CN103087999B - Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use - Google Patents
Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use Download PDFInfo
- Publication number
- CN103087999B CN103087999B CN201110343669.7A CN201110343669A CN103087999B CN 103087999 B CN103087999 B CN 103087999B CN 201110343669 A CN201110343669 A CN 201110343669A CN 103087999 B CN103087999 B CN 103087999B
- Authority
- CN
- China
- Prior art keywords
- gene
- tocopherol
- alfalfa
- mstmt
- alpha
- 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.)
- Expired - Fee Related
Links
Abstract
The invention discloses a protein coded by a medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene. The protein has an amino acid sequence shown in the formula SEQ ID No: 2, or has an amino acid sequence which is derived from the amino acid sequence shown in the formula SEQ ID No: 2 by displacement, deletion or addition of one or more amino acid residues and has a gamma-tocopherol methyltransferase activity. The invention also relates the MSTMT gene, an expression vector and a cell containing the MSTMT gene, a method for cultivating medicago sativa having high alpha-tocopherol content, and a use of the MSTMT gene, the protein, the expression vector and the cell. Through expression of the MSTMT gene in medicago sativa, alpha-tocopherol content of medicago sativa can be obviously improved. The MSTMT gene provides a gene resource for improvement of alpha-tocopherol content of main forage grass (leguminous plants such as medicago sativa) and has an important effect in the gene engineering-based forage grass improvement study.
Description
Technical field
The present invention relates to a kind of alfalfa gama-tocopherol methyl transferase genes involved, also relate to the albumen by this genes encoding, and the expression vector that contains described alfalfa gama-tocopherol methyl transferase gene and cell, also relate to a kind of method of cultivating alfalfa simultaneously, and the albumen of alfalfa gama-tocopherol methyl transferase gene and coding thereof and the expression vector that contains this gene and the application of cell in the plant of cultivating high content of alpha-tocopherol content.
Background technology
Vitamin-E is an anthropoid necessary liposoluble vitamin, has important physiological function.It can remove the free radical that lipid peroxidation produces and the biomembranous lipid bilayer of stable protection makes cell avoid the injury of superoxide.Thereby be used as a kind of good antioxidant and be widely used in the industries such as medicine, food, feed.A large amount of experimentation on animalies show, in feed, add vitamin-E have improve animal immunizing power, improve meat, improve animal reproductive performance, alleviate the unusual effects such as Animal stress reaction.
The production of vitamin-E mainly obtains by chemosynthesis and natural extract.The synthetic product of chemical process mainly exists with the form of acetic ester, and by product is many and biological activity is low.Natural VE is mainly present in plant, is obviously better than synthesising complex E in physiologically active and security.
Natural VE is made up of 8 kinds of tocopherol isomers.Can be divided into tocopherol and the large class of tocotrienol two according to side chain saturation ratio.Difference be divided into again α, β, γ, Delta-Tocopherol and α, β, γ, δ-tocotrienol according to methyl number on aromatic nucleus and position.Content and the relative reactivity of these 8 kinds of tocopherol isomers in plant is all not quite similar, the biological activity of alpha-tocopherol is the highest, and can be by human body and animal body preferential absorption and utilization, this is because the transfer protein that transports alpha-tocopherol in liver preferentially determines in conjunction with the characteristic of alpha-tocopherol.But the content of alpha-tocopherol in plant is relatively low, therefore, increase substantially high reactivity alpha-tocopherol content in plant (particularly forage grass), will there is good Social benefit and economic benefit.
Research plant vitamin E route of synthesis is found, by transmethylation, Gamma-Tocopherol is converted into the gama-tocopherol methyl transferase of alpha-tocopherol, can improve content and the ratio of alpha-tocopherol in plant.Gama-tocopherol methyl transferase gene is cloned and is obtained from Arabidopis thaliana, blue-green algae etc. at present, but clone and separate that so far there are no from forage grass (particularly clover) is to gama-tocopherol methyl transferase gene, and be transformed into the report that improves alpha-tocopherol content in forage grass.
Alfalfa is China and even most important leguminous forage in the world, is described as " King of Pasture ".If can clone the gama-tocopherol methyl transferase gene that obtains being derived from alfalfa, and forwarded in the forage grass including alfalfa, can be improving animal immunizing power, improve meat, the aspect such as reproductive performance that improves animal has great importance.
