CN101514346B - Lettuce gamma-tocopherol methyltransferase protein coded sequence - Google Patents
Lettuce gamma-tocopherol methyltransferase protein coded sequence Download PDFInfo
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Abstract
The invention relates to a lettuce gamma-tocopherol methyltransferase protein coded sequence, belonging to the technical field of gene engineering. The sequence has nucleotide sequence shown by 112-1008 bits in SEQ ID NO.3, and the polypeptide encoded by the sequence has amino acid sequence shown in SEQ ID NO.4; a method for increasing the content of plant vitamin E by utilizing the sequence comprises the steps: the lettuce gamma-tocopherol methyltransferase protein coded sequence is connected on plant expression regulator sequence, so that the corresponding plant expression vector is obtained; the expression vector in the step one is transferred agrobacterium to be converted to be arabidopsis; after being screened by antibiotics, transformed cells containing the lettuce gamma-tocopherol methyltransferase protein coded sequence can be obtained; finally, transgene plant or descendants thereof can be regenerated. The invention can increase the content of vitamin E in vegetables, improvethe nutritional quality of foods, ensure the transgene plant containing the gamma-tocopherol methyltransferase protein coded sequence to become a bioreactor, and further realize the mass production of the vitamin E.
Description
Technical field
The present invention relates to a kind of albumen coded sequence of gene engineering technology field, specifically is a kind of lettuce gamma-tocopherol methyltransferase protein coded sequence.
Background technology
Vitamin-E is a kind of liposoluble vitamin, its natural product is divided into eight types according to the difference of structure, be respectively alpha-tocopherol, 5,8-dimethyl tocol, Gamma-Tocopherol, Delta-Tocopherol (tocopherol) and alpha-tocotrienol, β-tocotrienols, γ-tocotrienols, δ-tocotrienols (tocotrienol), wherein the biological activity of alpha-tocopherol is the highest, is considered to the main active ingredient of vitamin-E.
Nineteen twenty-two, the biological function of vitamin-E found that medical research shows that vitamin-E is not only relevant with reproductive system, and with the unify eubolism of musculature of central nervous system, Digestive tract, cardiovascular system substantial connection arranged all.Vitamin-E plays an important role for human and animal's health.Vitamin-E participates in the structure of cytolemma and keeps, and is important antioxidant in animal and human's body.Vitamin-E is the important auxiliary medicaments of cardiovascular and cerebrovascular diseases such as treatment coronary heart disease, atherosclerosis, helps blood fat reducing and blood cholesterol content, anticoagulation, anti-oxidant and elimination free radical, thus play the effect that prevents and treat blood vessel embolism.Vitamin-E has antidotal function for enhance immunity ability important influence, can eliminate the cytopigment precipitation, improves skin elasticity, weakens the sexual gland atrophy, thereby is extensive use of in a large amount of healthcare products and cosmetic products.Vitamin-E can prevent and treat various gynecological diseases, treatment premature infant's hemolytic anemia and favism, treatment malnourished children's megaloblastic anemia.Vitamin-E can promote choleresis, bile duct to form and the bilirubin secretion, and acute, chronic hepatitis and liver cirrhosis are had therapeutic action.Vitamin-E can preventing cancer takes place, and for cancer patients's treatment important booster action is arranged also.
The route of synthesis of vitamin-E only is present in the green photosynthetic plant, comprises low wait one-celled plants blue-green algae and higher plant; There is not the vitamin-E route of synthesis in human body and the animal body, so the picked-up of the required vitamin-E of daily nutrition is from green plants, the seed of various oil crops particularly, and by vegetables oil that seed squeezed.
Directly the biological activity by the mixed liquid that contracts of the tocopherol that obtains in the plant oil deodorizing distillate is very low, and the content of alpha-tocopherol is lower.The industrial production vitamin-E mainly is that to mix the liquid that contracts with above-mentioned tocopherol be raw material, through semi-synthesis method, change non-alpha-tocopherol composition wherein into alpha-tocopherol, but cost is higher, and the alpha-tocopherol racemism that produces is different with natural alpha-tocofecol, and activity also is lower than natural alpha-tocofecol.Therefore, improve natural plant tocopherol content and alpha-tocopherol ratio thereof and have very important use value.
