CN1232639C - Gamma-tocopherol tranferase, gene and use thereof - Google Patents

Abstract

The present invention provides a whole-length cDNA sequence for gamma-tocopherol methyl transferase (gamma-TMT) cloned from the seedling of brassica oleracea L. Var, capitata L. Consequently, a new structure gene with a code of gamma-TMT and gamma-TMT are provided. Prokaryotic expression plasmids, plant constitutions and specific expression plasmids in plant seeds are configured. The gamma-TMT structure gene is converted into microbes with high efficiency and stable expression, and alpha-tocopherol is generated by methylation. In addition, plasmids containing plant constructive expression and a transgenic plant with specificity expression in vegetable seeds are obtained. The content of the alpha-tocopherol in transgenic plant tissue and the seeds is obviously enhanced.

Description

Gama-tocopherol methyl transferase and gene thereof and purposes

Technical field

The invention belongs to gama-tocopherol methyl transferase (γ-tocopherolmethyltransferase, the genetically engineered field of γ-TMT).Specifically, relate to a kind of gama-tocopherol methyl transferase from cabbage (Brassica oleracea L.Var.capitata L.), and gama-tocopherol methyl transferase gene and its structure gene and recombinant plasmid thereof; Relate to it has composing type or seed-specific expression in plant recombinant plasmid, with content that improves alpha-tocopherol in plant and the corresponding product and the distribution (particularly improving the content of alpha-tocopherol in seed and the oil) that changes alpha-tocopherol; Simultaneously, also relate to the recombinant plasmid that in microorganism, has specific expressed γ-TMT, utilize bio-reactor catalytic production alpha-tocopherol.

Background technology

Vitamin-E (claiming tocopherol again) divides natural and synthetic two kinds.In naturally occurring four kinds of tocopherols, the biological activity of alpha-tocopherol the highest (α-, β-, γ-, the relative reactivity of Delta-Tocopherol is respectively 100%, 50%, 10% and 3%).Natural VE chemically is being often referred to alpha-tocopherol, is only by fat-soluble antioxidant of photosynthetic organism synthetic one class and nutritious supplementary.The alpha-tocopherol that human body absorbs 7-9mg every day is that to keep muscle, the central nervous system vascular system normal physiological function of unifying necessary.Clinical study over nearly 20 years shows, the alpha-tocopherol that day absorbs 70-250mg can improve immunologic function, prevent or delay the process of many human degenerative disorders, reduce the danger of suffering from cardiovascular disorder and some cancer, can be used for treating senile dementia, hypertension, coronary heart disease, myocardial infarction, arteriosclerosis, thrombus and infertility etc. clinically.Along with the progressively favor of people to green food, Organic food, natural VE is widely used in medicine, healthcare products, food, nutritious prod, makeup and the fodder industry as nutritious supplementary and antioxidant.The raising of alpha-tocopherol level can prolong the shelf time of fresh and producing vegetable product and improve its stability in the crop.In food, add vitamin-E, not only can improve its nutritive value, and can prevent the oxidative rancidity of fat in the food, improve storage period greatly; In cosmetic industry, vitamin-E is the effective constituent in anti-sun shine cream and the essence, adds vitamin-E and have deodorising effect in shampoo water; In addition, in the feed of pig, ox, poultry, add vitamin-E, the shelf time of meat products after can significantly improving the quality of meat and prolonging processing.

Development along with modern medicine and trophology, a series of experimentation on animalies also confirm, alpha-tocopherol (natural VE) is by human body preferential absorption and utilization, no matter be all obviously to be better than the synthetic vitamin-E on the physiologically active or in security, its activity is about 1.3-1.4 times of synthesising complex E, and price in the international market is about synthetic 2-3 doubly.Along with the continuous expansion of vitamin-E in aspect purposes such as medicine, healthcare products, food, nutritious prod, makeup and feeds, the demand of natural VE constantly increases, and a year consumption reaches about 3,000 tons at present, and a year demand growth is 10%.And the present vitamin-E product of China almost is synthetics entirely.

Alpha-tocopherol mainly extracts from vegetables oil or its refining byproduct, and the content of alpha-tocopherol is determined by floristics.The total amount and the component difference of tocopherol are very big in the different plant tissues, and in the leaf tissue of green, alpha-tocopherol is the abundantest tocopherol of content, but total tocopherol levels of these tissues very low (10-50 μ g/g fresh weight).Different with photosynthetic tissue, the non-chlorenchyma of plant (as the seed of most of oil crops) often contains total tocopherol (500-2 of high density, 000 μ g/g fresh weight), but the content of alpha-tocopherol very low (84-200 μ g/g fresh weight) wherein, the overwhelming majority is its biosynthesizing precursor---Gamma-Tocopherol, the content of alpha-tocopherol is 7%-10% only in some main vegetables oil (as soya-bean oil, peanut oil, rapeseed oil), and its biosynthesizing precursor---Gamma-Tocopherol is up to 67%-70%.For this reason, alpha-tocopherol is to the ratio of Gamma-Tocopherol in change crop and the product thereof, promptly improve the content of alpha-tocopherol and changed the alpha-tocopherol distribution, particularly the content that improves alpha-tocopherol in seed and the oil has been had crucial society and economic benefit.U.S. scientist has only cloned γ-TMT gene from model plant Arabidopis thaliana and blue-green algae, alpha-tocopherol content increases in changeing γ-TMT gene Arabidopis thaliana.So far not seeing has the new gene that obtains γ-TMT from cash crop, and it is transformed in the cash crop of non-model plant goes, with improve alpha-tocopherol in crop and its product content and change the report of the distribution of alpha-tocopherol.

Summary of the invention

The object of the invention is to provide a kind of γ-TMT gene (containing structure gene) with the nucleotide sequence shown in the sequence table SEQ NO.1; Have a kind of gama-tocopherol methyl transferase of the aminoacid sequence shown in the sequence table SEQ NO.2 and recombinant plasmid p3END-T, p5END-T, pTMT.

Another object of the present invention provides a kind of γ from cabbage (Brassica oleracea L.Var.capitata L.)-TMT structure gene and makes up new plant constitutive expression recombinant plasmid pBin-TMTL or seed-specific expression recombinant plasmid p7S-TMTL, and in microorganism the recombinant plasmid pET-TMT of specifically expressing; A kind of high transgenic plant of serial alpha-tocopherol content of cultivating are provided, particularly oil crops such as soybean, rape, peanut etc. improve the content of alpha-tocopherol in the plants and plant product and the new way of change alpha-tocopherol distribution (being meant the content that has improved alpha-tocopherol in seed and the oil especially).The present invention not only will help people to obtain high-caliber alpha-tocopherol from daily meals, reach and build up health, the purpose that diseases prevention combines with health care, can also reduce simultaneously the cost of suitability for industrialized production natural vitamin E product greatly, improve the output and the quality of product, be with a wide range of applications at aspects such as medicine, healthcare products, food, nutritious prod, makeup and feeds.