Summary of the invention
The present invention is based on the research to the Molecular Biology Mechanism that in alfalfa, vitamin-E route of synthesis is relevant, the albumen of alfalfa gama-tocopherol methyl transferase gene and this genes encoding is provided on the one hand, the expression vector and the cell that contain described alfalfa gama-tocopherol methyl transferase gene are also provided on the other hand, and the method for the alfalfa of cultivation high content of alpha-tocopherol content, their application is also provided.
The invention provides a kind of albumen of alfalfa gama-tocopherol methyl transferase gene 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 gama-tocopherol methyl transferase activity.
The present invention also provides a kind of alfalfa gama-tocopherol methyl transferase gene, and 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 ID No:2.
The present invention also provides a kind of expression vector, and wherein, this expression vector contains alfalfa gama-tocopherol methyl transferase gene provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains alfalfa gama-tocopherol methyl transferase gene provided by the invention.
The present invention also provides a kind of method of cultivating alfalfa, wherein, the method comprises alfalfa gama-tocopherol methyl transferase gene provided by the invention is imported in alfalfa cell, obtains alfalfa cell and transfer-gen plant that alpha-tocopherol content is high.
The present invention also provide alfalfa gama-tocopherol methyl transferase gene 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 alpha-tocopherol content is high.
The gama-tocopherol methyl transferase gene that the present invention clones from alfalfa is for improving Dominant Species of Forage Grass plant (particularly leguminous plants, as alfalfa) in the content of alpha-tocopherol genetic resources is provided, in the research of genetically engineered Improvement plant, will play a significant role.
Brief description of the drawings
Fig. 1 has shown the structure of in embodiment 1, Medicago sativa being carried out the used pCAMBIA1302 carrier that is inserted with gama-tocopherol methyl transferase gene of agriculture bacillus mediated gama-tocopherol methyl transferase gene conversion.
Embodiment
The invention provides a kind of albumen of being encoded by alfalfa gama-tocopherol methyl transferase gene, 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 gama-tocopherol methyl transferase 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 gama-tocopherol methyl transferase gene, 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 ID No:2.Preferably, this gene has the nucleotide sequence shown in SEQ ID No:1.
Alfalfa gama-tocopherol methyl transferase gene provided by the invention is that contriver screens and obtains by the following method:
According to cutting the conservative territory design of the gama-tocopherol methyl transferase gene (Genbank registration number is: BT051703) of having cloned in type clover primer, (RT-PCR primer is: F:5 '-TCTCAGCCCTGTTCAAGC-3 '; R:5 '-CAAAGGGAGACCAATCTG-3 '), from alfalfa (Medicago sativa L.cv.Zhongmu No.1, No. one, middle lucerne, Zhong Xu east, Beijing grass cultivation science and technology limited Company) in increase, reclaim the fragment that expection size is 400bp (reclaiming the fragment of this length after leakage of electricity swimming), the fragment of recovery is connected into PMD18-T Vector (Takara), order-checking: sequencing result carries out BLASTx analysis in the nonredundancy albumen database of NCBI; 2 sequences relevant with gama-tocopherol methyl transferase class in analytical results are carried out to continuous open reading frame (ORF) analysis with DNAstar, find that 1 has continuous ORF (length is 404bp); With this sequences Design special primer (MsTMT-RT-F:5 '-ACGGCCAGTTTGATCTAGTGT-3 '; MsTMT-RT-R:5 '-CTTCATTCGGGGCAAG-3 '), and utilize this sequences Design 5 ' RACE primer and 3 ' RACE primer, be organized as material with alfalfa blade, obtain its full length sequence, concrete steps are:
Utilize SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) respectively this est sequence to be carried out to 5 ' RACE and 3 ' RACE clone, finally obtained this full length gene sequence (SEQ ID NO:1).Wherein, the normal experiment operation that the method for gene clone is biology field, concrete grammar is as follows:
RACE-ready cDNA's is synthetic
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;
For the preparation of the cDNA:3.75 μ l RNA of 3 ' RACE, 1.0 μ l 3 '-CDS Primer A;
C. ready liquid is put in 72 DEG C 3 minutes, then be put in 42 DEG C cooling 2 minutes, of short duration centrifugal collection liquid;
D. to the SMARTer IIA oligo that adds 1 μ l in the cDNA of 5 ' RACE in B;
E. prepare 5 ' RACE and 3 ' RACE-Ready cDNA reaction solution: 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 3 ' RACE-Ready cDNA synthesis reaction solution; Liquid in step e is joined in step D, complete the preparation of 5 ' RACE-Ready cDNA synthesis reaction solution;
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 Ready-cDNA.