Along with the development of Plant Genome in recent years, DellaPenna has proposed the notion of vegetative gene group (nutritional genomics) in 1999, promptly utilize the achievement in research of genomics fully, the key gene of separating plant Nutrition and Metabolism approach, resolve the plant micronutrient pathways metabolism, understand the control methods of pathways metabolism in depth, thereby utilize the ways and means improvement crop quality of metabolic engineering, improve nutritive value of crops.Based on the basis of genomics, the gene isolation of vitamin-E route of synthesis, clone and functional study have been obtained breakthrough progress.People have tentatively finished separation and the functional verification work to participation vitamin-E route of synthesis key gene, and have analyzed vitamin-E route of synthesis general picture.
The vitamin-E route of synthesis mainly is made up of following five steps: (1) 4-hydroxyphenylpyruvic acid (HPP) generates homogentisic acid (HGA) under the catalysis of 4-hydroxyphenylpyruvate dioxygenase (HPPD); (2) condensation takes place with phytyl bisphosphate (PDP) in homogentisic acid under homogentisic acid phytyl transferring enzyme (HPT) catalysis, generates 2-methyl-6-phytyl-benzoquinones; (3) 2-methyl-6-phytyl-benzoquinones generates 2,3-dimethyl-6-phytyl-benzoquinones under the catalysis of methyl phytyl benzoquinone methyl transferase (MPBQ MT); (4) 2,3-dimethyl-6-phytyl-benzoquinones generates Gamma-Tocopherol under the catalysis of tocopherol cyclase (TC); (5) Gamma-Tocopherol (under the catalysis of γ-TMT), generates alpha-tocopherol at gama-tocopherol methyl transferase.
Find the report that Shang Weijian is relevant with lettuce gamma-tocopherol methyltransferase protein coded sequence through literature search to prior art.
Summary of the invention
The object of the present invention is to provide a kind of lettuce gamma-tocopherol methyltransferase protein coded sequence.The present invention can improve the content of vitamin-E in the vegetables, improves the nutritional quality of food, can make the transgenic plant that contain gamma-tocopherol methyltransferase protein coded sequence become bio-reactor, and then realizes the scale operation of vitamin-E.Gama-tocopherol methyl transferase (γ-TMT), and with this gene transformation Arabidopis thaliana, prove that it has important effect to the raising of content of vitamin E in the plant really has been cloned in the present invention from the romaine lettuce (Lactuca sativa) of composite family.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of lettuce gamma-tocopherol methyltransferase protein coded sequence, this sequence has the nucleotide sequence shown in the 112-1008 position among the SEQID NO.3.
Has the aminoacid sequence shown in the SEQ ID NO.4 according to described gene order encoded polypeptides.
The invention still further relates to a kind of method of utilizing lettuce gamma-tocopherol methyltransferase protein coded sequence to improve plant vitamin E content, its step is as follows:
Step 1 is connected in lettuce gamma-tocopherol methyltransferase protein coded sequence on the expression of plants regulating and controlling sequence, obtains containing the plant expression vector of lettuce gamma-tocopherol methyltransferase protein coded sequence;
Step 2 changes the expression vector in the step 1 over to Agrobacterium, utilizes this Agrobacterium-mediated Transformation Arabidopis thaliana;
Step 3 by antibiotic-screening, obtains to contain the transformant of lettuce gamma-tocopherol methyltransferase protein coded sequence, final regeneration of transgenic plant and offspring thereof.
Among the present invention, can select various carrier known in the art for use when containing the plant expression vector of lettuce gamma-tocopherol methyltransferase protein coded sequence in the construction step one, the carrier as commercially available comprises plasmid, clay etc.When producing lettuce gamma-tocopherol methyltransferase protein polypeptide of the present invention, lettuce gamma-tocopherol methyltransferase protein coded sequence operationally can be connected in expression regulation sequence, thereby form the lettuce gamma-tocopherol methyltransferase protein expression vector in the step 1; Operationally be connected in finger, some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.
