CN101514346A - Lettuce gamma-tocopherol methyltransferase protein coded sequence - Google Patents

Abstract

A lettuce gamma-tocopherol methyltransferase protein coding sequence in the field of genetic engineering technology, the sequence has the nucleotide sequence shown in the 112th-1008th position in SEQ ID NO.3, and the polypeptide encoded by the sequence has the SEQ ID The amino acid sequence shown in NO.4; the method for increasing the vitamin E content of plants by utilizing the sequence, comprising the following steps: connecting the lettuce γ-tocopheryl methyltransferase protein coding sequence to the plant expression control sequence to obtain a plant expression vector; Transform the expression vector in step 1 into Agrobacterium, and use the Agrobacterium to transform Arabidopsis thaliana; obtain transformed cells containing lettuce γ-tocopherol methyltransferase protein coding sequence through antibiotic screening, and finally regenerate transgenic plants and their offspring . The invention can increase the content of vitamin E in vegetables, improve the nutritional quality of food, and can make transgenic plants containing gamma-tocopheryl methyltransferase protein coding sequences into bioreactors, thereby realizing large-scale production of vitamin E.

Description

Lettuce γ-tocopheryl methyltransferase protein coding sequence

technical field

The invention relates to a protein coding sequence in the technical field of genetic engineering, in particular to a lettuce gamma-tocopherol methyltransferase protein coding sequence.

Background technique

Vitamin E is a fat-soluble vitamin, and its natural products are divided into eight types according to the structure, namely α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol (tocopherol) and α-tocopherol Trienols, β-tocotrienols, γ-tocotrienols, and δ-tocotrienols (tocotrienol), among which α-tocotrienol has the highest biological activity and is considered to be the main active ingredient of vitamin E.

The biological function of vitamin E was discovered in 1922. Medical research shows that vitamin E is not only related to the reproductive system, but also closely related to the normal metabolism of the central nervous system, digestive system, cardiovascular system and muscular system. Vitamin E plays an important role in human and animal health. Vitamin E is involved in the construction and maintenance of cell membranes, and is an important antioxidant substance in animals and humans. Vitamin E is an important auxiliary drug for the treatment of coronary heart disease, atherosclerosis and other cardiovascular and cerebrovascular diseases. The role of embolism. Vitamin E has an important impact on improving the body's immunity. It has anti-aging functions, can eliminate cytochrome deposition, improve skin elasticity, and weaken gonadal atrophy. Therefore, it is widely used in a large number of health care products and beauty products. Vitamin E can prevent and treat a variety of gynecological diseases, treat hemolytic anemia and favism in premature infants, and treat megaloblastic anemia in malnourished children. Vitamin E can promote bile secretion, bile duct formation and bilirubin secretion, and has a therapeutic effect on acute and chronic hepatitis and cirrhosis. Vitamin E can prevent cancer, and it also plays an important auxiliary role in the treatment of cancer patients.

The synthesis pathway of vitamin E only exists in green photosynthetic plants, including low single-celled plants cyanobacteria and higher plants; there is no vitamin E synthesis pathway in humans and animals, so the vitamin E intake required for daily nutrition comes from green plants, especially Seeds of various oil crops, and vegetable oils extracted from the seeds.

The biological activity of the tocopherol concentrate obtained directly from the deodorized distillate of vegetable oil is very low, and the content of α-tocopherol is low. The industrial production of vitamin E is mainly based on the above-mentioned tocopherol condensation liquid as raw material, and the non-α-tocopherol components in it are converted into α-tocopherol through a semi-synthetic method, but the cost is relatively high, and the produced α-tocopherol is eliminated. The spin is different from natural α-tocopherol, and the activity is also lower than that of natural α-tocopherol. Therefore, increasing the plant natural tocopherol content and its α-tocopherol ratio has very important application value.

With the development of plant genomics in recent years, DellaPenna proposed the concept of nutritional genomics in 1999, that is, making full use of the research results of genomics, isolating key enzyme genes of plant nutrient metabolism pathways, and analyzing plant micronutrients Metabolic pathways, in-depth understanding of the regulation of metabolic pathways, so that the methods and means of metabolic engineering can be used to improve crop quality and increase crop nutritional value. Based on the basis of genomics, breakthroughs have been made in gene isolation, cloning and functional research of vitamin E synthesis pathway. People have preliminarily completed the isolation and functional verification of key genes involved in the vitamin E synthesis pathway, and analyzed the profile of the vitamin E synthesis pathway.