Synthesizing fast of cDNA end
H. prepare basic liquid: 34.5 μ l PCR waters, 5.0 μ l 10 × Advantage 2PCR damping fluids, 1.0 μ l dNTP Mix (10mM), 1.0 μ l 50 × Advantage 2 polysaccharase Mix;
I. react for the preparation of the PCR of 5 ' RACE: 2.5 μ l 5 ' RACE-ready cDNA, 5.0 μ lUPM (10 ×), 1.0 μ l GSP1 5 '-GTTGTAGGGATTCTTCATTCGGGGC-3 '; The damping fluid of preparing in the step H of 41.5 μ l.
PCR reaction for the preparation of 3 ' RACE: 2.5 μ l 3 ' RACE-ready cDNA, 5.0 μ lUPM (10 ×), 1.0 μ l GSP2 5 '-TTGTTGGTGAGTTAGCACGGGTAGC-3 '; The damping fluid of preparing in the step H of 41.5 μ l.
RACE reaction system is: 94 DEG C 30 seconds, 72 DEG C 3 minutes, totally 5 circulations: 94 DEG C 30 seconds, 70 DEG C 3 minutes, 72 DEG C 3 minutes, totally 5 circulations; 94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 20 circulations.
Get PCR product and on the sepharose of 1.0 % by weight, carry out electrophoresis detection, 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, carry out sequence assembly, result shows that this gene has the nucleotide sequence shown in SEQ ID:No:1 (1306bp).
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.
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 the alfalfa that alpha-tocopherol content is high, wherein, the method comprises the gene with the Nucleotide shown in SEQ ID No:1 is imported in alfalfa cell, obtains alfalfa cell and transfer-gen plant that alpha-tocopherol content 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 gama-tocopherol methyl transferase gene 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 alpha-tocopherol content is high.Preferably, described plant is alfalfa.
Embodiment 1
Agriculture bacillus mediated gama-tocopherol methyl transferase (MsTMT) gene transformation Medicago sativa
One, material and reagent
1, vegetable material
Be alfalfa (Medicago sativa L.cv.Zhongmu No.1, No. one, middle lucerne, 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: LBA4404 (sky, Beijing bounties Gene Tech. Company Limited)
Agrobacterium substratum:
Plasmid vector: pCAMBIA1302 (purchased from Shanghai Xi Piji Bioisystech Co., Ltd).MsTMT gene is inserted in pCAMBIA1302, concrete steps are: the upstream primer that comprises NcoI restriction enzyme site and the downstream primer (F:5 '-CATGCCATGGATGGCGGCGGTGAAAGAAGC-3 ' of SpeI restriction enzyme site are held in design 5 ', R:5 '-GGACTAGTTCATTGACCCTCTGCATTTTCAGGC-3 ', the total length reading frame of MsTMT increases from cDNA template with this primer, 1302 carriers and full length fragment reclaim product after NcoI and SpeI double digestion, connect through T4 DNA ligase, obtain being inserted with the pCAMBIA1302 carrier of MSTMT gene, it contains CaMV35s promotor, MSTMT gene (SEQ ID NO:1) and a hygromycin resistance selection markers, the transfer-gen plant that can obtain by hygromycin selection preliminary evaluation when carrying out genetic transformation.Shown in the structure iron 1 of the plasmid vector that contains object fragment.
The pCAMBIA1302 carrier that is inserted with MSTMT gene is imported in agrobacterium tumefaciens lba4404, and concrete steps are as follows:
A. in 100 μ l Agrobacterium competent cell LBA4404, add approximately 1 μ g plasmid DNA, mix gently ice bath 30min;
B. quick-frozen 1min in liquid nitrogen, is placed in 37 DEG C of water-bath incubation 5min immediately;
C. add 800 μ l YEB liquid nutrient mediums, 28 DEG C of 150rpm cultivate 4-6h;
D. it is dull and stereotyped upper that thalline is coated the YEB selection that contains 50mg/L kantlex (Kanamycin Sulfate) and 100mg/L Streptomycin sulphate (streptomycin), is inverted for 28 DEG C and cultivates two days.
