CN103087999A - 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
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Abstract
本发明公开了一种由紫花苜蓿γ-生育酚甲基转移酶基因编码的蛋白，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γ-生育酚甲基转移酶活性的氨基酸序列。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α-生育酚含量高的紫花苜蓿的方法和它们的应用。本发明提供的紫花苜蓿γ-生育酚甲基转移酶基因(MSTMT)在紫花苜蓿中的表达可以显著提高紫花苜蓿中α-生育酚的含量。该基因的发现为提高主要牧草植物(特别是豆科植物，如紫花苜蓿)中α-生育酚的含量提供了基因资源，在基因工程改良牧草的研究中将发挥重要作用。
The invention discloses a protein encoded by alfalfa gamma-tocopherol methyltransferase 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 The amino acid sequence of the amino acid sequence still has γ-tocopherol methyltransferase activity after one or several amino acid residues are substituted, deleted or added. The invention also relates to the coding gene of the protein, as well as the expression vector and cell containing the gene, and also relates to a method for cultivating alfalfa with high alpha-tocopherol content and their application. The expression of the alfalfa gamma-tocopherol methyltransferase gene (MSTMT) provided by the invention in the alfalfa can significantly increase the alpha-tocopherol content in the alfalfa. The discovery of this gene provides a gene resource for increasing the content of α-tocopherol in main forage plants (especially leguminous plants, such as alfalfa), and will play an important role in the research of genetic engineering improvement of forage.
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 and the cell that contain described alfalfa gama-tocopherol methyl transferase gene, also relate to simultaneously a kind of method of cultivating alfalfa, and the application in the plant of cultivating high content of alpha-tocopherol content with the expression vector that contains this gene and cell of the albumen of alfalfa gama-tocopherol methyl transferase gene and coding thereof.
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, add in feed vitamin-E have improve animal immunizing power, improve meat, improve animal reproductive performance, alleviate the unusual effect 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 mainly is present in plant, obviously is better than synthesising complex E in physiologically active and security.
Natural VE is comprised of 8 kinds of tocopherol isomers.Can be divided into tocopherol and the large class of tocotrienol two according to the 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 all is 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, increases substantially high reactivity alpha-tocopherol content in plant (particularly forage grass), will have 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 has been cloned from Arabidopis thaliana, blue-green algae etc. and has been obtained 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 it is 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 it is forwarded in the forage grass that comprises alfalfa, can improve animal immunizing power, improve meat, the aspects such as reproductive performance that improve animal have great importance.
Summary of the invention
The present invention is based on the research to the relevant the Molecular Biology Mechanism of vitamin-E route of synthesis in alfalfa, 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 also are provided on the other hand, and the method for cultivating the alfalfa of high content of alpha-tocopherol content, their application also is 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, and perhaps this albumen has the aminoacid sequence that still has the gama-tocopherol methyl transferase activity with after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.
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, and perhaps 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 the alfalfa cell, obtains alpha-tocopherol content high alfalfa cell and transfer-gen plant.
The present invention also provide alfalfa gama-tocopherol methyl transferase gene provided by the invention and coding thereof albumen, contain the expression vector of described gene, the application of transgenic cell in cultivating the high plant of alpha-tocopherol content that contains described gene.
The gama-tocopherol methyl transferase gene that the present invention clones from alfalfa is for improving Dominant Species of Forage Grass plant (leguminous plants particularly, as alfalfa) in the content of alpha-tocopherol genetic resources is provided, will play a significant role in the research of genetically engineered Improvement plant.
Description of drawings
Fig. 1 has shown the structure of in embodiment 1, Medicago sativa being carried out the agriculture bacillus mediated gama-tocopherol methyl transferase gene conversion pCAMBIA1302 carrier that is inserted with gama-tocopherol methyl transferase gene used.