Adopt 3 ', 5 '-RACE and RT-PCR technology, the full length cDNA sequence of from cabbage (Brassica oleraceaL.Var.Capitata L.) seedling terminal bud, having cloned the γ-TMT shown in sequence table No.1.This cDNA long 1,265bp, comprise one 1, the open reading frame of 044bp, coding comprises that chloroplast(id) leads the protein of peptide (47 amino acid) and two S-adenosylmethionine binding domainss (SAM-binding domain) 347 amino acid composition γ-TMT shown in sequence table No.2.This sequence is compared with the model plant Arabidopis thaliana of U.S. scientist report and γ-TMT gene of blue-green algae (Synechocystis PCC6803), nucleotide sequence homology is respectively 83.5% and 37.7%, the deduced amino acid homology is respectively 86.5% and 41.8%, the cDNA complete sequence of the present invention's γ-TMT gene that the clone obtains from non-model plant first.The main difference of amino acid on primary structure of the γ-TMT genes encoding of the Arabidopis thaliana of the amino acid of γ of the present invention-TMT genes encoding and U.S. scientist report: leading peptide moiety at 47 amino acid whose chloroplast(id)s has 22 amino acid different; (10 amino acid) has 1 amino acid difference second functional zone; With the blue-green algae γ-difference of TMT gene on the amino acid primary structure: it is 47 amino acid that chloroplast(id) of the present invention is led peptide, and it is 25 that the bacterium of blue-green algae is led peptide; Wherein have only 7 amino acid identical; (9 amino acid) has 3 amino acid differences in first functional zone; Second functional zone 4 amino acid differences are arranged.Generally speaking, the three leads peptide moiety at chloroplast(id) only has 3 amino acid identical; In first functional zone 3 amino acid differences are arranged, 4 amino acid differences are arranged second functional zone.Should be noted that, the amino acid of the expressed enzyme molecule of gama-tocopherol methyl transferase gene of the present invention is carried out one or more amino acid to be replaced, inserts or lack resulting functional analogue and also can reach purpose of the present invention, thereby the present invention also comprises having with the aminoacid sequence shown in the Seq NO.2 to have 80% homology at least, the homology that preferably has gama-tocopherol methyl transferase 90%, but have the active functional analogue of gama-tocopherol methyl transferase simultaneously.

Utilize plant constitutive expression carrier pBin438 (Li Taiyuan, Yingchuan, field, Qin Xiaofeng etc. 1994, the research of efficient insect-resistant transgenic tobacco, Chinese science (B collects) 24 (3): 276-282) made up recombinant plant constitutive expression carrier pBin-TMTL and the recombinant plant seed-specific expression carrier p7S-TMTL that contains this structure gene with seed-specific expression carrier p7S438.Through transforming recombination bacillus coli JM109 and reorganization Agrobacterium LBA4404-Bin-TMTL, LBA4404-7S-TMTL, GV3101-Bin-TMTL and the GV3101-7S-TMTL that obtains to contain pBin-TMTL and p7S-TMTL respectively.With soybean aseptic seedling cotyledonary node is explant, through agriculture bacillus mediated, obtains that PCR, Southern blot, RT-PCR and Western blot are male and the TO that can normally yield positive results for the genetically engineered soybean strain is.Alpha-tocopherol content has improved 4 times at least in the seed of HPLC analysis genetically engineered soybean.T1 also can normally be yielded positive results for the PCR positive plant of genetically engineered soybean.Therefore, the present invention improves its alpha-tocopherol content for transgenic plant and has opened up a new way.

Another purpose of the present invention provides a kind of enzyme that utilizes recombinant microorganism or utilize recombinant microorganism to produce, and produces the new way of alpha-tocopherol by bio-reactor.For reaching this purpose, adopt general genetic engineering means at present, γ-TMT structure gene is inserted into prokaryotic expression carrier pET30a (Novagen, Cat.No.69909-3) in prokaryotic micro-organisms, preferred intestinal bacteria (Escherichia coli), in e. coli bl21 (DE3), obtained to efficiently express, expression product accounts for 22% of bacterial protein, the determination of activity result of vitro enzyme shows γ-TMT tool gama-tocopherol methyl transferase activity, the catalysis gama-tocopherol methyl generation alpha-tocopherol effectively of expression.External enzyme activity determination comparison shows that, the reorganization bacterium be contain that unloaded contrast bacterium enzyme lives 10 surplus times.Equally, γ-TMT structure gene is inserted carrier for expression of eukaryon pPIC9, in eukaryotic microorganisms, the preferred yeast bacterium, effectively secrete in methanol yeast (being pichia spp, Pichia pastoris) and efficiently express, expression product has higher gama-tocopherol methyl transferase activity equally.

Description of drawings

Fig. 1: the γ-expression of TMT structure gene in intestinal bacteria.

M shows protein molecular weight standard; 1, show the empty carrier pET30a that induces 4h; 2, show the plasmid pET-TMT (arrow indication inductive specific protein leukorrhea) that induces 4h.

Fig. 2: the enzyme activity assay of the γ-TMT of escherichia coli expression.

1, the Gamma-Tocopherol standard substance; 2, the negative control of pET30a; 3, the pET-TMT reaction product; 4, the alpha-tocopherol standard substance.

Fig. 3: the soybean of the commentaries on classics γ-TMT structure gene of Agrobacterium LBA4404-Bin-TMTL mediation.

1, the cotyledonary node explant; 2, explant grows the bud of growing thickly on the MSB1 substratum; 3, taking out of growing on the MSB2 substratum sprouts; 4, taking out sprouts takes root on the MSB substratum; 5, the genetically engineered soybean plant that pods; 6, T1 is for the genetically engineered soybean plant; 7, the seed of genetically engineered soybean.

Fig. 4: the Western Blot of the transgenic arabidopsis of Agrobacterium GV3101-7S-TMTL mediation detects

1, the blade of wild-type Arabidopis thaliana; 2,3, the blade of transgenic arabidopsis; 4, the pod of wild-type Arabidopis thaliana; 5,6, the pod of transgenic arabidopsis; 7, the γ of escherichia coli expression-TMT albumen.

Embodiment

For further understanding above-mentioned purpose of the present invention, specified by following examples, but present embodiment is not as limitation of the invention.

The clone of embodiment 1. gama-tocopherol methyl transferase genes

1) extraction of the total RNA of cabbage

Get the terminal bud 2.5g of 9 days seedlings of sweet ride on Bus No. 11 in the cabbage (Brassica oleracea L.Var.Capitata L.), after the liquid nitrogen grinding, add 15mL rapidly and extract damping fluid (100mmol/L Tris-HCl at the RNA of 65 ℃ of preheatings, pH8.0,2%CTAB, 2%PVP4000,25mmol/LEDTA, 2.0mol/L NaCl, 0.5g/L Spermidine and 2% beta-mercaptoethanol), put upside down mixing.Use isopyknic chloroform, (V/V=24: 1) extracting is twice for the primary isoamyl alcohol mixing solutions, get last supernatant solution, add 1/4 volume 10mol/L LiCl, 4 ℃ of placements are spent the night, 4 ℃ of next day, 10, RNA precipitation in the above-mentioned solution of the centrifugal recovery of 000rpm, precipitation is dissolved in SSTE solution (the 10mmol/L Tris-HCl pH8.0 of 500 μ L again, 1.0mol/L NaCl, 0.5%SDS, 1mmol/LEDTA pH8.0), use chloroform, twice of the extracting again of primary isoamyl alcohol mixing solutions, supernatant liquor adds the dehydrated alcohol of two volumes, behind-70 ℃ of placement 2h, in 4 ℃, 10, the centrifugal 20min of 000rpm, reclaim the RNA precipitation, with 70% washing with alcohol precipitation, dry postprecipitation dissolves with the deionized water of twice sterilization.For preventing the pollution of DNA, total RNA of per 100 μ g adds 2U DNase, and (Promega is USA) with 40U RNase inhibitor, in 37 ℃ of processing 30min, with dna digestion.The ultraviolet spectrometry degree measurement result of RNA solution is A ₂₆₀/ A ₂₈₀1.80, A ₂₆₀/ A ₂₃₀=2.0, electrophoretic analysis estimates that the ratio of the rRNA amount of 28S, 18S is approximately 28S: 18S=2: 1, and this RNA solution can be used as the template of RT-PCR reaction.