E. picking list bacterium colony, is inoculated in (containing 50mg/L Kan and 100mg/LStr) in YEB liquid nutrient medium, 28 DEG C of concussion overnight incubation.
Extract the plasmid the order-checking that import the Agrobacterium that has MSTMT 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 MSTMT.
Two, experimental technique
1, the cultivation of Agrobacterium
Importing there is is the Agrobacterium of MSTMT gene on the solid medium that contains 50mg/LKan and 100mg/L Str, 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 YEB liquid nutrient medium that contains 50mg/L Kan and 100mg/L Str 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 MSTMT gene
Picking list bacterium colony on flat board, is inoculated in the YEB liquid nutrient medium that 20ml contains 50mg/L Kan and 100mg/L Str, and on constant-temperature table, in 28 DEG C, 180rpm cultivates.The bacterium liquid that later takes a morsel for two days, with 1: 50-1: 100 dilution proportion, in the YEB liquid nutrient medium containing 50 μ g/ml Kan and 100 μ g/ml Str, is cultivated 6-12h to logarithmic phase for 28 DEG C.By microorganism collection, in centrifuge tube, the centrifugal 10min enrichment of 4,000rpm thalline, abandons supernatant, does not more contain the resuspended thalline of SH liquid nutrient medium of antibiotic improvement with about 20ml, makes the OD of bacterium liquid
600value is for 0.6-0.8, stand-by.
3, the preparation of explant:
The alfalfa cotyledon of 4-5 days sizes is cut from aseptic seedling with scalpel, be cut into the long fritter of 3-4mm, be put in the SH liquid nutrient medium of improvement, prevent 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, reclaim explant.The explant of recovery is put in step 2 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, sucks unnecessary bacterium liquid, 28 DEG C of dark cultivations 4 days in the common culture medium of blank.
Explant after cultivating altogether 4 days, transfers to and contains 2mg/L 2, and on the SH solid medium of the improvement of 4-D, 0.2mg/L KT, 40mg/L Totomycin and 300mg/L Cef, induction produces callus; After cultivating 20 days, the callus that induction is produced is transferred to and is contained 0.2mg/L KT, 2g/L caseinhydrolysate, and on the UM substratum of 50mg/L Totomycin and 300mg/L Cef, 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 of improvement;
Callus induction substratum: the SH substratum+2mg/L 2 of improvement, 4-D+0.2mg/L KT;
Embryoid induction substratum: UM substratum+0.2mg/L KT+2g/L caseinhydrolysate+50mg/L Hyg+300mg/L Cef.
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 1.
Embodiment 2
Detect the alpha-tocopherol content in the alfalfa that turns the pCAMBIA1302 carrier that is inserted with MSTMT gene obtaining in the embodiment 1 of the alfalfa that turns empty carrier that obtains in the comparative example 1 of 10 strains and 10 strains, concrete grammar is as follows:
A. the preparation of sample: after drying and pulverize for alfalfa, cross 40 order mesh screens, accurately take 0.5g in hydrolysis pipe, add 10mL 6% ethanol pyrogallol solution, ultrasonic extraction 10min, adds 2mL 60% potassium hydroxide solution, and adds the tocol solution of 100 μ L 1mg/ml, as interior mark, the tocol content that final sample is measured in liquid is 10 μ g/ml.Be placed in 70 DEG C of water-bath saponification 30min, after taking-up as for cooling in ice bath.Cooled sample saponification liquor is all transferred in separating funnel, added 20mL 2% sodium chloride solution, then use twice of 10mL n-hexane/ethyl acetate (85/15, V/V) solution extraction.In organic phase, add a small amount of anhydrous sodium sulphate.Draw organic phase 1ml in centrifuge tube, the centrifugal 15min of 14000rpm/min, gets supernatant liquor 100 μ l upper machine in HPLC bottle and measures.
B. drawing standard curve: accurately take alpha-tocopherol standard substance 0.0500g, in 50mL volumetric flask, make standard stock solution with n-hexane dissolution constant volume.By alpha-tocopherol standardized solution with being made into the alpha-tocopherol standard operation liquid that mass concentration is 0.10,1.00,2.00,4.00,6.00,8.00,10.00,20.00 μ g/mL after normal hexane stepwise dilution, sample introduction 20 μ L measure, taking tocopherol absolute content as X-coordinate, peak area is ordinate zou, drawing standard curve;
C. measure: get the sample test solution of preparing in 20 μ L steps A and measure under following chromatographic condition, each material is surveyed 3 times and repeated.