The vitamin E synthesis pathway is mainly composed of the following five steps: (1) 4-hydroxyphenylpyruvate (HPP) generates homogentisic acid (HGA) under the catalysis of 4-hydroxyphenylpyruvate dioxygenase (HPPD); (2) Under the catalysis of homogentisate phytotransferase (HPT), homogentisic acid condenses with phytyl diphosphate (PDP) to generate 2-methyl-6-phytyl-benzoquinone; (3) 2- Methyl-6-phytyl-benzoquinone generates 2,3-dimethyl-6-phytyl-benzoquinone under the catalysis of methylphytylbenzoquinone methyltransferase (MPBQ MT); (4)2 , 3-dimethyl-6-phytyl-benzoquinone generates γ-tocopherol under the catalysis of tocopherol cyclase (TC); (5) γ-tocopherol is activated by γ-tocopherol methyltransferase ( Under the catalysis of γ-TMT), α-tocopherol is generated.

After searching the literature of the prior art, it is found that there is no report related to the coding sequence of lettuce γ-tocopheryl methyltransferase protein.

Contents of the invention

The purpose of the present invention is to provide a lettuce gamma-tocopherol methyltransferase protein coding sequence. The invention can increase the content of vitamin E in vegetables, improve the nutritional quality of food, and can make the transgenic plant containing the coding sequence of gamma-tocopherol methyltransferase protein become a bioreactor, thereby realizing the large-scale production of vitamin E. The present invention clones γ-tocopheryl methyltransferase (γ-TMT) from lettuce (Lactuca sativa) of Compositae, and transforms the gene into Arabidopsis thaliana, which proves that it is indeed important for the improvement of vitamin E content in plants role.

The present invention is achieved through the following technical solutions:

The present invention relates to a lettuce gamma-tocopherol methyltransferase protein coding sequence, which has the nucleotide sequence shown in the 112th-1008th position in SEQ ID NO.3.

The polypeptide encoded according to the gene sequence has the amino acid sequence shown in SEQ ID NO.4.

The present invention also relates to a method for increasing the vitamin E content of plants by utilizing the lettuce gamma-tocopherol methyltransferase protein coding sequence, the steps of which are as follows:

Step-, connecting the lettuce gamma-tocopherol methyltransferase protein coding sequence to the plant expression control sequence to obtain a plant expression vector containing the lettuce gamma-tocopherol methyltransferase protein coding sequence;

In step 2, the expression vector in step 1 is transformed into Agrobacterium, and the Agrobacterium is used to transform Arabidopsis;

The third step is to obtain transformed cells containing lettuce gamma-tocopherol methyltransferase protein coding sequence through antibiotic screening, and finally regenerate transgenic plants and their progeny.

In the present invention, various vectors known in the art, such as commercially available vectors, including plasmids and cosmids, can be used to construct the plant expression vector containing lettuce γ-tocopherol methyltransferase protein coding sequence in step 1. When producing the lettuce γ-tocopherol methyltransferase protein polypeptide of the present invention, the lettuce γ-tocopherol methyltransferase protein coding sequence can be operably linked to the expression control sequence, thereby forming the lettuce γ-tocopherol methyltransferase protein polypeptide in step 1. Tocopherol methyltransferase protein expression vector; operably linked to fingers, some parts of the linear DNA sequence can affect the activity of other parts of the same linear DNA sequence.

In the present invention, the transformed cells containing lettuce gamma-tocopherol methyltransferase protein coding sequence in step 3 are eukaryotic cells, and commonly used eukaryotic host cells include yeast cells, germ cells or callus of Arabidopsis and other plants cell.

The present invention has the following beneficial effects: the present invention can increase the content of vitamin E in vegetables and improve the nutritional quality of food; at the same time, the present invention can make transgenic plants containing γ-tocopherol methyltransferase protein coding sequence into bioreactors, Achieving large-scale production of vitamin E; using the lettuce γ-tocopherol methyltransferase protein of the present invention, through various conventional screening methods, substances that interact with the γ-tocopherol methyltransferase protein can be screened out, or receptors, inhibitors, or antagonists.