2) rapid amplifying (3 ' RACE) of cDNA 3 ' end

In the solution that contains the total RNA of 5 μ g (11 μ L), add the Oligo (dT) of 1 μ L ₁₇(0.5mol/L) primer (5 '-GAGGATCC (T) ₁₇-3 '), place 3min on ice fast behind 70 ℃ of placement 10min, moment is centrifugal.In above-mentioned solution, add 5 * the first chain damping fluids, 4 μ L successively, DTT 2 μ L (0.1mol/L), dNTPs (every kind of dNTP is 10mmol/L) 1 μ L, slight mixing is placed on 42 ℃ of 2min, adds the Superscript of 1 μ L then in this solution ^TMII ReverseTranscriptase (Gibco-BRL, USA).This reaction system is behind 42 ℃ of placement 50min, and is again 70 ℃ of water-bath 15min termination reactions, last instantaneous centrifugal.

Above-mentioned reaction solution takes out 1 μ L and makes pcr amplification for the first time.The pcr amplification system comprises dNTP 1 μ L (every kind of dNTP respectively is 10mmol/L), 5 ' end primer Psa3:5 '-GAAAGTAGTGGATGTTGGGTG-3 ' and 3 ' end primer Oligo (dT) ₁₇Each 2 μ L (10pmol/ μ L), 10 * Taq Plus enzyme buffer liquid, 5 μ L, Taq Plus enzyme 0.5 μ L (5u/ μ L), water are supplemented to 50 μ L.The condition of pcr amplification is: 94 ℃ of 4min; 94 ℃ of 1min, 42 ℃ of 2min, 72 ℃ of 2min (35 circulations); 72 ℃ of 10min.Getting for the first time, PCR product 1 μ L does the nest-type PRC amplification.The pcr amplification system comprises dNTP 1 μ L (every kind of dNTP respectively is 10mmol/L), 5 ' end primer Psa4:5 '-CAGGAGGTAGGATAATAATAGTGA-3 ' and 3 ' end primer Oligo (dT) ₁₇Each 2 μ L (10pmol/ μ L), 10 * Taq Plus enzyme buffer liquid, 5 μ L, Taq Plus enzyme 0.5 μ L (5u/ μ L), water are supplemented to 50 μ L.The condition of pcr amplification is: 94 ℃ of 3min; 94 ℃ of 1min, 48 ℃ of 1min, 72 ℃ of 1min30sec (35 circulations); 72 ℃ of 10min.The agarose electrophoretic analysis of pcr amplification product shows the comparatively significantly DNA band of about 583bp.

(Promega, Cat.No.A3600 USA) do the reaction of 10 μ L linked systems, wherein contain the T4DNA ligase enzyme of 5 μ L (2 * connect damping fluid), 1 μ L to get the nest-type PRC amplified production of 3 μ L and 1 μ L pGEM-T Easy Vector.Linked system is spent the night 4 ℃ of reactions, get 2 μ L electric shock transformed competence colibacillus e. coli jm109 (Stratagen, Cat.No.200235) after, be applied on the LB solid medium that contains Amp (penbritin).Picking list bacterium colony utilizes above PCR system, directly is that template is carried out PCR with the bacterium colony.To PCR male bacterium colony, with boiling method upgrading grain.Utilize two EcoRI restriction enzyme sites on the carrier, the plasmid of 5 μ L is made the single endonuclease digestion of EcoRI and identified.Selection contains the bacterium colony (JM109-3END) of about 583bp dna fragmentation recombinant plasmid according to electrophoresis result, extracts plasmid (p3END-T), is checked order by Shanghai Bo Ya Bioisystech Co., Ltd.

3) the terminal rapid amplifying (5 ' RACE) of cDNA5 '

5 μ g total rna solutions are used for 5 ' RACE reaction.Reactions steps is carried out with reference to 5 ' the RACE test kit explanation of Gibco-BRL company.Be template at first, use primer GSP1:5 '-TTCCACGTTAATGCGGTTC-3 ' at Superscript with total RNA ^TMCarry out reverse transcription reaction under the effect of II Reverse Transcriptase, the synthetic first chain cDNA.Handle through RNaseH, behind the Glass MAXDNA isolation spin cartridge purifying, add poly (dC) tail at the synthetic first chain cDNA3 ' end with terminal enzyme (DNA).Use nested primer GSP3:5 '-GCAGATTCTGTCCAAGAGGTTC-3 ' and anchor primer AAP:5 '-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3 ' to carry out pcr amplification, the pcr amplification program is: 94 ℃ of 4min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min20sec (35 circulations); 72 ℃ of 10min.The agarose electrophoretic analysis of pcr amplification product demonstrates the DNA band of a tangible 800bp.This fragment is cloned as stated above on pGEM-T Easy Vector, transformed into escherichia coli JM109 competent cell, the single endonuclease digestion method of the EcoRI by PCR and recombinant plasmid, picking contains JM109-5END thalline and the order-checking of recombinant plasmid p5END-T.

From 2) and 3) sequencing result draws the full length cDNA sequence of the γ-TMT shown in sequence table No.1.

4) coding region sequence of RT-PCR amplification cDNA

Total RNA of 3 μ g is used for reverse transcription reaction, and reactive system is with 3 ' RACE.Get the reverse transcription product of 1 μ L and make pcr amplification.The system of pcr amplification has dNTP 1 μ L (every kind of dNTP respectively is 10mmol/L), upstream primer RT6:5 '-CGGGATCCACCATGAAAGCGACTCTCG-3 ' (containing a BamH I restriction enzyme site) and downstream primer RT3:5 '-TGAACTTAGAGAGGCTTCTGGCAA-3 ' each 2 μ L (10pmol/ μ L), 10 * Taq Plus enzyme buffer liquid, 5 μ L, Taq Plus enzyme 0.5 μ L (5u/ μ L), water to be supplemented to 50 μ L.The pcr amplification program is: 94 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of min30sec (35 circulations); 72 ℃ of 10min.The agarose electrophoretic analysis of PCR product shows the DNA band of a bright about 1060bp.

The pcr amplification product and the 1 μ L pGEM-T Easy Vector that get 3 μ L do ligation according to above method, connect product electric shock transformed into escherichia coli JM109 competent cell with 2 μ L, are applied to then on the LB solid medium that contains Amp.Pick out positive bacterium colony by PCR, extract plasmid with boiling method.Utilize the restriction enzyme site of BamH I on SalI on the carrier and the PCR product, the plasmid of 5 μ L is done the double digestion reaction of BamH I, SalI.Select to cut out about 1 according to electrophoresis result, the bacterium colony (JM109-TMTL) of 060bp dna fragmentation recombinant plasmid extracts plasmid (pTMTL) and order-checking.

Embodiment 2. contains the preparation of gama-tocopherol methyl transferase structure gene recombinant plant constitutive expression carrier, recombinant plant seed-specific expression carrier and reorganization Agrobacterium thereof

BamH I, Sal I double digestion plasmid pTMTL reclaim the DNA small segment.Same double digestion plant constructive expression carrier pBin438 and seed-specific expression carrier p7S438 (YE Liang, LICong and Song Yanru.2000, Construction of plant seed-specificexpressionvectors pSCB and pSCAB and the obtainment of transgenicBrassica napus H165 expressing poly-3-hydroxybutyrate syntheticgenes.Chinese Science Bulletin 45:1206-1210; Z.L.Chen, N.S.Panand R.N.Beachy.1988.A DNA sequence element that confersseed-specific enhancement to a constitutive promoter.The EMBCJournal 7:297-302; Z.L.Chen, M.A Schulerand R.N.Beachy.1986, Functional analysis of regulatory elements in a plantembryo-specific gene.Proc.Natl.Acad.Sci.USA 83:8560-8564), reclaim the big fragment of dna vector.Getting the big fragment of these dna vectors is connected with above-mentioned DNA small segment respectively.Behind the electric shock transformed competence colibacillus e. coli jm109, be applied on the LB solid medium that contains Kan (kantlex).Utilize the evaluation transformant of PCR and BamH I, Sal I double digestion respectively, and picking contains the JM109 thalline of recombinant plasmid pBin-TMTL or p7S-TMTL.