Chromatographic condition: chromatographic column: Hypersil SI 200 silicagel columns (200mm × 2.1mm.i.d., m) moving phase of 5 μ: normal hexane: Virahol=98: 2 (V/V); Column temperature: 25 DEG C; Flow velocity: 0.3ml/min; Fluorimetric detector wavelength: Ex=195nm, Em=330nm; Sample size: 20 μ L.
Result shows, the alpha-tocopherol content turning in the alfalfa of pCAMBIA1302 empty carrier is below 0.10mg/100g, and turns more than alpha-tocopherol content in the alfalfa of the pCAMBIA1302 carrier that is inserted with MSTMT gene on average reaches 3.08mg/100g.
This shows, the expression of alfalfa gama-tocopherol methyl transferase gene provided by the invention (MSTMT) in alfalfa can significantly improve the content of alpha-tocopherol in alfalfa.The content that is found to be the alpha-tocopherol in raising Dominant Species of Forage Grass plant (particularly leguminous plants, as alfalfa) of this gene provides genetic resources, in the research of genetically engineered Improvement plant, will play a significant role.
Claims (6)
1. an albumen of being encoded by alfalfa gama-tocopherol methyl transferase gene, is characterized in that, the aminoacid sequence of this albumen is as shown in SEQ ID No:2.
2. the gene of alfalfa gama-tocopherol methyl transferase of encoding, it is characterized in that, the nucleotide sequence of this gene is as shown in SEQ ID No:1, or the nucleotides sequence of this gene is classified the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2 as.
3. an expression vector, is characterized in that, this expression vector contains gene claimed in claim 2.
4. a method of cultivating alfalfa, is characterized in that, the method comprises gene claimed in claim 2 is imported in alfalfa cell, obtains alfalfa cell and transfer-gen plant that alpha-tocopherol content is high.
5. albumen claimed in claim 1, gene claimed in claim 2 or expression vector claimed in claim 3 are in the application of cultivating in the plant that alpha-tocopherol content is high.
6. application according to claim 5, wherein, described plant is alfalfa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110343669.7A CN103087999B (en) | 2011-11-03 | 2011-11-03 | Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110343669.7A CN103087999B (en) | 2011-11-03 | 2011-11-03 | Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103087999A CN103087999A (en) | 2013-05-08 |
| CN103087999B true CN103087999B (en) | 2014-09-03 |
Family
ID=48201132
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110343669.7A Expired - Fee Related CN103087999B (en) | 2011-11-03 | 2011-11-03 | Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103087999B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472324A (en) * | 2002-08-01 | 2004-02-04 | 中国农业科学院生物技术研究所 | Encoding DNA sequence and amino sequence of sunflower gama tocopherol methyl transferases and use thereof |
| CN1578835A (en) * | 2001-10-25 | 2005-02-09 | 孟山都技术有限公司 | Aromatic methyltransferases and uses thereof |
| CN1807608A (en) * | 2006-01-24 | 2006-07-26 | 中国农业科学院生物技术研究所 | Gama-tocopherol methyl transferase gene, its coding vector and uses |
| WO2007059077A2 (en) * | 2005-11-14 | 2007-05-24 | E.I.Du Pont De Nemours And Company | Compositions and methods for altering alpha- and beta-tocotrienol content |
| CN101514346A (en) * | 2009-02-19 | 2009-08-26 | 上海交通大学 | Lettuce gamma-tocopherol methyltransferase protein coded sequence |
| CN101842488A (en) * | 2007-10-04 | 2010-09-22 | 纳幕尔杜邦公司 | Compositions and methods for altering alpha- and beta-tocotrienol content using multiple transgenes |
-
2011
- 2011-11-03 CN CN201110343669.7A patent/CN103087999B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1578835A (en) * | 2001-10-25 | 2005-02-09 | 孟山都技术有限公司 | Aromatic methyltransferases and uses thereof |
| CN1472324A (en) * | 2002-08-01 | 2004-02-04 | 中国农业科学院生物技术研究所 | Encoding DNA sequence and amino sequence of sunflower gama tocopherol methyl transferases and use thereof |
