days in common culture medium, transfers to and contains 2mg/L 2, and on the SH solid medium of the improvement of 4-D, 0.2mg/L KT, 30mg/L Totomycin and 300mg/L Cef, induction produces callus; After cultivating 20 days, the callus that induction is produced is transferred on the UM substratum that contains 0.2mg/L KT, 30mg/L Totomycin and 250mg/L Cef, and induction produces embryoid; After embryoid maturation, (approximately cultivate 30 days), embryoid is transplanted on 1/2MS substratum, root induction, thus complete the regeneration of plant.
In regenerative process, each step substratum is composed as follows:
Culture medium altogether: the SH substratum+2mg/L 2 of improvement, 4-D+0.2mg/L KT;
Callus induction substratum: the SH substratum+2mg/L 2 of improvement, 4-D+0.2mg/L KT+300mg/LCef+30mg/L Hyg;
Embryoid induction substratum: UM substratum+0.6mg/L KT+250mg/L Cef+30mg/LHyg;
Root media: 1/2MS substratum+250mg/L Cef+30mg/L Hyg.
Comparative example 1
Turn the acquisition of empty carrier control plant
With plasmid pCAMBIA1302 conversion Agrobacterium, obtain restructuring Agrobacterium, by restructuring Agrobacterium-mediated Transformation alfalfa, obtain turning the adjoining tree of empty carrier, method is as embodiment 2.
Embodiment 4
The alfalfa that turns the pCAMBIA1302 carrier that is inserted with bZIP gene obtaining in the embodiment 3 of the alfalfa that turns empty carrier obtaining in the comparative example of 30 strains 1 and 30 strains is carried out to drought resisting experiment, concrete grammar is as follows: 1) plant is taken out from soil, water washes the soil on root; 2) cleaned plant is put into the Hoagland Complete plant liquid growth media that contains 30% (w/v) PEG8000 and carry out simulating drought processing 2 days.
Result shows, the alfalfa that turns pCAMBIA1302 empty carrier is all withered, and the alfalfa that turns the pCAMBIA1302 carrier that is inserted with bZIP gene has 28 strains survivals, and most of blade keeps green, and plant strain growth is good.
This shows, the expression of alfalfa gene related to drought tolerance provided by the invention (bZIP) in alfalfa can significantly strengthen the drought resistance of alfalfa.The drought resisting research that is found to be staple crops (particularly alfalfa) of this gene provides genetic resources, in the drought resistance research of genetically engineered improvement plant, will play a significant role.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059928B (en) * | 2014-05-30 | 2016-03-02 | 廊坊师范学院 | The albumen improving the gene GhASS1 of intestinal bacteria salt tolerances and preparation thereof and utilize it to encode |
| CN109486838A (en) * | 2018-12-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343131B (en) * | 2013-06-28 | 2015-07-15 | 廊坊师范学院 | Gene GmCTRL2 used for improving osmotic stress resistance of alfalfa, preparation and protein coded by gene GmCTRL2 |
| CN110016479B (en) * | 2019-05-17 | 2022-08-26 | 河南省农业科学院畜牧兽医研究所 | Alfalfa MsGPF gene |
| CN116064568B (en) * | 2022-07-25 | 2025-02-07 | 兰州大学 | Alfalfa MsASG166 gene and its use in improving plant drought tolerance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472223A (en) * | 2002-07-30 | 2004-02-04 | 中国农业科学院生物技术研究所 | A maize bZIP-like transcription factor and its coding gene and application |
| CN102140443A (en) * | 2010-02-03 | 2011-08-03 | 中国科学院遗传与发育生物学研究所 | Plant stress-resistant associated protein, and encoding gene and application thereof |
-
2011
- 2011-10-14 CN CN201110310769.XA patent/CN103044534B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472223A (en) * | 2002-07-30 | 2004-02-04 | 中国农业科学院生物技术研究所 | A maize bZIP-like transcription factor and its coding gene and application |
| CN102140443A (en) * | 2010-02-03 | 2011-08-03 | 中国科学院遗传与发育生物学研究所 | Plant stress-resistant associated protein, and encoding gene and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| Carlos Molina et al.SuperSAGE- the drought stress-responsive transcriptome of chickpea roots.《BioMed Central》.2008, |
| Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.);W Walter Lorenz et al;《BMC Genomics》;20110524;1-17 * |
| SuperSAGE- the drought stress-responsive transcriptome of chickpea roots;Carlos Molina et al;《BioMed Central》;20081124;1-28 * |
| W Walter Lorenz et al.Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.).《BMC Genomics》.2011, |
| 植物抗旱的分子机制研究;沈元月等;《中国生态农业学报》;20020331;第10卷(第1期);30-34 * |
| 沈元月等.植物抗旱的分子机制研究.《中国生态农业学报》.2002,第10卷(第1期), |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059928B (en) * | 2014-05-30 | 2016-03-02 | 廊坊师范学院 | The albumen improving the gene GhASS1 of intestinal bacteria salt tolerances and preparation thereof and utilize it to encode |
| CN109486838A (en) * | 2018-12-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis |
| CN109486838B (en) * | 2018-12-21 | 2021-09-17 | 中国农业科学院北京畜牧兽医研究所 | Transcription factor gene for regulating plant flavonoid synthesis and application thereof |
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