Extract plasmid pBin-TMTL and p7S-TMTL through alkaline process, respectively transformed competence colibacillus Agrobacterium LBA4404 (Gibco BRL, Cat.No.18313-015; Hoekema A, Hirsch PR, Hooykaas PJJ, et al., 1983, A binary plant vector strategy based onseparation of vir-and T-region of the Agrobacterium tumefacinensTi-plasmid.NAture 303:179-180.), be applied on the YEP solid medium that contains 50 μ g/ml Kan and 50 μ g/mlRif (Rifampin).Picking list bacterium colony upgrading grain, PCR and BamH I, Sal I double digestion are identified and are shown Agrobacterium LBA4404-Bin-TMTL and the LBA4404-7S-TMTL that has obtained to contain pBin-TMTL and p7S-TMTL respectively.

Equally, with plasmid pBin-TMTL and p7S-TMTL difference transformed competence colibacillus Agrobacterium GV3101 (Csaba Koncz and Jeff Schell.1986, The promoter of Tl-DNA gene5 controls the tissue-specific expression of chimaeric genescarried by a novel type of Agrobacterium binary vector.Mol GenGenet 204:383-396; N.Van Larebeke, G.Engler, M.Holsters et al., 1974, Large plasmid in Agrobacterium tumefaciens essential forcrown gall-inducing ability.Nature 252:169-170.), be applied on the YEP solid medium that contains 50 μ g/mlKan and 50 μ g/ml Rif (Rifampin).Picking list bacterium colony extracts plasmid, and PCR and BamH I, Sal I double digestion are identified and shown Agrobacterium GV3101-Bin-TMTL and the GV3101-7S-TMTL that has obtained to contain pBin-TMTL and p7S-TMTL respectively.

Picking contains Agrobacterium LBA4404 or the single bacterium colony of GV3101 of pBin-TMTL or p7S-TMTL, places the YEP substratum that contains 50 μ g/ml Kan and 50 μ g/ml Rif of 5mL, in 28 ℃, 250rpm overnight incubation.This bacterium liquid can be used for soybean and transforms after the MSB liquid nutrient medium is done 10 times of dilutions.

Embodiment 3. contains the preparation of gama-tocopherol methyl transferase structure gene recombinant microorganisms express carrier and recombinant microorganism, the expression of γ-TMT, the enzyme activity assay of expressing γ-TMT and the Antibody Preparation of γ-TMT

1) contains the preparation of gama-tocopherol methyl transferase structure gene expression of recombinant e. coli carrier and recombination bacillus coli and the abduction delivering of γ-TMT

In order to make gama-tocopherol methyl transferase structure gene in intestinal bacteria, can correct acquisition expression and expressed proteins can have activity, we have designed a upstream primer RT5:5 '-CGGGATCCACCATGACAACGACGGCAAC-3 ' according to the cabbage gama-tocopherol methyl transferase structural gene sequence that is obtained, this primer contains a BamH I restriction enzyme site, when it and RT3 primer carried out the RT-PCR amplification, one of the 930bp product coding that is obtained had been removed most of NH ₂The protein that-end chloroplast(id) is led the brachymemma of peptide.The RT-PCR amplification condition is with embodiment 1, pcr amplification product is cloned on pGEM-T Easy Vector, transformed into escherichia coli JM109 competent cell, by the BamH I of PCR and recombinant plasmid, the double digestion method of Sal I, picking contains JM109-TMT thalline and the order-checking of recombinant plasmid pTMT.The plasmid pTMT that gets 80 μ L carries out the double digestion reaction of BamH I, Sal I in 100 μ L systems, after 37 ℃ of temperature were bathed 3h, the reaction solution of getting 5 μ L carried out electrophoretic examinations.Cut completely to enzyme that reaction solution all carries out electrophoresis, reclaim the dna fragmentation that test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) reclaims 930bp, be dissolved in the 30 μ L water with DNA glue.Get the big fragment of pET30a that the 930bp dna fragmentation solution of 3 μ L and 10 μ L reclaim and do the ligation of 20 μ L behind same double digestion, system also comprises the T4DNA ligase enzyme of 2 μ L (10 * connect damping fluid), 2 μ L, and water is supplied 20 μ L.(Stratagen, Cat.No.200133), converted product is applied on the LB solid medium that contains Kan (kantlex) to get 2 μ L connection product electric shock transformed competence colibacillus e. coli bl21.By BamH I, the Sal I double digestion method of PCR and recombinant plasmid, picking contains the BL21-ET-TMT thalline of recombinant plasmid pET-TMT.

The BL21 thalline that contains recombinant plasmid pET-TMT is cultured to 0D600=0.5 in the LB liquid nutrient medium that contains 50 μ g/mLKan after, add IPTG to final concentration 0.4mmol/L, 4h makes its abduction delivering in the 250rpm shaking culture.Get 1.5mL bacterium liquid in 12, the centrifugal 30sec of 000rpm reclaims thalline.Thalline is resuspended in 2 * SDS damping fluid (containing 10% beta-mercaptoethanol) of 100 μ L sterilized waters and 100 μ L, boils 3min again 12, the centrifugal 5min of 000rpm.Get supernatant liquor 10 μ L and carry out the SDS-PAGE protein electrophoresis, show the differential protein band of a 39kD.After inducing 4h, its expression amount accounts for 22% (Fig. 1) of bacterial protein.

2) induce the vitro enzyme work of γ-TMT to analyze

The BL21-ET-TMT thalline that contains recombinant plasmid pET-TMT during to OD600=0.6, adds IPTG to final concentration 0.4mmol/L in 28 ℃ of shaking culture, and inducible protein is expressed 3.5h.8, the thalline of the centrifugal collection of 000rpm 500mL nutrient solution, be resuspended in the ultrasonic damping fluid of 5mL (10mmol/L HEPES pH7.8,5mmol/L DTT, the 0.24mol/L sorbyl alcohol, 1mmol/LPMSF) in, use the ultrasonic cell disruption instrument smudge cells on ice.In homogenate, add TritonX-100 to final concentration be 1%, place 30min on ice after, 4 ℃, 30, the centrifugal 30min of 000g after getting supernatant liquor and measuring proteinic concentration with BioRad analysis of protein method, carries out enzyme activity assay.With Escherichia coli BL21 (DE3) bacterial strain that contains empty carrier pET30a as negative control.The reaction system of 125 μ L contains 50mmol/L Tris (pH8.5) 5mmol/L DTT, 0.64mmol/L Gamma-Tocopherol, 1.28mmol/L S-adenosylmethionine (SAM), sample 60 μ L, behind 25 ℃ of incubation 1.5h, (the V: chloroform V): methyl alcohol (BHT that contains 1mg/mL) and 125 μ L, 0.9% physiological saline termination reaction that added 500 μ L 2: 1.Behind the violent vortex 1min of each sample, centrifugal phase-splitting changes lower floor's chloroform in the one new centrifuge tube mutually, and vacuum method is removed chloroform, and with the fluid behind the heavy molten removal chloroform of the ethyl acetate (BHT that contains 1mg/mL) of 20 μ L, point sample is in TLC plate (silica gel G F ₂₅₄Plate) on, exhibition layer in methylene dichloride.Make TLC plate drying at room temperature, observe chromatogram down in UV-light (253nm).The reaction system that contains γ-TMT expressing protein a chromatogram spot of alpha-tocopherol very clearly occurred on the TLC plate, and this spot (Fig. 2) does not appear in control group behind 25 ℃ of reaction 1.5h.In addition,, during 120min, take out the reaction solution of 20 μ L respectively in enzymatic reaction 0,20,40,60,80,100, as stated above termination reaction with carry out thin-layer chromatography and separate, under UV-light, indicate position corresponding to the alpha-tocopherol bands of a spectrum.Scrape and get each bands of a spectrum thing, the dehydrated alcohol that adds 2mL, the vortex vibration is left standstill 5min, 12 then, the centrifugal 5min of 000rpm, supernatant liquor is changed in the cuvette, add 0.5% α, α '-two pyridine ethanolic soln 0.5mL and 0.2% iron trichloride ethanolic soln 0.5mL to each glass, mixing, the absorption value of mensuration 520nm after adding the lucky 2min of iron trichloride.The absorbancy curve display at 520nm place is in the reaction system that contains γ-TMT expressing protein, along with the carrying out of reaction, A ₅₂₀Be worth increase gradually, very fast in preceding 60min reaction, reach the highest during to 80min, become mild subsequently gradually, and control group there is no this variation generation.γ in the reaction system that contains γ-TMT expressing protein-TMT activity is more than 10 times of contrast when reaction 80min.