Among the present invention, the transformant that step 3 contains lettuce gamma-tocopherol methyltransferase protein coded sequence is an eukaryotic cell, and eukaryotic host cell commonly used comprises sexual cell or the callus cell of yeast cell, Arabidopis thaliana and other plant.
The present invention has following beneficial effect: the present invention can improve the content of vitamin-E in the vegetables, improves the nutritional quality of food; The present invention simultaneously can make the transgenic plant that contain gamma-tocopherol methyltransferase protein coded sequence become bio-reactor, realizes the scale operation of vitamin-E; Utilize lettuce gamma-tocopherol methyltransferase protein of the present invention,, can filter out with gamma-tocopherol methyltransferase protein interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
Related Agrobacterium is agrobacterium tumefaciens bacterial strain GV3101 among the present invention, and this bacterial strain is at " CsabaKoncz, Jeff Schell; The promoter of TL-DNA gene 5 controls the tissue-specificexpression of chimaeric genes carried by a novel type of Agrobacterium binaryvector, Mol Gen Genet, 1986,204:383-396 " open in the document.Agrobacterium tumefaciens GV3101 can obtain by disclosing commercially available commercial channel, and as buying from Australian CAMBIA company, strain number is Gambar3.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment
Step 1, the clone of lettuce gamma-tocopherol methyltransferase gene
1, the extraction of RNA
Get the romaine lettuce leaf tissue, place liquid nitrogen to grind, add and fill in 1.5mL Eppendorf (EP) centrifuge tube of lysate, fully after the vibration, specification sheets extracted total RNA according to the TIANGEN test kit, identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
2, the full-length clone of gene
The amino acid conserved sequence coded according to genes involved in the Arabidopis thaliana, utilize homologous genes clone principle, adopt RACE (Rapid Amplification of cDNA Ends) method to carry out the cDNA full-length clone, the clone uses the Clontech test kit, and the clone divides three phases to carry out:
(1) first chain cDNA's is synthetic
The 5 '-CDS primer A and the SMART II Aoligo primer that utilize the Clontech test kit to be provided are template with the total RNA that is extracted, synthetic 5 '-RACE-Ready cDNA under the effect of PowerScript ThermoScript II; The 3 '-CDS primer A that utilizes the Clontech test kit to be provided is a primer, is template with the total RNA that is extracted, synthetic 3 '-RACE-ReadycDNA under the effect of PowerScript ThermoScript II.
(2)3’-RACE
The universal primer sequence (UPM) that provides with the Clontech test kit and utilize the gene specific primer 2 (GSP2 of Arabidopis thaliana homologous region design, see SEQ ID NO.1) carry out 3 '-RACE PCR reaction, detect and product is carried out glue reclaim with agarose gel electrophoresis, the purpose fragment that glue is reclaimed is connected on the pMD18-T carrier and checks order.
(3)5’-RACE
The universal primer sequence (UPM) that provides with the Clontech test kit and utilize the gene specific primer 1 (GSP1 sees SEQ ID NO.2) of Arabidopis thaliana homologous region design to carry out 5 '-RACE PCR reaction.Detect and product is carried out glue reclaim with agarose gel electrophoresis, the purpose fragment that glue is reclaimed is connected on the pMD18-T carrier and checks order.With the overlap splicing of sequencing result, obtain the complete coding region sequence of this gene.The gene that BLAST analytical results proof obtains from romaine lettuce really is the genes involved of a gama-tocopherol methyl transferase.
By above-mentioned steps, obtained to participate in the romaine lettuce complete encoding sequence (SEQ ID NO.3) of vitamin-E synthetic gamma-tocopherol methyltransferase protein.
Step 2, the sequence information of lettuce gamma-tocopherol methyltransferase genes involved and homology analysis
The full-length cDNA length of the romaine lettuce γ-TMT that obtains in the step 1 is 1131bp (SEQ ID NO.3), wherein open reading frame is positioned at 112-1008 Nucleotide, derive the aminoacid sequence (seeing SEQ ID NO.4) of lettuce gamma-tocopherol methyltransferase according to the cDNA that is obtained, totally 298 amino-acid residues, molecular weight is 33057.04, and iso-electric point is 5.86.
