CN101586108A - Lettuce HPPD protein coded sequence - Google Patents

Abstract

The invention relates to a lettuce HPPD protein coded sequence in the technical field of gene engineering, which comprises that: a polypeptide nucleotide sequence with lettuce HPPD protein activity is coded; and the nucleotide sequence consists of nucleotide sequences at 197-1,534 positions in SEQ ID NO.3 with at least 70 percent of homology. A method for transforming plants to increase the content of pant vitamin E comprises the following steps: connecting coded polypeptide purified nucleotide sequence with the lettuce HPPD protein activity to a plant expression regulatory sequence to form a plant expression vector containing lettuce HPPD protein gene; transforming the expression vector in the step (1) into Agrobacterium; transforming the Agrobacterium containing the expression vector into Arabidopsis by an immigration method; and screening the Arabidopsis by antibiotics to obtain transgenic cells containing the lettuce HPPD protein gene, and finally regenerating transgenic plants and descendants comprising plant seeds and plant tissues. In the invention, the gene is expressed in the plant, so the content of the vitamin E in the obtained transgenic plants is obviously improved.

Description

Lettuce HPPD protein coded sequence

Technical field

What the present invention relates to is a kind of albumen coded sequence of gene engineering technology field, particularly, the present invention relates to a kind of lettuce HPPD protein coded sequence.

Background technology

From nineteen twenty-two vitamin-E biological function be found after, long-term medical research shows, 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 (Traber and Sies, 1996) is arranged all.Vitamin-E is a kind of lipid VITAMIN, its natural product is divided into eight types according to the difference of structure, be respectively α, β, γ, Delta-Tocopherol (tocopherol) and α, β, γ, δ-tocotrienols (tocotrienol), wherein the biological activity of alpha-tocopherol is the highest, is considered to the main active ingredient of vitamin-E.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.

Vitamin-E plays an important role for human and animal's health, but 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, changes wherein non-alpha-tocopherol composition 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 participating in the separation and the functional verification work of vitamin-E route of synthesis key gene, initial analysis vitamin-E (tocopherol) route of synthesis general picture.

Vitamin-E (tocopherol) route of synthesis mainly is made up of following five steps.(1) 4-hydroxyphenylpyruvic acid (HPP) generates homogentisic acid (HGA) (Norriset al., 1998) under the catalysis of 4-hydroxyphenylpyruvate dioxygenase (HPPD); (2) condensation takes place with phytyl bisphosphate (PDP) in homogentisic acid under homogentisic acid phytyl prenyltransferase (HPT) catalysis, generates 2-methyl-6-phytyl-benzoquinones (Collakova and DellaPenna, 2001); (3) 2-methyl-6-phytyl-benzoquinones generates 2,3-dimethyl-6-phytyl-benzoquinones (van Eenennaam et al., 2003) under the catalysis of methyl phytyl benzoquinone methyl transferase (MPBQ MT); (4) 2,3-dimethyl-6-phytyl-benzoquinones generates Gamma-Tocopherol (Porfirovaet al., 2002) under the catalysis of tocopherol cyclase (TC); (5) Gamma-Tocopherol (under the catalysis of γ-TMT), generates alpha-tocopherol at gama-tocopherol methyl transferase.

In analysis to the prior art document, still find no so far and utilize the technical measures of genetic engineering technique content of vitamin E in the romaine lettuce raising plant, also find lettuce HPPD protein sequence and the relevant report of nucleotide sequence thereof mentioned with the present invention.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of lettuce HPPD protein coded sequence is provided.This genes encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD), it participates in the metabolic pathway of synthesizing of vitamin-E; Lettuce HPPD protein is in the application that utilizes on the transgenic technology raising plant vitamin E.

The present invention is achieved by the following technical solutions:

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of lettuce HPPD protein, and described nucleotide sequence constitutes at least 70% homology by the nucleotide sequence of 197-1534 position among the SEQ ID NO.3.