3) preparation, the γ-TMT that contains gama-tocopherol methyl transferase structure gene recombinant yeast expression vector and recombination yeast expresses and the analysis alive of the vitro enzyme of expressing γ-TMT

The above-mentioned 930bp product that obtains is inserted the polylinker of carrier for expression of eukaryon pPIC9, α-factor signal peptide sequence translation in goal gene and the plasmid is merged, built up with alcohol oxidase 1 (AOX1) promotor and terminator guiding and expressed, α-factor signal peptide sequence guiding secretion is the recombinant plasmid of methanol yeast (the being pichia spp) expression-secretion of selective marker with his4.Recombinant plasmid is cut with the BglII enzyme, makes linearizing, and electric shock transforms methanol yeast GS115 (his4, mut ⁺), select his ⁺Transformant, picking transformant corresponding points are received on MM and the MD substratum, normal growth on the MD substratum, and the abnormal transformant of growth is positive colony on the MM substratum.Through high density fermentation, in fermented liquid, there is purpose product gama-tocopherol methyl transferase.

Methanol yeast GS115 (negative control) that will contain empty carrier pPIC9 respectively and the methanol yeast GS115 that contains γ-TMT structure gene carry out high density fermentation, by 2) described method measures the activity of gama-tocopherol methyl transferase.Contain γ-TMT structure gene methanol yeast GS115 fermented liquid reaction system 25 ℃ the reaction 1.5h after, a chromatogram spot of alpha-tocopherol very clearly on the TLC plate, occurred, and this spot does not appear in control group.The result shows in the methanol yeast GS115 fermented liquid contain γ-TMT structure gene and has expressed the reorganization gama-tocopherol methyl transferase really, and has the activity of gama-tocopherol methyl transferase.

4) Antibody Preparation of gama-tocopherol methyl transferase is to carry out the antibody that Western blot detects needs the preparation gama-tocopherol methyl transferase, adopt above-mentioned 1) middle method, with the gel behind the SDS-PAGE protein electrophoresis, after 4 ℃ of 0.1N KCl soak 5min, take the gel of 39kD specific expression protein leukorrhea, twice of sterile water wash of gel, each 5min that soaks, grind with mortar behind the liquid nitrogen flash freezer, suspend with physiological saline again, with suspension direct immunization rabbit, gather the antiserum(antisera) of rabbit after one month.Detect this sero-fast tiring with the ELISA method, more than 000 at 1: 200.

Embodiment 4. changes the cultivation of cabbage gama-tocopherol methyl transferase structure gene soybean

1) conversion of soybean and regeneration

Make big pea green No. 1 seed in getting, in 70% alcohol-pickled 5sec, 0.1%HgCl is used in aseptic washing 3 times again ₂Surface sterilization 15min with aseptic water washing 5 times, soaks 6-14h and is placed on the MSB substratum and sprouts, 25 ℃ of illumination every day 16 hours.Get the soybean aseptic seedling of 6 days seedling ages, remove kind of a skin, 1/2 cotyledon is removed in crosscut, keeps the 2-4mm hypocotyl, vertically partial application makes and becomes two symmetric parts, scratches at the cotyledonary node position behind the excision plumule and with blade and is the cotyledonary node (explant) that is used to transform.Explant is dipped in pre-treatment 30min in the 0.35mol/L mannitol solution, place above Agrobacterium diluent again, take out explant after 30 minutes, absorb the bacterium liquid on explant surface with aseptic filter paper, explant is the 45 oblique cutting on the MSB1 solid medium, make otch insert substratum up, 28 ℃ of dark cultivations three days.After three days, with the explant cultivated altogether with aseptic washing 4 times to remove epontic Agrobacterium, soak 30min with the sterilized water that contains 500mg/L Cef (cephamycin), transfer on the MSB1 substratum that contains 500mg/L Cef again, the cultivation of sprouting was induced in 25 ℃ of illumination every day in 16 hours.When growing the growing thickly during bud of 0.5 ~ 1cm on the cotyledonary node, explant is changed on the MSB2 solid medium that contains 50mg/L Kan, 500mg/L Cef, 25 ℃ of illumination every day were carried out selective screening in 16 hours and are cultivated.Take out when growing to 3-4mm when young shoot, young shoot is downcut root induction in the MSB solid medium.Treat that young shoot sends out roots, and take out seedling during the about 6-7cm of height of seedling, thoroughly remove the root substratum, seedling is moved into cultivate (Fig. 3) in the soil.

2) the branch factory Biological Detection of genetically engineered soybean

A) pcr amplification and Southern blot detect and get above-mentioned genetically engineered soybean blade 30mg, CTAB buffered soln (the 100mmol/L Tris-HCl that adds 150 μ L, pH8.0,1.4mol/LNaCl, 20mmol/L EDTA, 2%CTAB) grind the back, adds 350 μ L CTAB buffered soln more in addition, mixing gently, 65 ℃ of temperature are bathed 30min.Add the chloroform of 500 μ L then 4 ℃ of precoolings: the primary isoamyl alcohol mixed solution (v: v=24: 1), carefully shake up, room temperature 10, the centrifugal 5min of 000rpm, sucking-off 400 μ L waters change in another Eppendorf pipe.Aqueous phase adds the Virahol of 400 μ L 4 ℃ of precoolings, shakes up gently, places 30min in-20 ℃.10, the centrifugal 15min of 000rpm reclaims the DNA precipitation.Abandon supernatant liquor, twice of the washing with alcohol precipitation with 70%.Be dissolved in again in the deionized water of 100 μ L after the precipitation drying.Get 1 this solution of μ L and make template, carry out pcr amplification (primer is respectively 35S:5 '-TGATGTGATGGTCCGATTG-3 ' and GSP2:5 '-CACCAGGCCGGGAGATAA-3 ') by above PCR system.Electrophoresis result is shown as a 800bp DNA band, illustrates that this plant is the positive strain of PCR.