Embodiment
The invention provides a kind of albumen by alfalfa gama-tocopherol methyl transferase gene coding, wherein, this albumen has the aminoacid sequence shown in SEQ ID No:2, and perhaps this albumen has the aminoacid sequence that still has the gama-tocopherol methyl transferase activity with after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.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, and perhaps 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 the contriver screens by the following method and obtains:
(the Genbank registration number is: BT051703) (the RT-PCR primer is conservative territory design primer: F:5 '-TCTCAGCCCTGTTCAAGC-3 ' according to cutting the gama-tocopherol methyl transferase gene of having cloned in the type clover; R:5 '-CAAAGGGAGACCAATCTG-3 '), from alfalfa (Medicago sativa L.cv.Zhongmu No.1, No. one, middle lucerne, poultry east grass cultivation science and technology limited Company in Beijing) increase in, reclaim the fragment that the expection size is 400bp (reclaiming the fragment of this length after the leakage of electricity swimming), the fragment that reclaims is connected into PMD18-T Vector (Takara), order-checking: sequencing result carries out BLASTx and analyzes in the nonredundancy albumen database of NCBI; 2 sequences relevant with the gama-tocopherol methyl transferase class in analytical results are carried out 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 the 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 5 ' RACE and 3 ' RACE clone, obtained at last this full length gene sequence (SEQ ID NO:1).Wherein, the method for gene clone is the normal experiment operation of biology field, and 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 ℃ 3 minutes, then be put in 42 ℃ cooling 2 minutes, of short duration centrifugal collection liquid;
D. add the SMARTer IIA oligo of 1 μ l in the B in the cDNA of 5 ' RACE;
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 for preparing is put in 42 ℃ 90 minutes, in last 70 ℃ 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. the PCR for the preparation of 5 ' RACE reacts: 2.5 μ l 5 ' RACE-ready cDNA, 5.0 μ lUPM (10 *), 1.0 μ l GSP1 5 '-GTTGTAGGGATTCTTCATTCGGGGC-3 '; 41.5 the damping fluid for preparing in the step H of 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 '; 41.5 the damping fluid for preparing in the step H of μ l.
The RACE reaction system is: 94 ℃ 30 seconds, 72 ℃ 3 minutes, totally 5 circulations: 94 ℃ 30 seconds, 70 ℃ 3 minutes, 72 ℃ 3 minutes, totally 5 circulations; 94 ℃ 30 seconds, 68 ℃ 30 seconds, 72 ℃ 3 minutes, totally 20 circulations.
Get the PCR product and carry out electrophoresis detection on the sepharose of 1.0 % by weight, reclaim the purpose band, connect and reclaim on product and pEGM T-Easy carrier, transform the bacillus coli DH 5 alpha competent cell, extract plasmid and 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, the nucleotide sequence of the aminoacid sequence shown in SEQ ID No:2 of perhaps encoding.Gene provided by the invention can be building up in expression vector by existing method, can add any enhancing promotor or inducible promoter before its transcription initiation Nucleotide.
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, the nucleotide sequence of the aminoacid sequence shown in SEQ ID No:2 of perhaps encoding.Described cell can be monocotyledonous cell, also can be the cell of dicotyledons, as the cell of paddy rice, wheat, corn, cucumber, tomato or clover etc.Be preferably the clover cell, more preferably the alfalfa cell.
The present invention also provides a kind of method of cultivating the high alfalfa of alpha-tocopherol content, wherein, the method comprises that the gene that will have the Nucleotide shown in SEQ ID No:1 imports in the alfalfa cell, obtains alfalfa cell and transfer-gen plant that alpha-tocopherol content improves.
To there be gene provided by the invention to import to the method that method in the alfalfa cell is genetically engineered field routine according to of the present invention, such as conventional biological method transformed plant cells or the tissue such as leading by Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity, agriculture bacillus mediated, afterwards the vegetable cell that transforms is cultivated into plant.
The present invention also provide alfalfa gama-tocopherol methyl transferase gene provided by the invention and coding thereof albumen, contain the expression vector of described gene, the application of transgenic cell in cultivating the high plant of alpha-tocopherol content that contains described gene.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, poultry east grass cultivation science and technology limited Company in Beijing) for the examination alfalfa variety.
The aseptic seedling cotyledon that germinateed 7 days is the acceptor material of genetic transformation.