Utilize vectorNTI 9.0 softwares that the related amino acid sequence of the gama-tocopherol methyl transferase that derives from each kind of plant is carried out the homology comparison, found that lettuce gamma-tocopherol methyltransferase gene of being cloned into and Arabidopis thaliana (Arabidopsis thaliana), rape (Brassica napus), soybean (Glycine max), corn (Zea mays), cotton (Gossypium hirsutum), wheat (Triticum aestivum), potato (Solanum tuberosum), the coded amino acid sequence similarity of homologous gene is respectively in tomato (Solanum lycopersicum) and the Sunflower Receptacle (Helianthus annuus): 65%, 64%, 67%, 64%, 68%, 62%, 64%, 66%, 89% (table 2).This shows, the low grade except the unicellular organism chlamydomonas, there is higher homology in γ in romaine lettuce γ-TMT gene and other higher plants-TMT genes involved on coded amino acid levels, can think that their product also has higher similarity on function.
Step 3, lettuce gamma-tocopherol methyltransferase associated protein or polypeptide are carried out the content of vitamin E of eukaryotic cell expression and transfer-gen plant and are identified in arabidopsis cell
1, contains the goal gene (structure of the expression vector of romaine lettuce γ-TMT)
According to the complete encoding sequence (SEQ ID NO.3) of romaine lettuce γ-TMT, design amplifies the primer that complete coding is read frame, and introduces BamHI and SacI restriction endonuclease sites respectively on positive anti-primer, so that construction of expression vector.5 '-RACE-Ready cDNA with acquisition among the embodiment 1 is a template, behind pcr amplification, the coding region sequence of romaine lettuce γ-TMT is connected among the intermediate carrier pMD18-T checks order, the coding region sequence of correct γ-TMT of will checking order again further is cloned among the expression vector pHB, then change it over to agrobacterium tumefaciens, the performing PCR of going forward side by side checking.The result shows that the plant expression vector that contains romaine lettuce γ-TMT gene successfully is building up in the agrobacterium tumefaciens bacterial strain.
2, adopt the flower-dipping method arabidopsis thaliana transformation
(1) gets growth one month, upgrowth situation good stand, can carry before transforming and previous week plant be gone to the top, make plant produce more petal, improve transformation efficiency, transform and water the day before yesterday;
(2) will contain the Agrobacterium of transgene carrier in 28 ℃ of overnight incubation, to OD
600≈ 2.0,4, the centrifugal 10min of 500rpm;
(3) bacterial sediment is suspended in the freshly prepared conversion fluid, to final concentration OD
600≈ 0.8;
Little basin is inverted when (4) transforming, and guarantees that the whole petals of Arabidopis thaliana over-ground part all are submerged into about 5sec in the prior bacterium liquid that suspends with conversion Buffer;
(5) inhale with thieving paper and go excess liquid, plant is lain against in the capsule of a sealing to keep humidity, lucifuge is spent the night;
The plant taking-up vertically, was transferred under the normal condition and grown in (6) second days.After treating that plant grows to a certain degree, it is depended on bamboo let tie up, so that solid better;
(7) T
0For behind the seed maturity its whole strain being cut, sieve.Seed is laid on the screening culture medium that contains 50 μ g/mL Totomycin, and 4 ℃ of vernalization 48h move on to one week of growth under the phytotron 24h continuous illumination condition;
(8) transplant the continued growth of burying after waiting to grow green transgenosis resistance seedling, transformant be that the seedling and the root of green is longer; Non-transformant is etiolated seedling substantially, or green unrooted seedling;
(9) individual plant results transfer-gen plant (because of genetically modified insertion site difference, each all is different) mark becomes different strain systems;
(10) T of individual plant results
2Proterties having occurred for seed separates.Seed is taped against on the 9cm flat board equably, because of will determining whether it is that unit point inserts, so added with antibiotic not.After waiting to grow to 6-7 sheet true leaf, spray herbicide, the strain of screening unit point insertion in 3: 1 is that the individual plant results continue the transfer-gen plant that screening is isozygotied until acquisition.