Prepare the total DNA of transgenic soybean gene group of 70 μ g by the method for said extracted soybean gene group DNA, the Hind III that adds 200u does the endonuclease reaction of 400 μ L systems.Behind 37 ℃ of insulation 12h, the Hind III that adds 200u enzyme again cuts 6h, and whether enzyme cuts entirely to get 5 μ L electrophoresis detection.If enzyme adds the distilled water of 50 μ L 3mol/L NaAc (pH5.8), 50 μ L and the dehydrated alcohol of 2 times of volumes after cutting entirely successively, mixing is placed 30min in-20 ℃.10, the centrifugal 15min of 000rpm reclaims the DNA precipitation that enzyme is cut.Abandon supernatant liquor, twice of the washing with alcohol precipitation with 70%.Precipitation is dissolved in the DNA sample-loading buffer of the deionized water of 30 μ L and 10 μ L after drying, 1% agarose gel electrophoresis, the about 10h of 30V.Shift in the big culture dish of sepharose to, add depurination liquid (0.25mol/L HCl) and just flooded gel to liquid level, 20min decolours on shaking table.Outwell depurination liquid, distilled water flushing 5 times.Add 0.4N NaOH solution, 20min vibrates on shaking table.Adopt the alkali transfer method to utilize capillary theory that the DNA in the glue is transferred on the nylon membrane, changeing film liquid is 0.4NNaOH, changes film 12-16h.Take out film, rinsing 1min gently in 2 times SSC solution dries film (about 30min) slightly.The air dried film is put into the crosslinked 4min of UV-crosslinked instrument medium ultraviolet slightly.Film is put into hybrid pipe, in DNA faces.The prehybridization solution (7%SDS, 1%BSA, 1mmol/L EDTA, the 250mmol/L NaPO that add 65 ℃ of preheatings of 15mL ₄The pH7.2 damping fluid, 1% milt DNA), 65 ℃ of prehybridization 5-6h.Change prehybridization solution, behind the sex change probe by the probe of 50 μ L: the 15mL hybridization solution joins in the hybrid pipe, and 65 ℃ of hybridization are spent the night.Add the film washing liquid I (0.1%SDS, 2 times of SSC) of about 60mL, wash film 1min under the room temperature.Outwell film washing liquid I, add the film washing liquid II (0.1%SDS, 1 times of SSC) of about 60mL, wash film twice for 65 ℃, each 10min.Outwell film washing liquid II, determine the number of times and the time of washing film according to radioactive intensity on the film with film washing liquid III.Film is dried the back wrap, in the darkroom, press the X-ray sheet with preservative film.The radioautograph result shows that a specific DNA band has appearred in genetically engineered soybean, illustrates that the gama-tocopherol methyl transferase structure gene of cabbage has been incorporated in the transgenic soybean gene group.

B) RT-PCR and Western blot detect and get above-mentioned Southern blot male genetically engineered soybean plant leaf 0.2g, it is last to pulverize in liquid nitrogen, change one over to and contain 0.6mL4mol/L guanidinium isothiocyanate solution (4mol/L guanidinium isothiocyanate, 25mmol/L two hydration trisodium citrates, 0.5% sarcosyl, 10% ethanol) in the 1.5mL centrifuge tube, put upside down mixing.Add 0.06mL 2mol/L NaAc (pH4.0-5.0) successively, water saturated sour phenol of 0.6mL and 0.2mL chloroform are put upside down mixing, place 20min on ice.4 ℃, 8, the centrifugal 13min of 000rpm.The sucking-off supernatant liquor, the dehydrated alcohol of 3 times of volumes of adding ,-70 ℃ of precipitation 1h or longer time.4 ℃, 6, the centrifugal 13min of 000rpm.Abandon supernatant liquor, precipitation is resuspended in the 0.2mL 4mol/L LiCl solution, places 10min on ice, and then 13, the centrifugal 15min of 000rpm.Precipitation heavily is dissolved in 0.08mL in the distilled water of twice sterilization, with isopyknic chloroform extracting once, 10, the centrifugal 5min of 000rpm.Get supernatant liquor, add the 3mol/L NaAc (pH5.8) of 0.1 times of volume and the dehydrated alcohol of 2 times of volumes, in-70 ℃ of precipitation 30min.13, the centrifugal 15min of 000rpm abandons supernatant, washes precipitation twice with 70% ethanol, and dried precipitation is dissolved in 40 μ L in the distilled water of twice sterilization.Get the total RNA of 20 μ L and carry out the DNase processing as stated above, after reverse transcription became cDNA, the cDNA that gets 2 μ L was a template, was primer with RT5 and RT3, carried out the pcr amplification first time by above-mentioned reaction system, and the pcr amplification program is: 94 ℃ of 4min; 94 ℃ of 8sec, 50 ℃ of 8sec, 72 ℃ of 8sec (35 circulations); 72 ℃ of 10min.The PCR product first time of getting 1 μ L is a template, is primer with Psa4 and RT3, carries out the pcr amplification second time, and the PCR response procedures is: 94 ℃ of 4min; 94 ℃ of 8sec, 53 ℃ of 8sec, 72 ℃ of 8sec (35 circulations); 72 ℃ of 10min.Electrophoresis result is shown as the specific band of a 380bp, and the wild-type soybean should band.RT-PCR presentation of results cabbage gama-tocopherol methyl transferase structure gene not only has been incorporated in the transgenic soybean gene group, and can successfully transcribe.

Get 0.1g genetically engineered soybean blade,, change over to and contain 150 μ L albumen extraction buffer (50mmol/L Na with behind the liquid nitrogen grinding powdered ₂HPO ₄-NaH ₂PO ₄, 0.5mol/L NaCl, 1mmol/L EDTA, 14mmol/L mercaptoethanol, 1mmol/L PMSF) centrifuge tube in.Abundant mixing, 4 ℃, 12, the centrifugal 20min of 500rpm.Supernatant liquor changes in the new centrifuge tube, measures protein concentration with the BioRad method under 595nm.According to the protein concentration of being measured, each sample is got the solubility total protein of 80 μ g, the protein example damping fluid (20% glycerine, 0.05% tetrabromophenol sulfonphthalein, the 0.125mol/L Tris-HCl that add 2 times of equal-volumes, pH6.8,4%SDS, 10% beta-mercaptoethanol), 100 ℃ of water-bath 5min, then 10, the centrifugal 8min of 000rpm.Sample carries out 12% SDS-PAGE electrophoresis, treats that tetrabromophenol sulfonphthalein swims out behind the gel entirely, and the half dry type albumen that adopts Bio-Rad company to produce shifts instrument the albumen in the gel is transferred on the nitrocellulose filter, and constant voltage 9V shifts 30min.Take out nitrocellulose filter, spend the night in the room temperature sealing with Buffer III (Buffer I adds 5% skim-milk).Outwell BufferIII, add Buffer I (1mol/L Tris-HCl (pH7.4) 10mL that contains 5% skim-milk, 5mol/L NaCl 15mL, 0.5mol/L EDTA (pH8.0) 1mL, Tween-20 0.5mL, power water is to 500mL) dilution an anti-solution (the anti-γ of rabbit-TMT serum, 1: 5000 times of dilution), 37 ℃ the effect 2.5h.Buffer I washes film 3 times, each 5min.The two anti-solution (goat anti-rabbit igg of alkaline phosphate ester enzyme labelling, 1: 3000 times of dilution) that add the Buffer I dilution that contains 5% skim-milk, 37 ℃ of effect 45min.Buffer I washes film 3 times, each 5min.Add the solution II (1mol/L Tris (uncomfortable pH) 10mL, 5mol/L NaCl 2mL, the 50mmol/L MgCl that contain 66 μ L chlorination nitro blue tetrazoliums (NBT) and 33 μ L 5-bromo-4-chloro-3-indoles tricresyl phosphate amine indigo plants (BCIP) ₂0.5mL, add water to 100mL) and 10mL, put the dark place colour developing, occur until the purpose band.With tap water rinsing termination reaction.Western Blot result shows that genetically engineered soybean a specific immunity band that comparison is obviously deepened according to (wild-type soybean) occurred at 34kD size place, and this result shows the successful expression of gama-tocopherol methyl transferase in changeing gama-tocopherol methyl transferase structure gene soybean.Alpha-tocopherol content has improved 4 times at least in the seed of HPLC analysis genetically engineered soybean.T1 is for the PCR positive plant of genetically engineered soybean also can normally yield positive results (Fig. 3).