Described nucleotide sequence is by polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative constitutes.

Described polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, also provide a kind of utilization to have the lettuce HPPD protein nucleotide sequence and be transformed into the method that plant is improved plant vitamin E content, its step is as follows:

(1) nucleotide sequence that coding is had a purifying of the active polypeptide of lettuce HPPD protein is connected in the expression of plants regulating and controlling sequence, forms the plant expression vector that contains the lettuce HPPD protein gene;

(2) change the expression vector in the step (1) over to Agrobacterium, the Agrobacterium that will contain expression vector is by the flower-dipping method arabidopsis thaliana transformation;

(3), obtain to contain the transformant and the final regeneration of transgenic plant of lettuce HPPD protein gene and comprise plant seed and the offspring of plant tissue again by antibiotic-screening.

Containing in the transfer-gen plant of lettuce HPPD protein gene the content of vitamin-E has had and has significantly improved.

Preferably, the nucleotide sequence that uses in the method has the sequence of 197-1534 position among the SEQ ID NO.3.

In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, refer to encode has the nucleotide sequence of the active polypeptide of lettuce HPPD protein to term " lettuce HPPD protein (or polypeptide) encoding sequence ", as 197-1534 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 197-1534 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.

This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ ID NO.3 with natural lettuce HPPD identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, term " lettuce HPPD protein (or polypeptide) " refers to have the active SEQ IDNO.4 of lettuce HPPD polypeptide of sequence.This term also comprises having and the variant form lettuce HPPD identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.

The variant form of lettuce HPPD protein of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with coded albumen of the DNA of the DNA hybridization of coding lettuce HPPD protein and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of antibiosis dish HPPD to obtain.

In the present invention, " lettuce HPPD protein conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.As following table 1

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr

Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Invention also comprises the analogue of lettuce HPPD protein or polypeptide.The difference of these analogues and natural lettuce HPPD related polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing lettuce HPPD protein polypeptide of the present invention, lettuce HPPD protein coded sequence operationally can be connected in expression regulation sequence, thereby form the lettuce HPPD protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, arabidopsis cell and other vegetable cell.

In addition, according to the nucleotide sequence and the aminoacid sequence of lettuce HPPD of the present invention, can be on the homology basis of nucleic acid homology or marking protein, relevant homologous gene of screening lettuce HPPD protein or homologous protein.

Lettuce HPPD associated nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the nucleotide sequence of lettuce HPPD associated protein in the code book invention.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

Utilize lettuce HPPD protein of the present invention,, can filter out with HPPD albumen interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.

Related Agrobacterium is agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain GV3101 among the present invention, and this bacterial strain is open purchase (derive from Australian CAMBIA company, strain number is Gambar 3) from the market.

Description of drawings

Fig. 1 relatively tabulates for the homology of the aminoacid sequence of HPPD associated protein in the aminoacid sequence of lettuce HPPD protein of the present invention and the other plant species.Wherein, identical amino acid marks with the amino acid monocase between different peptide sequences.

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 1

The clone of lettuce HPPD gene

1.RNA extraction (RNA extraction)

Get the romaine lettuce leaf tissue, place liquid nitrogen to grind, adding fills in 1.5mL Eppendorf (EP) centrifuge tube of lysate, fully after the vibration, according to the specification sheets extracted total RNA of 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 (Cloning of Full-length cDNA)

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 (Clontech) test kit) carry out the cDNA full-length clone, divide three phases to carry out:

(1) first chain cDNA's is synthetic:

The 5 '-CDS primer A and the SMART2 A oligo primer that utilize the Clontech test kit to be provided are template with the total RNA that is extracted, synthetic 5 '-RACE-ReadycDNA 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-Ready cDNA 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) (SEQ ID NO.1) of Arabidopis thaliana homologous region design to 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) (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 analyzes and the sequencing result proof obtains from romaine lettuce really is the HPPD gene.