The Agrobacterium involved in the present invention is Agrobacterium tumefaciens strain GV3101, which has been published in "CsabaKoncz, Jeff Schell; The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector , Mol Gen Genet, 1986, 204: 383-396" disclosed in the literature. Agrobacterium tumefaciens GV3101 can be obtained through publicly available commercial channels, for example, it can be purchased from CAMBIA Company in Australia, and the strain number is Gambar3.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example

Step 1, cloning of lettuce γ-tocopheryl methyltransferase gene

1. Extraction of RNA

Lettuce leaf tissue was taken, ground in liquid nitrogen, added to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, and fully oscillated, the total RNA was extracted according to the instructions of the TIANGEN kit, and the total RNA was identified by formaldehyde denaturing gel electrophoresis. RNA quality was then measured on a spectrophotometer for RNA content.

2. Full-length cloning of genes

According to the conserved sequence of amino acids encoded by related genes in Arabidopsis, using the principle of homologous gene cloning, the RACE (Rapid Amplification of cDNA Ends) method was used to clone the full-length cDNA. Cloning was carried out using Clontech kits, and the cloning was carried out in three stages. :

(1) Synthesis of first-strand cDNA

Use the 5'-CDS primer A and SMART II A oligo primers provided by the Clontech kit, and use the extracted total RNA as a template to synthesize 5'-RACE-Ready cDNA under the action of PowerScript reverse transcriptase; use the Clontech reagent The 3'-CDS primer A provided in the box was used as a primer, and the extracted total RNA was used as a template to synthesize 3'-RACE-Ready cDNA under the action of PowerScript reverse transcriptase.

(2) 3'-RACE

Use the universal primer sequence (UPM) provided by the Clontech kit and the gene-specific primer 2 (GSP2, see SEQ ID NO.1) designed using the Arabidopsis homology region to carry out 3'-RACE PCR reaction, and use agarose gel electrophoresis Detection and gel recovery were performed on the product, and the target fragment recovered from the gel was connected to the pMD18-T vector for sequencing.

(3) 5'-RACE

Use the universal primer sequence (UPM) provided by the Clontech kit and the gene-specific primer 1 (GSP1, see SEQ ID NO.2) designed using the Arabidopsis homology region to carry out 5'-RACE PCR reaction. Detection was performed by agarose gel electrophoresis and gel recovery was performed on the product, and the target fragment recovered from the gel was connected to the pMD18-T vector for sequencing. The overlapping regions of the sequencing results were assembled to obtain the complete coding region sequence of the gene. The results of BLAST analysis proved that the gene obtained from lettuce was indeed a gene related to γ-tocopherol methyltransferase.

Through the above steps, the full-length coding sequence (SEQ ID NO.3) of the gamma-tocopheryl methyltransferase protein involved in vitamin E synthesis in lettuce was obtained.

Step 2: Sequence information and homology analysis of genes related to lettuce γ-tocopherol methyltransferase

The full-length cDNA length of the lettuce γ-TMT obtained in step 1 is 1131bp (SEQ ID NO.3), wherein the open reading frame is located at 112-1008 nucleotides, and the lettuce γ-tocopherol is deduced according to the obtained cDNA The amino acid sequence of the methyltransferase (see SEQ ID NO.4) has a total of 298 amino acid residues, a molecular weight of 33057.04, and an isoelectric point of 5.86.

Using the vectorNTI 9.0 software to perform homologous comparison of the relevant amino acid sequences of γ-tocopherol methyltransferases from various plants, it was found that the cloned lettuce γ-tocopherol methyltransferase gene was similar to that of Arabidopsis ( Arabidopsis thaliana), rapeseed (Brassica napus), soybean (Glycine max), corn (Zea mays), cotton (Gossypium hirsutum), wheat (Triticum aestivum), potato (Solanum tuberosum), tomato (Solanum lycopersicum ), and sunflower (Helianthus annuus The amino acid sequence similarities encoded by the homologous genes in ) were: 65%, 64%, 67%, 64%, 68%, 62%, 64%, 66%, 89% (Table 2). It can be seen that, except for the lower unicellular organism Chlamydomonas, the lettuce γ-TMT gene has high homology with the γ-TMT related genes in other higher plants at the level of encoded amino acids, and it can be considered that their products are in There is also a high similarity in function.