Embodiment 5. cabbage gama-tocopherol methyl transferases specific expressed in the transgenic arabidopsis seed

Picking contains the single bacterium colony of Agrobacterium GV3101 of p7S-TMTL, places the YEP substratum that contains 50 μ g/mL Kan and 50 μ g/mL Rif of 5mL, in 28 ℃, 250rpm overnight incubation.Change this bacterium liquid in the 500mL YEP substratum over to next day, and 28 ℃ of joltings are cultured to OD600=1.2.5, the centrifugal collection thalline of 000rpm, abandoning supernatant is with transforming suspension (1/2MS salt, 1 times vitamin B5,5% sucrose, the 6-BA of 10 μ L/L suspension, 0.02% Silwet L-77) resuspended thalline, and be sub-packed in the cuvette that vacuumizes every glass of bacterium liquid of putting 2/3 volume approximately.To remove the Arabidopis thaliana that bears pods and bloom, with bungee flowerpot is made " ten " word and tie up, be inverted on the cuvette, inflorescence part immerses in the bacterium liquid, places in the vacuum reservoir.0.05Mpa vacuum-treat 5min under the pressure.Take out Arabidopis thaliana and with its horizontal positioned 2 days in the cool.Arabidopis thaliana is put 23 ℃ then, every day illumination cultivation 16h.Collect the seed of transgenic arabidopsis after 25-30 days.

The seed of getting an amount of exsiccant transgenic arabidopsis adds 1mL 70% Ethanol Treatment 2min in the 1.5mL centrifuge tube.Sucking-off ethanol, after aseptic washing 2 times, the colored king's thimerosal that adds 1mL 10-20% is handled 15min.The sucking-off thimerosal is with sterilized water washing 5 times.Use 0.1% the resuspended seed of agar at last, in the 1/2MS substratum, (contain Kan 50 μ g/mL) with the density of 200-300 grain seed/culture dish and cultivate.Place 48h, change 23 ℃ of illumination cultivation then over to for 4 ℃.Treat that Kan resistance seedling grows to move in the soil behind the 4-8 sheet lotus throne leaf and cultivate.

Anti-Kan seedling leaves 50mg grinds to form homogenate in 200 μ L extraction damping fluids (1 times DNA extraction damping fluid, 1 times karyorhexis damping fluid, the sarcosyl of 0.4 times of volume 5%).Add the extraction damping fluid of 65 ℃ of preheatings of 500 μ L, put upside down mixing.65 ℃ of water-bath 30-60min.The chloroform that adds 750 μ L: primary isoamyl alcohol (24: 1) mixed solution, fully mixing.10, the centrifugal 5min of 000rpm.Supernatant liquor is changed in the new centrifuge tube, add the cold Virahol of 2/3 to 1 times of volume, put upside down mixing and precipitate to DNA.10, the centrifugal 10min of 000rpm abandons supernatant liquor, washes precipitation twice with 70% ethanol, and is drying precipitated, with the heavy dissolution precipitation of sterilized water of 50 μ L.Getting 1 μ L DNA as template, is primer with 7S5 (5 '-CCCAAGCTTCCTATCTGTCACTTC-3 ') and 7S3 (5 '-CGGGATCCGAGAGACTGGTGATT-3 '), carries out pcr amplification by above-mentioned system.The result shows that a specific 530bp band has appearred in transgenic arabidopsis, shows successful the changing in the Arabidopis thaliana of cabbage gama-tocopherol methyl transferase structure gene.

Get the positive transgenic arabidopsis blade of PCR and each 50mg of pod, by among the embodiment 4 2) the b method carries out Western Blot, and the result shows that the pod of the transgenic arabidopsis of the specific expressed plasmid p7S-TMTL of transferred species a specific immunity band (Fig. 5) of obviously deepening than wild-type Arabidopis thaliana pod occurred at 34kD size place.This shows that seed specific promoters guiding gama-tocopherol methyl transferase structure gene has obtained to efficiently express in the transgenic arabidopsis pod.

The enzyme activity determination of the gama-tocopherol methyl transferase of in the transgenic arabidopsis seed, expressing: take by weighing 5mg Western Blot male transgenic arabidopsis T2 for seed, enzyme extraction damping fluid (the 50mmol/L Tris pH8.5 that adds 100 μ L, 5mmol/L DTT, 1%Triton-100 grinds to form homogenate after 1mmol/LPMSF).10, the centrifugal 6min of 000rpm draws supernatant liquor, gets 25 μ L detection γ-TMT enzyme and lives.Enzyme reaction system alive and detection thereof are with embodiment 32).TLC result shows the chromatogram spot that has occurred alpha-tocopherol in the sample of transgenic arabidopsis T2 for seed of the specific expressed plasmid p7S-TMTL of transferred species, and this spot does not appear in the seed of wild-type Arabidopis thaliana, γ-TMT that the transgenic arabidopsis T2 that illustrates at the specific expressed plasmid p7S-TMTL of transferred species son expresses in for seed has the enzymic activity of catalysis gama-tocopherol methyl formation alpha-tocopherol.

Note:

LB substratum: 5g/L NaCl; 10g/LTrypton; 10g/LYeast ExtractpH7.0 (3NNaOH accent); 15g/L agar; Sterilized 30 minutes for 15 pounds.

YEP substratum: 5g/L NaCl; 10g/L Polypepton; 10g/LYeast ExtractpH7.0 (3NKOH accent); 15g/L agar; Sterilized 30 minutes for 15 pounds.

MSB substratum: macroelement (KNO ₃1.9g/L, NH ₄NO ₃1.6g/L, KH ₂PO ₄0.17g/L, CaCl ₂2H ₂O 0.44g/L, MgSO ₄7H ₂O 0.37g/L), trace element (KI 0.83mg/L, HBO 36.2mg/L, MnSO ₄4H ₂O 22.3mg/L, ZnSO ₄7H ₂O 8.6mg/L, Na ₂MoO ₄2H ₂O 0.25mg/L, CuSO ₄5H ₂O 0.025mg/L, CoCl ₂6H ₂O0.025mg/L), molysite (FeSO ₄7H ₂O 27.8mg/L, NaEDTA2H ₂O 37.3mg/L), organic composition (VITMAIN B1 10mg/L, vitamin B6 1.0mg/L, nicotinic acid 1.0mg/L, inositol 100mg/L), sucrose 3%, adjust pH to 5.8, solid medium add 0.7% agar, sterilize 30 minutes for 8 pounds.

The MSB1 substratum: the IBA and the 6BA final concentration that add filtration sterilization in the MSB substratum are respectively 0.2mg/L and 10mg/L.

MSB2 substratum: the MS substratum that contains 0.05mg/L IBA and 0.5mg/L 6BA.

DNA extraction damping fluid: 127.54g sorbyl alcohol, 24.2g Tris pH8.2,3.722g EDTA.Na ₂, add the water constant volume to 2L.

Karyorhexis damping fluid: 200mL 1mol/L Tris, pH7.5; 200mL 0.25mol/L EDTA; 400mL5mol/L NaCl; 20g CTAB; The 200mL deionized water.