3, the PCR of transgenic arabidopsis plant detects
According to the sequences Design forward primer of CaMV 35S, goal gene is detected according to the sequences Design reverse primer of γ-TMT.The result shows, can amplify the specific DNA fragment of goal gene size with CaMV 35S forward primer and γ-TMT reverse primer.And when being template, do not amplify any fragment with non-arabidopsis thaliana transformation genomic dna.
Step 4 utilizes HPLC-UVD to measure content of vitamin E in the transgenic arabidopsis
1, the preparation of HPLC-UVD condition and system suitability and standardized solution
HPLC: adopt Waters 2695 separations module systems, chromatographic column is C-18 reverse phase silica gel post (Calesil ODS-100C18), concrete specification is: 4.6mm internal diameter * 250mm length, moving phase are methyl alcohol and water, and wherein the volume ratio of methyl alcohol and water is 98: 2, at this moment, the appearance time of tocopherol is 26.3min, and the peak type is good, and column temperature is 30 ℃, flow velocity 1.0mL/min, sample size 30 μ L.
UVD: adopt Waters 2996 photodiode array detector systems, UV-detector detects wavelength 292nm.The tocopherol standard substance 2.0mg that accurately takes by weighing Sigma company dissolves fully with 1mL methyl alcohol, obtains 2mg/mL tocopherol standard solution, be stored in-20 ℃ standby.
2, the making of typical curve
With described reference substance solution difference sample introduction 5 μ l, 10 μ l, 15 μ l, 20 μ l, 30 μ l record collection of illustrative plates and chromatographic parameter under corresponding chromatographic condition, with peak area (Y) standard substance content (X, μ g) is carried out regression analysis respectively.By research, tocopherol presents good log-log linear relationship among the present invention in 10 μ g-60 μ g scopes.The log-log equation of linear regression of tocopherol reference substance is: Y=7.26e+002X+1.09e+004; R=0.999740.
3, the preparation of sample and tocopherol Determination on content
The leaching process of tocopherol is as follows: get fresh Arabidopsis leaf 1g-2g (fresh weight), liquid nitrogen grinding is used the 4mL normal hexane extraction, ultrasonic 30 minutes.Centrifugal, collect supernatant liquor, dry up with Nitrogen evaporator, with an amount of dissolve with methanol extract, be used for HPLC and detect.
Adopt HPLC-UVD to measure tocopherol content, the sample feeding volume is 30 μ l, go out tocopherol content (mg) in the sample according to the linear regression equation calculation of peak area substitution, amount to into amount of substance (pmol), again divided by the Arabidopsis leaf fresh weight (mg) of sample, thereby calculate the content of tocopherol in the Arabidopis thaliana plant.
The conversion of γ-TMT gene of gama-tocopherol methyl transferase of encoding has in the present invention significantly improved content of vitamin E in the Arabidopsis leaf, and this makes that the content of vitamin-E surpasses more than 50% of contrast in the transfer-gen plant.This shows that γ-TMT gene can be used as a kind of candidate gene that plant nutrition is worth that improves, the research and the industrialization that are used for utilizing transgenic technology to improve the crop content of vitamin E are produced.