2, agrobacterium strains and plasmid vector
Agrobacterium strains used is agrobacterium tumefaciens: LBA4404 (sky, Beijing bounties Gene Tech. Company Limited)
The Agrobacterium substratum:
Plasmid vector: pCAMBIA1302 (available from the uncommon lucky Bioisystech Co., Ltd in Shanghai).the MsTMT gene is inserted in pCAMBIA1302, concrete steps are: design 5 ' end comprise the upstream primer of NcoI restriction enzyme site and the downstream primer of SpeI restriction enzyme site (F:5 '-CATGCCATGGATGGCGGCGGTGAAAGAAGC-3 ', R:5 '-GGACTAGTTCATTGACCCTCTGCATTTTCAGGC-3 ', the total length reading frame of MsTMT increases from the cDNA template with this primer, 1302 carriers and full length fragment reclaim product after NcoI and SpeI double digestion, connect through the T4 DNA ligase, obtain being inserted with the pCAMBIA1302 carrier of MSTMT gene, it contains the CaMV35s promotor, MSTMT gene (SEQ ID NO:1) and a hygromycin resistance selection markers, can be by the transfer-gen plant of hygromycin selection preliminary evaluation acquisition when carrying out genetic transformation.The structure of plasmid vector that contains the purpose fragment is shown in Figure 1.
The pCAMBIA1302 carrier that is inserted with the MSTMT gene is imported in agrobacterium tumefaciens lba4404, and concrete steps are as follows:
A. add approximately 1 μ g plasmid DNA in 100 μ l Agrobacterium competent cell LBA4404, mixing gently, ice bath 30min;
B. quick-frozen 1min in liquid nitrogen, be placed in 37 ℃ of water-bath incubation 5min immediately;
C. add 800 μ l YEB liquid nutrient mediums, 28 ℃ of 150rpm cultivate 4-6h;
D. thalline is coated on the YEB selection flat board that contains 50mg/L kantlex (Kanamycin Sulfate) and 100mg/L Streptomycin sulphate (streptomycin), is inverted for 28 ℃ and cultivates two days.
E. picking list bacterium colony is inoculated in the YEB liquid nutrient medium and (contains 50mg/L Kan and 100mg/LStr), 28 ℃ of concussion overnight incubation.
Extract the plasmid and the order-checking that import the Agrobacterium that the MSTMT gene is arranged, 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 the Agrobacterium of MSTMT gene draw flat board on the solid medium that contains 50mg/LKan and 100mg/L Str, is put in incubator 28 ℃ of cultivations.Two days later, picking list bacterium colony from the flat board is inoculated in the 20ml YEB liquid nutrient medium that contains 50mg/L Kan and 100mg/L Str 180rpm, 28 ℃ of cultivations.Draw flat board with the bacterium liquid that shakes, 28 ℃ of cultivations after growing single bacterium colony, are put in 4 ℃ of preservations with 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 in 28 ℃, 180rpm cultivates on constant-temperature table.The bacterium liquid that took a morsel later in two days, with 1: 50-1: 100 dilution proportion is cultivated 6-12h to logarithmic phase for 28 ℃ in the YEB liquid nutrient medium that contains 50 μ g/ml Kan and 100 μ g/ml Str.In centrifuge tube, the centrifugal 10min enrichment of 4,000rpm thalline is abandoned supernatant with microorganism collection, 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, and is stand-by.
3, the preparation of explant:
The alfalfa cotyledon of 4-5 days sizes is downcut 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, with the explant that is soaked in the SH liquid nutrient medium of improvement, fall in the filter of the bacterium of having gone out, reclaim explant.The explant that reclaims is put in step 2 in ready bacterium liquid, carries out During Agrobacterium.Every for a moment, shake several under, help 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 ℃ of dark cultivations 4 days on the common culture medium of blank.
Explant after cultivating altogether 4 days is transferred to and is contained 2mg/L 2, on the SH solid medium of the improvement of 4-D, 0.2mg/L KT, 40mg/L Totomycin and 300mg/L Cef, induces the generation callus; After cultivating 20 days, the callus of inducing generation is transferred to contained 0.2mg/L KT, 2g/L caseinhydrolysate, on the UM substratum of 50mg/L Totomycin and 300mg/L Cef, induce the generation embryoid; (approximately cultivated 30 days) after the embryoid maturation, embryoid is transplanted on the 1/2MS substratum, root induction, thus complete the regeneration of plant.