Sequence table

＜110〉Institute of Microorganism, Academia Sinica

＜120〉gama-tocopherol methyl transferase and gene thereof and purposes

<130>cai2000305

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1265

<212>DNA

<213>Brassica?oleracea

<400>1

tttctccaac?caacctctca?ttataaatga?aagcgactct?cgcaccaccc?tcctctctca 60

taagcctccc?caggcacaaa?gtatcttctc?tccgttcacc?gtcgcttctc?cttcagtccc 120

agcggccatc?ctcagcctta?atgacaacga?cggcaacacg?tggaagcgta?gctgtgacgg 180

ctgctgctac?ctcctccgct?gaggcgctgc?gagaaggaat?agcggaattc?tacaacgaga 240

cgtcgggatt?atgggaggag?atttggggag?atcatatgca?tcacggcttc?tacgatcccg 300

attcctctgt?tcaactttca?gattccggtc?accgggaagc?tcagatccgg?atgattgaag 360

agtctctacg?tttcgccggc?gttactgaag?aggagaaaaa?gataaagaga?gtggtggatg 420

ttgggtgtgg?gatcggagga?agctcaaggt?atattgcctc?taaatttggt?gccgaatgca 480

ttggcatcac?actcagtccc?gttcaagcca?agagagccaa?tgatctcgcc?gccgctcaat 540

cactctctca?taaggtttcc?ttccaagttg?cagatgcatt?ggaccaacca?tttgaagatg 600

gtattttcga?tcttgtttgg?tcaatggaaa?gcggtgagca?tatgcctgac?aaggccaagt 660

tcgtgaagga?attggtacgt?gtgacggctc?caggaggaag?gataataata?gtgacatggt 720

gccacagaaa?tctatcccaa?ggggaagaat?ctttgcagcc?atgggagcag?aacctcttgg 780

acagaatctg?caaaacattt?tatctcccgg?cctggtgctc?cacctctgat?tatgtcgagt 840

tgcttcaatc?cctctcgctc?caggatatta?agtgtgcaga?ttggtcagag?aacgtagctc 900

ctttctggcc?ggcggttata?cgaaccgcat?taacgtggaa?gggccttgtg?tctctgcttc 960

gtagtggtat?gaagagtata?aaaggagcat?tgacaatgcc?attgatgatt?gaagggtaca 1020

agaaaggtgt?cattaaattt?ggcatcatcg?cttgccagaa?gcctctctaa?gttcaatcta 1080

aacaataaaa?ttgtcgtact?tttcagcgaa?ttgatttcta?tctatgatat?aggagattga 1140

ataagagtca?cgtgagaaat?gtggatgcat?gaaatccctt?aaacgtcatt?aatgttcgtt 1200

catggctacg?ttgtctattt?tagataaata?tacaagttga?aaggtgtcaa?aaaaaaaaaa 1260

aaaaa 1265

<210>2

<211>347

<212>PRT

<213>Brassica?oleracea

<400>2

Met?Lys?Ala?Thr?Leu?Ala?Pro?Pro?Ser?Ser?Leu?Ile?Ser?Leu?Pro?Arg

1 5 10 15

His?Lys?Val?Ser?Ser?Leu?Arg?Ser?Pro?Ser?Leu?Leu?Leu?Gln?Ser?Gln

20 25 30

Arg?Pro?Ser?Ser?Ala?Leu?Met?Thr?Thr?Thr?Ala?Thr?Arg?Gly?Ser?Val

35 40 45

Ala?Val?Thr?Ala?Ala?Ala?Thr?Ser?Ser?Ala?Glu?Ala?Leu?Arg?Glu?Gly

50 55 60

Ile?Ala?Glu?Phe?Tyr?Asn?Glu?Thr?Ser?Gly?Leu?Trp?Glu?Glu?Ile?Trp

65 70 75 80

Gly?Asp?His?Met?His?His?Gly?Phe?Tyr?Asp?Pro?Asp?Ser?Ser?Val?Gln

85 90 95

Leu?Ser?Asp?Ser?Gly?His?Arg?Glu?Ala?Gln?Ile?Arg?Met?Ile?Glu?Glu

100 105 110

Ser?Leu?Arg?Phe?Ala?Gly?Val?Thr?Glu?Glu?Glu?Lys?Lys?Ile?Lys?Arg

115 120 125

Val?Val?Asp?Val?Gly?Cys?Gly?Ile?Gly?Gly?Ser?Ser?Arg?Tyr?Ile?Ala

130 135 140

Ser?Lys?Phe?Gly?Ala?Glu?Cys?Ile?Gly?Ile?Thr?Leu?Ser?Pro?Val?Gln

145 150 155 160

Ala?Lys?Arg?Ala?Asn?Asp?Leu?Ala?Ala?Ala?Gln?Ser?Leu?Ser?His?Lys

165 170 175

Val?Ser?Phe?Gln?Val?Ala?Asp?Ala?Leu?Asp?Gln?Pro?Phe?Glu?Asp?Gly

180 185 190

Ile?Phe?Asp?Leu?Val?Trp?Ser?Met?Glu?Ser?Gly?Glu?His?Met?Pro?Asp

195 200 205

Lys?Ala?Lys?Phe?Val?Lys?Glu?Leu?Val?Arg?Val?Thr?Ala?Pro?Gly?Gly

210 215 220

Arg?Ile?Ile?Ile?Val?Thr?Trp?Cys?His?Arg?Asn?Leu?Ser?Gln?Gly?Glu

225 230 235 240

Glu?Ser?Leu?Gln?Pro?Trp?Glu?Gln?Asn?Leu?Leu?Asp?Arg?Ile?Cys?Lys

245 250 255

Thr?Phe?Tyr?Leu?Pro?Ala?Trp?Cys?Ser?Thr?Ser?Asp?Tyr?Val?Glu?Leu

260 265 270

Leu?Gln?Ser?Leu?Ser?Leu?Gln?Asp?Ile?Lys?Cys?Ala?Asp?Trp?Ser?Glu

275 280 285

Asn?Val?Ala?Pro?Phe?Trp?Pro?Ala?Val?Ile?Arg?Thr?Ala?Leu?Thr?Trp

290 295 300

Lys?Gly?Leu?Val?Ser?Leu?Leu?Arg?Ser?Gly?Met?Lys?Ser?Ile?Lys?Gly

305 310 315 320

Ala?Leu?Thr?Met?Pro?Leu?Met?Ile?Glu?Gly?Tyr?Lys?Lys?Gly?Val?Ile

325 330 335

Lys?Phe?Gly?Ile?Ile?Ala?Cys?Gln?Lys?Pro?Leu

340 345

Claims (14)

1. gama-tocopherol methyl transferase that derives from cabbage (Brassica oleracea L.Var.capitata L.), its aminoacid sequence are the aminoacid sequence after one or several amino acid conservative property in the aminoacid sequence shown in aminoacid sequence shown in the SEQ ID NO.2 or the SEQ ID NO:2 substitutes.

2. the gene of the aminoacid sequence of the described gama-tocopherol methyl transferase of claim 1 of encoding.

3. gene of the described gama-tocopherol methyl transferase of claim 1 of encoding, it is the nucleotide sequence shown in the SEQ NO.1.

4. recombinant plasmid contains the gene of claim 2 or 3 described gama-tocopherol methyl transferases.

5. recombinant plasmid according to claim 4 has p3END-T, p5END-T, pTMTL, pBin-TMTL, p7S-TMTL and pET-TMT.

6. a recombinant microorganism contains claim 4 or 5 described recombinant plasmids.

7. recombinant microorganism intestinal bacteria according to claim 6.

8. recombinant microorganism Agrobacterium according to claim 6.

9. recombinant microorganism pichia spp according to claim 6.

10. recombinant microorganism according to claim 6, or by the gama-tocopherol methyl transferase of its generation in the application for preparing by Gamma-Tocopherol in the alpha-tocopherol.

11. the application of gama-tocopherol methyl transferase according to claim 1 in the transgenic plant of cultivating composing type or seed-specific expression gama-tocopherol methyl transferase.

12. the application in the transgenic plant of cultivating composing type or seed-specific expression gama-tocopherol methyl transferase according to claim 2 or 3 described genes.

13. the application in the transgenic plant of cultivating composing type or seed-specific expression gama-tocopherol methyl transferase according to claim 4 or 5 described recombinant plasmids.

14. the application in the transgenic plant of cultivating composing type or seed-specific expression gama-tocopherol methyl transferase according to claim 6 or 7 described recombinant microorganisms.

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