Sequence table
＜110〉Shanghai Communications University
＜120〉lettuce gamma-tocopherol methyltransferase protein coded sequence
<160>4
<210>1
<211>25
<212>DNA
＜213〉romaine lettuce (Lactuca sativa)
<400>1
tacctcccga?aaagtcccta?cgccc?25
＜110〉Shanghai Communications University
＜120〉lettuce gamma-tocopherol methyltransferase protein coded sequence
<160>4
<210>2
<211>25
<212>DNA
＜213〉romaine lettuce (Lactuca sativa)
<400>2
ctgggcgtag?ggacttttcg?ggagg?25
＜110〉Shanghai Communications University
＜120〉lettuce gamma-tocopherol methyltransferase protein coded sequence
<160>4
<210>3
<211>1131
<212>DNA
＜213〉romaine lettuce (Lactuca sativa)
<400>3
gattgttgac?gcaataccac?caccaccacc?acgaggcagc?ttctgcagtc?actcaacact?60
caaggagtta?tgagtacggt?ggttgctgat?gcaacagttc?ctccgatgac?aatggcgact?120
gcagcggatg?agcagcagca?acaacagcta?aaaaaaggaa?tagcagaatt?ctacgatgaa?180
tcttcgggga?tgtgggagaa?tatatgggga?gaacacatgc?atcacggatt?ctacgacacc?240
gatgccgtcg?tagaactctc?cgaccaccgc?gctgctcaga?tccgtatgat?cgaacaaagc?300
ctacttttcg?cctctgttcc?tgatgatcca?gtaaagaagc?cgaaaaccat?agttgatgtt?360
gggtgtggta?taggaggtag?ctcaaggtac?ctagcaagaa?aatatggagc?tgaatgccat?420
ggcatcaccc?tcagccctgt?tcaagctgaa?agggctcaag?ccctagctgc?tacccaagga?480
ttagctgaca?aggtttcatt?tcaagttgcg?gatgctttga?accagccttt?tcctgatgga?540
aagtttgacc?tagtttggtc?aatggagagt?ggagaacaca?tgcctgacaa?actaaagttt?600
gttagtgagt?tggctcgagt?ggctgctcca?ggagccacaa?ttatcatagt?cacatggtgt?660
catagggacc?ttttacctcc?cgaaaagtcc?ctacgcccag?aggaagaaaa?aatcttgaac?720
aagatttgtt?caggattttt?tcttcctgct?tggtgttcta?ccgctgatta?tgtaaaatta?780
ctcgaatcca?tttcccttca?ggacatcaaa?gcagaagact?ggtcaggaaa?tgtggcacca?840
ttttggccag?ctgtgataaa?aacagccttg?tcttggaagg?gcattacgtc?attactaagg?900
agtggatgga?agactataag?aggagcaatg?gtaatgccat?caatgattga?aggatttaag?960
aaagatgtaa?taaaattctc?catcattaca?tgtaaaaagc?ctgaataaaa?ataggtgtgt?1020
aatgtacttt?tgattgttgt?actttctttc?atagactgct?tcattttggg?ttcttgtttt?1080
aatgtctaat?aactcttttt?tcaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?a 1131
＜110〉Shanghai Communications University
＜120〉lettuce gamma-tocopherol methyltransferase protein coded sequence
<160>4
<210>4
<211>298
<212>PRT
＜213〉romaine lettuce (Lactuca sativa)
<400>4
Met?Ala?Thr?Ala?Ala?Asp?Glu?Gln?Gln?Gln?Gln?Gln?Leu?Lys?Lys?Gly
1 5 10 15
Ile?Ala?Glu?Phe?Tyr?Asp?Glu?Ser?Ser?Gly?Met?Trp?Glu?Asn?Ile?Trp
20 25 30
Gly?Glu?His?Met?His?His?Gly?Phe?Tyr?Asp?Thr?Asp?Ala?Val?Val?Glu
35 40 45
Leu?Ser?Asp?His?Arg?Ala?Ala?Gln?Ile?Arg?Met?Ile?Glu?Gln?Ser?Leu
50 55 60
Leu?Phe?Ala?Ser?Val?Pro?Asp?Asp?Pro?Val?Lys?Lys?Pro?Lys?Thr?Ile
65 70 75 80
Val?Asp?Val?Gly?Cys?Gly?Ile?Gly?Gly?Ser?Ser?Arg?Tyr?Leu?Ala?Arg
85 90 95
Lys?Tyr?Gly?Ala?Glu?Cys?His?Gly?Ile?Thr?Leu?Ser?Pro?Val?Gln?Ala
100 105 110
Glu?Arg?Ala?Gln?Ala?Leu?Ala?Ala?Thr?Gln?Gly?Leu?Ala?Asp?Lys?Val
115 120 125
Ser?Phe?Gln?Val?Ala?Asp?Ala?Leu?Asn?Gln?Pro?Phe?Pro?Asp?Gly?Lys
130 135 140
Phe?Asp?Leu?Val?Trp?Ser?Met?Glu?Ser?Gly?Glu?His?Met?Pro?Asp?Lys
145 150 155 160
Leu?Lys?Phe?Val?Ser?Glu?Leu?Ala?Arg?Val?Ala?Ala?Pro?Gly?Ala?Thr
165 170 175
Ile?Ile?Ile?Val?Thr?Trp?Cys?His?Arg?Asp?Leu?Leu?Pro?Pro?Glu?Lys