Respectively go on foot the composed as follows of substratum in regenerative process:
Be total to culture medium: 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 Examples 1
Turn the acquisition of empty carrier control plant
Transform Agrobacterium with plasmid pCAMBIA1302, obtain the restructuring Agrobacterium, with restructuring Agrobacterium-mediated Transformation alfalfa, obtain turning the adjoining tree of empty carrier, method such as embodiment 1.
Embodiment 2
Detect the alpha-tocopherol content in the alfalfa that turns the pCAMBIA1302 carrier that is inserted with the MSTMT gene that obtains in the embodiment 1 of the alfalfa that turns empty carrier that obtains in the Comparative Examples 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 the 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 ℃ of water-bath saponification 30min, take out rear as for cooling in ice bath.Cooled sample saponification liquor is all transferred in separating funnel, added 20mL 2% sodium chloride solution, (85/15, V/V) solution extraction is twice then to use the 10mL n-hexane/ethyl acetate.Add a small amount of anhydrous sodium sulphate in organic phase.Draw organic phase 1ml in centrifuge tube, the centrifugal 15min of 14000rpm/min gets supernatant liquor 100 μ l upper machine in the HPLC bottle and measures.
B. drawing standard curve: accurately take alpha-tocopherol standard substance 0.0500g, in the 50mL volumetric flask, make the standard stock solution with the n-hexane dissolution constant volume.The alpha-tocopherol standardized solution is 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 with the normal hexane stepwise dilution, sample introduction 20 μ L measure, take the tocopherol absolute content as X-coordinate, peak area is ordinate zou, the drawing standard curve;
C. measure: get the sample test solution for preparing in 20 μ L steps A and measure under following chromatographic condition, each material is surveyed 3 times and is repeated.
Chromatographic condition: chromatographic column: Hypersil SI 200 silicagel columns (200mm * 2.1mm.i.d., 5 μ m) moving phase: normal hexane: Virahol=98: 2 (V/V); Column temperature: 25 ℃; Flow velocity: 0.3ml/min; Fluorimetric detector wavelength: Ex=195nm, Em=330nm; Sample size: 20 μ L.
Result shows, the alpha-tocopherol content that turns 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 the 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 being found to be of this gene improved the alpha-tocopherol in Dominant Species of Forage Grass plant (particularly leguminous plants, as alfalfa) provides genetic resources, will play a significant role in the research of genetically engineered Improvement plant.
Claims (10)
1. albumen by alfalfa gama-tocopherol methyl transferase gene coding, it is characterized in that, this albumen has the aminoacid sequence shown in SEQ ID No:2, and perhaps this albumen has the aminoacid sequence that still has the gama-tocopherol methyl transferase activity with after replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID No:2 through one or several amino-acid residue.
2. albumen according to claim 1, wherein, this albumen has the aminoacid sequence shown in SEQ ID No:2.
3. an alfalfa gama-tocopherol methyl transferase gene, is characterized in that, this gene has the nucleotide sequence shown in SEQ ID No:1, and perhaps this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.
4. gene according to claim 3, wherein, this gene has the nucleotide sequence shown in SEQ ID No:1.
5. an expression vector, is characterized in that, this expression vector contains gene claimed in claim 3.
6. a transgenic cell, is characterized in that, this transgenic cell contains gene claimed in claim 3.
7. transgenic cell according to claim 6, wherein, described transgenic cell is the alfalfa transgenic cell.
8. a method of cultivating alfalfa, is characterized in that, the method comprises gene claimed in claim 3 is imported in the alfalfa cell, obtains alpha-tocopherol content high alfalfa cell and transfer-gen plant.
9. albumen claimed in claim 1, gene claimed in claim 3, expression vector claimed in claim 5, the application of transgenic cell claimed in claim 6 in cultivating the high plant of alpha-tocopherol content.
10. application according to claim 9, wherein, described plant is alfalfa.
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