By above-mentioned steps, obtained to participate in the romaine lettuce the proteic complete encoding sequence of vitamin-E synthetic HPPD (SEQ ID NO.3).Wherein, (ATG) in SEQ ID NO.3, mark with underscore with terminator codon (TGA).

Embodiment 2

The sequence information of lettuce HPPD genes involved and homology analysis

The full-length cDNA length of the new lettuce HPPD that obtains among the present invention is 1743bp, and detailed sequence is seen SEQ IDNO.3, and wherein open reading frame is positioned at 197-1534 position Nucleotide (1338 Nucleotide).Derive the aminoacid sequence of lettuce HPPD according to the cDNA that is obtained, totally 446 amino-acid residues, molecular weight is 49kD, iso-electric point is 5.34.Detailed sequence is seen SEQ ID NO.4.

Utilize vectorNTI 9.0 softwares that the related amino acid sequence of the HPPD that derives from each kind of plant is carried out the homology comparison, found that, lettuce HPPD gene of being cloned into and puncture vine clover (Medicago truncatula), rape (Brassica rapa), Radix Dauci Sativae (Daucus carota), soybean (Glycine max), Arabidopis thaliana (Arabidopsis thaliana), barley (Hordeum vulgare), the coded amino acid sequence similarity of homologous gene is respectively in the wheat (Triticum aestivum): 77%, 73%, 73%, 73%, 68%, 60%, 59% (Fig. 1).This shows that there is higher homology in the HPPD genes involved in the species such as lettuce HPPD gene and puncture vine clover on coded amino acid levels, can think that their product also has very high similarity on function.

Embodiment 3

Lettuce HPPD associated protein or polypeptide carry out the content of vitamin E of eukaryotic cell expression and transfer-gen plant in arabidopsis cell identifies

1. the structure that contains the expression vector of goal gene (lettuce HPPD gene)

According to the complete encoding sequence (SEQ ID NO.3) of lettuce HPPD, 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 lettuce HPPD is connected among the intermediate carrier pMD18-T checks order, the coding region sequence of correct HPPD of will checking order again further is cloned among the expression vector pHB, then change it over to agrobacterium tumefaciens (as GV3101, usedly in the present embodiment given by Univ Nottingham UK), the performing PCR of going forward side by side checking.The result shows that the plant expression vector that contains the lettuce HPPD gene successfully is building up in the agrobacterium tumefaciens bacterial strain.Flower (floral-dip) method arabidopsis thaliana transformation is soaked in employing.

2. adopt and soak flower (floral-dip) method arabidopsis thaliana transformation

1) gets growth about month, upgrowth situation good stand (can carry previous week before transforming plant is 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;

6) plant taken out in second day, vertically, transfer under the normal condition and grow.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 (Hyg), and 4 ℃ of vernalization 48h move on to one week of growth under the phytotron 24h continuous illumination condition;

8) (transformant is longer for green seedling and root to wait to grow green transgenosis resistance seedling; Non-transformant is etiolated seedling substantially, or green unrooted seedling) back transplants the continued growth of burying;

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 the individual plant results.Continue screening until the transfer-gen plant that obtains to isozygoty.

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 HPPD.The result shows, can amplify the specific DNA fragment of goal gene size with CaMV 35S forward primer and HPPD reverse primer.And when being template, do not amplify any fragment with non-arabidopsis thaliana transformation genomic dna.

Embodiment 4

Utilize HPLC-UVD to measure content of vitamin E in the transgenic arabidopsis

1.HPLC-UVD the preparation of 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-100 C18,4.6mm I.D. * 250mm length), moving phase is methyl alcohol: water, methyl alcohol: the volume ratio of water is 98: 2, column temperature is 30 ℃, flow velocity 1.0mL/min, sample size 30 μ L.

UVD: adopt Waters 2996 photodiode array dectector systems, UV-detector detects wavelength 292nm.