Step 3, eukaryotic expression of lettuce γ-tocopherol methyltransferase-related protein or polypeptide in Arabidopsis cells and identification of vitamin E content in transgenic plants

1. Construction of expression vector containing target gene (lettuce γ-TMT)

According to the full-length coding sequence of lettuce γ-TMT (SEQ ID NO.3), primers were designed to amplify the complete coding reading frame, and BamHI and SacI restriction endonuclease sites were introduced on the forward and reverse primers respectively, so as to construct Expression vector. Using the 5'-RACE-Ready cDNA obtained in Example 1 as a template, after PCR amplification, the coding region sequence of lettuce γ-TMT was connected to the intermediate vector pMD18-T for sequencing, and then the sequenced correct γ-TMT The coding sequence of TMT was further cloned into the expression vector pHB, then transformed into Agrobacterium tumefaciens, and verified by PCR. The results showed that the plant expression vector containing lettuce γ-TMT gene had been successfully constructed into the Agrobacterium tumefaciens strain.

2. Transformation of Arabidopsis thaliana by flower dipping method

(1) Take a plant that has grown for one month and is in good growth condition. Before the transformation, the plants can be decapped one week in advance to make the plants produce more flower buds, improve the transformation efficiency, and water the day before the transformation;

(2) Cultivate the Agrobacterium containing the transgene vector overnight at 28°C until the OD ₆₀₀ ≈2.0, and centrifuge at 4,500rpm for 10min;

(3) The cell pellet was suspended in the freshly prepared transformation solution to a final concentration of OD ₆₀₀ ≈0.8;

(4) When transforming, the small pot is turned upside down to ensure that all the flower buds of the aerial part of Arabidopsis are submerged in the bacterial solution suspended in the transformation buffer for about 5 sec;

(5) Absorb excess liquid with absorbent paper, place the plants flat in a small airtight box to maintain humidity, and keep away from light overnight;

(6) The plants were taken out the next day, erected, and transferred to grow under normal conditions. After the plant grows to a certain extent, tie it up with bamboo sticks for better firmness;

(7) After the T ₀ generation seeds are mature, the whole plant is cut off and sieved. The seeds were spread on the screening medium containing 50 μg/mL hygromycin, vernalized at 4°C for 48 hours, and then moved to an artificial climate chamber for 24 hours of continuous light to grow for one week;

(8) After the green transgenic resistant seedlings are grown, they are transplanted into the soil to continue to grow, and the transformants are green seedlings with long roots; the non-transformants are basically etiolated seedlings, or green rootless seedlings;

(9) Harvest the transgenic plants (because the insertion sites of the transgenes are different, each is different) from a single plant and mark them as different strains;

(10) The T ₂ generation seeds harvested from a single plant have segregated traits. Spread the seeds evenly on a 9cm plate, and do not add antibiotics because it is necessary to determine whether it is inserted at a single point. After growing to 6-7 true leaves, spray herbicides, screen the lines with 3:1 single-site insertion, harvest single plants, and continue to screen until homozygous transgenic plants are obtained.

3. PCR detection of transgenic Arabidopsis plants

The forward primer was designed according to the sequence of CaMV 35S, and the reverse primer was designed according to the sequence of γ-TMT to detect the target gene. The results showed that the specific DNA fragment of the size of the target gene could be amplified by CaMV 35S forward primer and γ-TMT reverse primer. However, when non-transformed Arabidopsis genomic DNA was used as a template, no fragment was amplified.

Step 4, using HPLC-UVD to measure vitamin E content in transgenic Arabidopsis

1. HPLC-UVD conditions and system applicability and preparation of standard solutions

HPLC: Waters 2695 separations module system is adopted, the chromatographic column is a C-18 reversed-phase silica gel column (Calesil ODS-100C18), the specific specification is: 4.6mm inner diameter × 250mm length, the mobile phase is methanol and water, and the volume of methanol and water The ratio is 98:2. At this time, the peak elution time of tocopherol is 26.3 minutes, the peak shape is good, the column temperature is 30°C, the flow rate is 1.0mL/min, and the injection volume is 30μL.