| WO2007059077A2 (en) * | 2005-11-14 | 2007-05-24 | E.I.Du Pont De Nemours And Company | Compositions and methods for altering alpha- and beta-tocotrienol content |
| CN1807608A (en) * | 2006-01-24 | 2006-07-26 | 中国农业科学院生物技术研究所 | Gama-tocopherol methyl transferase gene, its coding vector and uses |
| CN101842488A (en) * | 2007-10-04 | 2010-09-22 | 纳幕尔杜邦公司 | Compositions and methods for altering alpha- and beta-tocotrienol content using multiple transgenes |
| CN101514346A (en) * | 2009-02-19 | 2009-08-26 | 上海交通大学 | Lettuce gamma-tocopherol methyltransferase protein coded sequence |
Non-Patent Citations (5)
| Title |
|---|
| Cloning and analysis of a Y-tocopherol methyltransf erase gene from Brassica oleracea and the function of its recombinant protein.《Progress in Natural Science》.2003,(第07期), |
| Cloning and analysis of a Y-tocopherol methyltransf erase gene from Brassica oleracea and the function of its recombinant protein;《Progress in Natural Science》;20030710(第07期);511-516 * |
| Y.Hu et al.gamma tocopherol methyltransferase[medicago truncatula].《GenBank:AAX63740.1》.2005, * |
| 拟南芥γ-生育酚甲基转移酶启动子在转基因烟草中的表达特性分析;朱永兴等;《中国农学通报》;20060828(第08期);65-68 * |
| 朱永兴等.拟南芥γ-生育酚甲基转移酶启动子在转基因烟草中的表达特性分析.《中国农学通报》.2006,(第08期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103087999A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101855355B (en) | There is the plant of the Correlated Yield Characters of raising and the method for preparing this plant | |
| Zou et al. | Expression analysis of nine rice heat shock protein genes under abiotic stresses and ABA treatment | |
| CN101812124A (en) | Plant stress-resistance-associated protein TaSnRK2.8, coding genes and application thereof | |
| CN108047320A (en) | The application of Protein G mMYB12 and encoding gene in isoflavones accumulation and salt tolerance is improved | |
| CN104988140A (en) | Promoter from rice and application thereof | |
| CN103103166B (en) | Plant stress tolerance associated protein TaNCED1, and coding gene and application thereof | |
| CN103031282B (en) | Medicago sativa L. anthocyanin reductase gene, protein coded by same and application | |
| AU2014287556A1 (en) | Abiotic stress resistance | |
| CN105420245A (en) | Salt-stress induced gene OsEAR1 of plants and encoding protein and application thereof | |
| CN107267521A (en) | A kind of cabbage type rape and NAC87 transcription factor genes and its application in arabidopsis | |
| CN101748144B (en) | Torch pear haloduric gene PpGST and application thereof | |
| CN104046639B (en) | Wheat methionine sulfoxide reductase gene TaMsrB3.1 and application thereof | |
| Muthukumar et al. | Transcriptional activation and localization of expression of Brassica juncea putative metal transport protein BjMTP1 | |
| CN110964740B (en) | Preparation method and application of tobacco with high flavonol content | |
| CN112430584A (en) | Du pear ubiquitin ligase gene, encoding protein and application thereof in plant drought-resistant genetic improvement | |
| CN103951740A (en) | Bermuda grass CCAAT transcription factor CdtNF-YC1 as well as coding gene and application thereof | |
| CN101358193A (en) | Identification of specificity promoter for rice leaf senescence | |
| CN103087999B (en) | Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use | |
| CN101001956B (en) | Preparation of organisms with faster growth and/or higher yield | |
| CN105505956B (en) | A kind of application of alfalfa Single-chip microcomputer dioxygenase gene | |
| CN102604963A (en) | Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene | |
| CN105087587A (en) | Peanut AhFAD2-2A gene promoter and application of peanut AhFAD2-2A gene promoter | |
| CN113024645B (en) | Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development | |
| CN110218248B (en) | Floral firewood film Na+ /H+Reverse transport protein, coding gene and application thereof | |
| CN111996197A (en) | Salt-tolerant gene and protein of pyrus betulaefolia, recombinant vector and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140903 Termination date: 20201103 |