Precision takes by weighing tocopherol standard substance (Sigma company) 2.0mg and dissolves fully with 1mL methyl alcohol, obtains 2mg/mL tocopherol standard solution, be stored in-20 ℃ standby.

Moving phase is methyl alcohol (methanol) among the present invention: water, ratio are 98%: 2% o'clock, and the appearance time of tocopherol is 26.3min, and the peak type is good.

2. the making of typical curve

With described reference substance solution difference sample introduction 5 μ L under corresponding chromatographic condition, 10 μ L, 15 μ L, 20 μ L, 30 μ L record collection of illustrative plates and chromatographic parameter carry out regression analysis with peak area (Y) to standard substance content (X, μ g) respectively.By research, tocopherol presents good log-log linear relationship among the present invention in 10-60 μ g scope.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: the fresh Arabidopsis leaf that takes a morsel (1-2g 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 HPPD gene 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 the HPPD 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 that the present invention relates to and mark apportion are as follows:

(1) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 25bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.1

5’-ATCCGCCGTCTTGATCACGCCGTAG-3’

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 27bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.2

5’-CCGGCGCCTTCGTTGTGTTCCAGATAC-3’

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 1743bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: Nucleotide

(iii) sequence description: SEQ ID NO.3

ATCTAATTTCTGGTTTGAATCTTTGAGATAACCAGTGTTACGTGTCAATATCATCACTCCAAACCAATCG

TTGATACTTCCGTGTGGTTCATATCCTGTTTCTTTTGGAGACGTGCTCCTACTCGCTTCTATACATCTAT

ATTTGATCTACACCTAATTCACGATTTCAAAATCCAAATCCAAATCCAAATCCAAA ATGGGAACGGAAGC

TAAACCCTCCAATGTTGCCGGAGAACAAACCGGCTCCGCCTTCAAGCTAGTTGGCTTCAACAACTTCATC

CGAACAAATCCCATGTCCGACAAATTCTCAGTCAAAAGATTCCACCACATTGGGTTCTGGTGCTCCGACG

CCACCAACACCGCCCGACGTTTCTCCTGGGGCCTCGGCATGCCCATCGTTCAGAAATCCGATCTATCAAC

TGGAAACGCAACTCACGCCTCCTACCTCCTCCGCTCCGGCCAACTCAACTTCCTCTTCACTGCTCCTTAT

TCCCCCTCCATCTCCTCCGCCGTCACCTCCACCACCGCTTCCATCCCGTCGTTTTCCCACACTGACTGCC

GCAACTTCACCGCTTCCCACGGCCTCGCAGTCCGCTCAGTCGCCATTGAAGTCGAAGACGCCGAAATCGC

TTTCTCCGTCAGCGTCTCCCACGGCGCTAAACCCTCTTCTTCCCCGATCACCCTTGGAAATAACGACGTC

GTACTGTCTGAAGTTAAACTCTACGGCGATGTCGTTTTGCGTTACATAAGCTACAAGAATCCCAACTTTG

ATGGCATATCTAATTTCTTGCCCGGGTTTGAACCAGTTGAAAAGACGTCGTCGTTTCCTGACCTTGACTA

CGGTATCCGCCGTCTTGATCACGCCGTAGGCAACGTCCCGGAACTCGCTCCGGCTGTAGACTACGTGAAA

TCGATCACCGGATTCCATGAGTTCGCCGAATTCACGGCGGAGGATGTCGGCACGAGCGAGAGCGGACTCA

ATTCGGTTGTTTTAGCGTGCAATAGCGAGATGGTTTTGATCCCTATGAATGAGCCGGTGTACGGAACAAA

GAGGAAGAGCCAGATTCAGACGTATCTGGAACACAACGAAGGCGCCGGAGTTCAGCATTTGGCGCTGGCA

AGTGAGGACATATTTCGGACCTTGAGAGAGATGAGGAAGCGCAGCGGTATTGGTGGTCTTGAGTTTATGC

CGTCGCCACCGCCTACTTATTATCGGAATTTGAAGAATAGAGTCGGAGATGTATTGACAGATGAAGAGAT

TAAGGAGTGTGAGGAGTTGGGAATATTAGTCGATAGAGATGATCAGGGGACTCTGCTTCAAATCTTCACC