UVD: Waters 2996 photodiode array detector system is adopted, and the detection wavelength of ultraviolet light detector is 292nm. Accurately weigh 2.0 mg of tocopherol standard product from Sigma Company and dissolve it completely in 1 mL of methanol to obtain a 2 mg/mL tocopherol standard product solution, which is stored at -20°C for later use.

2. Preparation of standard curve

Inject 5 μl, 10 μl, 15 μl, 20 μl, and 30 μl of the reference substance solution under corresponding chromatographic conditions to record the spectrum and chromatographic parameters, and perform regression analysis on the standard substance content (X, μg) with the peak area (Y) respectively. Through research, the tocopherol in the present invention exhibits a good log-log linear relationship within the range of 10 μg-60 μg. The log-log linear regression equation of the tocopherol reference substance is: Y=7.26e+002X+1.09e+004; R=0.999740.

3. Sample preparation and determination of tocopherol content

The extraction process of tocopherol is as follows: 1g-2g (fresh weight) of fresh Arabidopsis leaves are taken, ground with liquid nitrogen, extracted with 4mL of n-hexane, and ultrasonicated for 30 minutes. Centrifuge, collect the supernatant, dry it with a nitrogen blower, dissolve the extract with an appropriate amount of methanol, and use it for HPLC detection.

HPLC-UVD was used to measure the tocopherol content, the sample injection volume was 30 μl, and the tocopherol content (mg) in the sample was calculated according to the peak area into the linear regression equation, which was converted into the amount of substance (pmol), and then divided by the approximate amount of the sample The fresh weight (mg) of Arabidopsis leaves was used to calculate the content of tocopherol in Arabidopsis plants.

In the present invention, the transformation of the gamma-TMT gene encoding gamma-tocopherol methyltransferase significantly increases the vitamin E content in the leaves of Arabidopsis thaliana, which makes the vitamin E content in the transgenic plants exceed more than 50% of the control. It can be seen that the γ-TMT gene can be used as a candidate gene for improving the nutritional value of plants, and can be used in the research and industrial production of using transgenic technology to increase the vitamin E content of crops.

sequence listing

<110> Shanghai Jiaotong University

<120> lettuce γ-tocopheryl methyltransferase protein coding sequence

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<211>25

<212>DNA

<213> Lettuce (Lactuca sativa)

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tacctcccga aaagtcccta cgccc 25

<110> Shanghai Jiaotong University

<120> lettuce γ-tocopheryl methyltransferase protein coding sequence

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<210>2

<211>25

<212>DNA

<213> Lettuce (Lactuca sativa)

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ctgggcgtag ggacttttcg ggagg 25

<110> Shanghai Jiao Tong University

<120> lettuce γ-tocopheryl methyltransferase protein coding sequence

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<212>DNA

<213> Lettuce (Lactuca sativa)

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gcagcggatg agcagcagca acaacagcta aaaaaaggaa tagcagaatt ctacgatgaa 180

tcttcgggga tgtggggagaa 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

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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

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aaagatgtaa taaaattctc catcattaca tgtaaaaagc ctgaataaaa ataggtgtgt 1020

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<110> Shanghai Jiao Tong University

<120> lettuce γ-tocopheryl methyltransferase protein coding sequence

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<212>PRT

<213> Lettuce (Lactuca sativa)

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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 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

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

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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

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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 (3)

1. A lettuce gamma-tocopherol methyltransferase protein coding sequence, characterized in that it has the nucleotide sequence shown in the 112th-1008th position in SEQ ID NO.3. 2. The polypeptide encoded by the lettuce γ-tocopherol methyltransferase protein coding sequence according to claim 1, characterized in that it has the amino acid sequence shown in SEQ ID NO.4. 3. A method for improving vitamin E content in plants by using the lettuce gamma-tocopherol methyltransferase protein coding sequence as claimed in claim 1, comprising the steps of: Step 1, linking the lettuce γ-tocopherol methyltransferase protein coding sequence to the plant expression control sequence to obtain a plant expression vector containing the lettuce γ-tocopherol methyltransferase protein coding sequence; In step 2, the expression vector in step 1 is transformed into Agrobacterium, and the Agrobacterium is used to transform Arabidopsis; The third step is to obtain transformed cells containing lettuce gamma-tocopherol methyltransferase protein coding sequence through antibiotic screening, and finally regenerate transgenic plants and their progeny.