180 185 190
Ser?Leu?Arg?Pro?Glu?Glu?Glu?Lys?Ile?Leu?Asn?Lys?Ile?Cys?Ser?Gly
195 200 205
Phe?Phe?Leu?Pro?Ala?Trp?Cys?Ser?Thr?Ala?Asp?Tyr?Val?Lys?Leu?Leu
210 215 220
Glu?Ser?Ile?Ser?Leu?Gln?Asp?Ile?Lys?Ala?Glu?Asp?Trp?Ser?Gly?Asn
225 230 235 240
Val?Ala?Pro?Phe?Trp?Pro?Ala?Val?Ile?Lys?Thr?Ala?Leu?Ser?Trp?Lys
245 250 255
Gly?Ile?Thr?Ser?Leu?Leu?Arg?Ser?Gly?Trp?Lys?Thr?Ile?Arg?Gly?Ala
260- 265 270
Met?Val?Met?Pro?Ser?Met?Ile?Glu?Gly?Phe?Lys?Lys?Asp?Val?Ile?Lys
275 280 285
Phe?Ser?Ile?Ile?Thr?Cys?Lys?Lys?Pro?Glu
290 295
Claims (2)
1. the polypeptide of a lettuce gamma-tocopherol methyltransferase protein is characterized in that, described amino acid sequence of polypeptide is shown in SEQ ID NO.4, and the nucleotide sequence of described zymoprotein is shown in 112-1008 position among the SEQID NO.3.
2. a method of utilizing lettuce gamma-tocopherol methyltransferase protein as claimed in claim 1 to improve plant vitamin E content is characterized in that, comprises the steps:
Step 1 is connected in lettuce gamma-tocopherol methyltransferase protein coded sequence on the expression of plants regulating and controlling sequence, obtains containing the plant expression vector of lettuce gamma-tocopherol methyltransferase protein coded sequence;
Step 2 changes the expression vector in the step 1 over to Agrobacterium, utilizes this Agrobacterium-mediated Transformation Arabidopis thaliana;
Step 3 by antibiotic-screening, obtains to contain the transformant of lettuce gamma-tocopherol methyltransferase protein coded sequence, final regeneration of transgenic plant and offspring thereof.
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| CN103087999B (en) * | 2011-11-03 | 2014-09-03 | 中国农业科学院北京畜牧兽医研究所 | Medicago sativa gamma-tocopherol methyltransferase (MSTMT) gene and its coded protein and use |
| CN103756971A (en) * | 2014-01-17 | 2014-04-30 | 江苏省中国科学院植物研究所 | Method for preparing blackberry cell suspension culture solution |
| CN108239654A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | A kind of method for improving rice grain vitamin content |
| CN110423732B (en) * | 2019-08-14 | 2022-01-07 | 浙江大学 | Enzyme expressed in saccharomyces cerevisiae, genetic engineering bacteria for high yield of alpha-and gamma-tocotrienols and construction method thereof |
| CN113817705B (en) * | 2021-10-27 | 2023-03-24 | 陕西海斯夫生物工程有限公司 | Gamma-tocopherol methyltransferase mutant and application thereof in improving yields of alpha-tocopherol and alpha-tocotrienol |
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| 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 |
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2009
- 2009-02-19 CN CN2009100463534A patent/CN101514346B/en not_active Expired - Fee Related
Patent Citations (1)
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 唐克轩等.RACE的研究及其在植物基因克隆上的应用.《复旦学报（自然科学版）》.2002,第41卷(第6期), * |
| 邹礼平.番茄γ-生育酚甲基转移酶基因的克隆与分析.《安徽农业科学》.2008,第36卷(第2期), * |
| 邹礼平等.水稻γ-生育酚甲基转移酶基因全长cDNA的克隆与分析.《湖北农业科学》.2008,第47卷(第11期), * |
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