AAACCCGTGGGTGACAGGCCAACCATATTCATAGAGATAATTCAGAGAGTTGGGTGTATGGTGAAAGATG

GTGAAGGCAAGGTGCAGCAGAAGGCAGGGTGTGGAGGATTCGGGAAAGGGAACTTCTCTGAGCTCTTCAA

ATCTATCGAGGAATATGAGGAGACGCTTGAAGCACGAACCACTACTGAACCAACTGCTGCTGCA

AAG

CTGCCCATACTATGGTAGTAATATCCATGTCTTCTTCATTGAAATCCTATGATTCGAACATATTACATAT

GTACATGAGGATTCTGTAGAAAGTGAGTCCTGAAAATGTTTGATCTTGACAAGAAGATATTGGTAACATG

TTTTAATTTTCTATACGGAATAAATACTGTTGGGTTAAAAAAAAAAAAAAAAAAAAAAAAAAA

(4) information of SEQ ID NO.4

(i) sequence signature:

(A) length: 446 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) sequence description: SEQ ID NO.4

M G T E A K P S N V A G E Q T G S A F K L V G F N N F I R T N P M S D K

F S V K R F H H I G F W C S D A T N T A R R F S W G L G M P I V Q K S D

L S T G N A T H A S Y L L R S G Q L N F L F T A P Y S P S I S S A V T S

T T A S I P S F S H T D C R N F T A S H G L A V R S V A I E V E D A E I

A F S V S V S H G A K P S S S P I T L G N N D V V L S E V K L Y G D V V

L R Y I S Y K N P N F D G I S N F L P G F E P V E K T S S F P D L D Y G

I R R L D H A V G N V P E L A P A V D Y V K S I T G F H E F A E F T A E

D V G T S E S G L N S V V L A C N S E M V L I P M N E P V Y G T K R K S

Q I Q T Y L E H N E G A G V Q H L A L A S E D I F R T L R E M R K R S G

I G G L E F M P S P P P T Y Y R N L K N R V G D V L T D E E I K E C E E

L G I L V D R D D Q G T L L Q I F T K P V G D R P T I F I E I I Q R V G

C M V K D G E G K V Q Q K A G C G G F G K G N F S E L F K S I E E Y E E

T L E A R T T T E P T A A A

Claims (4)

1. lettuce HPPD protein coded sequence, it is characterized in that, comprise: coding has the nucleotide sequence of the active polypeptide of lettuce HPPD protein matter, and described nucleotide sequence constitutes at least 70% homology by the nucleotide sequence of 197-1534 position among the SEQ ID NO.3.

2. lettuce HPPD protein coded sequence as claimed in claim 1 is characterized in that, described nucleotide sequence is by polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative constitutes.

3. lettuce HPPD protein coded sequence as claimed in claim 1 is characterized in that, described polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

4. lettuce HPPD protein coded sequence as claimed in claim 1 is transformed into the method that plant is improved plant vitamin E content, it is characterized in that step is as follows:

(1) nucleotide sequence that coding is had a purifying of the active polypeptide of lettuce HPPD protein is connected in the expression of plants regulating and controlling sequence, forms the plant expression vector that contains the lettuce HPPD protein gene;

(2) change the expression vector in the step (1) over to Agrobacterium, the Agrobacterium that will contain expression vector is by the flower-dipping method arabidopsis thaliana transformation;

(3), obtain to contain the transformant and the final regeneration of transgenic plant of lettuce HPPD protein gene and comprise plant seed and the offspring of plant tissue again by antibiotic-screening.