CN104404063B - One cultivate peanut vitamin E synthesis key gene and application - Google Patents

Abstract

The present invention relates to one cultivate peanut vitamin E synthesis key gene and application, this laboratory by peanut transcript profile data results design primer, using improvement RACE technologies, clone and separate peanutHPPDThe cDNA sequence of gene.Build overexpression vector p35S::AhHPPD, transformation mode plant tobacco analyzes the content of alpha tocopherol in transgene tobacco and wild-type tobacco through RP HPLC, as a result shows that the content of alpha tocopherol in transgene tobacco improves 23 times.Show what is cloned from peanutAhHPPDTool functional p _ Hydroxyphenyl pyruvic acid dioxygenase can be encoded；The activity of AhHPPD can improve the important function of tocopherol such as alpha tocopherol synthesis.This is beneficial to improve the content of peanut vitamin E, extension grease storage period and shelf life, and tool is of great significance.

Description

One cultivate peanut vitamin E synthesis key gene and application

Technical field

This present invention relates to one cultivate peanut vitamin E synthesis key gene and application, belong to plant genetic engineering field.

Background technology

Vitamin E (Vitamin E) is that a class only synthesizes in photosynthetic organism and all plays an important roll to animal and plant Fat-soluble antioxidant.Vitamin E can strengthen the anti-environment stress ability of plant, improve the stability of grease and extend oil plant The storage period of seed, additionally it is possible to maintain human homergy, the generation of cancer is reduced, is suppressed cardiovascular and cerebrovascular disease and is improved body The functions such as immunocompetence.Vitamin E is also used in the fields such as food, medicine, health products and cosmetics extensively.

Vitamin E is made up of the fragrant head and a nonpolar isoprenoid side chain afterbody of a polarity, according to The saturation degree of side chain is divided into tocopherol and the class of tocotrienols two, per class again according to the position of methyl on fragrant head chromanol With quantity be divided into α-, β-, γ-, δ-four types.The precursor substance of polarity fragrance head is from shikimic acid pathway tyrosine Metabolite alcapton (HGA), the precursor substance of isoprenoid side chain afterbody is the plant from non-mevalonate pathway (MEP) Base diphosphonic acid (PDP) and Mang ox base Mang ox base diphosphonic acid (GGPP).In the vitamin E of eight types, in human liver Alpha-tocopherol transport protein (α-TTP) to the affinity highest of alpha-tocopherol, therefore alpha-tocopherol active highest in human body Also it is easier to absorb.

Alcapton is to provide unique precursor substance of the fragrant head of vitamin E synthesis, p _ Hydroxyphenyl pyruvic acid dioxygenase (HPPD) catalysis p _ Hydroxyphenyl pyruvic acid (HPP) generation alcapton is considered as crucial first of synthetic tocopherol in plant Step, is also the crucial first step of synthetic plasmid quinone.In the Arabidopsis Mutants of HPPD enzymes inactivationpds1In, tocopherol and plastoquinone Can not all synthesize.Tsegaye etc. (2002) overexpression HPPD genes in arabidopsis, make the protein content increase by 10 More than times, the content of tocopherol improves 28% in seed.Falk etc. (2003) mistakes in tobacco by the HPPD genes of barley Amount expression, the synthesis capability of HGA increases by 10 times in transfer-gen plant, and the content of tocopherol increases by 2 times in seed. Weiwei Ren etc. (2011) Transient Expression lettuces in lettuce bladeHPPDGene,HPPDExpression quantity increased 12 times, α- The content of tocopherol increased 4 times.Gemma etc. (2012) overexpressions in paddy rice by the HPPD genes of arabidopsis, make to turn base Because Gamma-Tocopherol is largely converted into alpha-tocopherol in rice paddy seed.As can be seen here, p _ Hydroxyphenyl pyruvic acid dioxygenase exists Played an important role during plant synthetic tocopherol.

Peanut is the main oil crops and industrial crops of China, accounts for the 1/2 of the oil crops gross area.Peanut oil is for I One of main edible oil of state resident, but peanut oil unsaturated fatty acid content is higher, air, light, high temperature and metal from Under the effect such as son, easily become sour, nutrient is destroyed, and peculiar smell occurs.The grease that long-term use is become sour, will make the flesh of people There is deficiency disease in body, also can cause serious poisoning and canceration because of the intake product that becomes sour because lacking required aliphatic acid.Flower Raw vitamin E (mainly tocopherol) content and activity are relatively low, therefore improve the content and activity of vitamin E in peanut, right In improving the Oil stability of peanut, extending grease storage period and meeting the demand of human health, with highly important meaning Justice.

Till now, the clone of relevant vitamin E synthesis key gene and Study on Genetic Transformation are limited to a small number of moulds On formula plant, and the paractical research of crops particularly oil crops is concentrated mainly on soybean.To peanut vitamin E Research report is simultaneously few, and only several vitamin E synthesis key genes are cloned out.So, clone gives birth to dimension from peanut Other related genes of plain E synthesis, and bioinformatic analysis, expression pattern analysis and transgenosis Function Identification are carried out to it, Peanut vitamin E metabolic Study of way can be improved, the content and activity to improving peanut vitamin E play an important roll, To carry out genetic improvement to peanut later, the quality for improving peanut lays the first stone.

The present invention be directed to background above technology, the peanut cDN Α libraries being had been built up using this laboratory, with reference to RACE technologies, clone the total length cDN Α sequences of these vitamin E synthesis related genes from peanut, make sequence correlation analysis And expression pattern analysis, transformation mode plant such as tobacco carries out Function Identification.To build seed specific expression carrier genetic transformation flower Life makes its overexpression in peanut provide scientific basis, to be intended to create vitamin E content（Mainly tocopherol content） And activity（Mainly alpha-tocopherol）The peanut new germ plasm for significantly improving provides material.The present invention will be raising peanut vitamin The storage stability of the content of E and activity and grease lays the foundation, all significant in theory and in practice.

The content of the invention

This discovery there is provided one cultivate peanut vitamin E synthesis key gene and application, be build seed specific expression carrier Genetic transformation peanut makes its overexpression in peanut provide scientific basis, to be intended to create vitamin E content（Mainly give birth to Educate phenol content）And activity（Mainly alpha-tocopherol）The peanut new germ plasm for significantly improving provides material.

To achieve the above object, the present invention is adopted the following technical scheme that：

One cultivates peanut vitamin E synthesis key gene, and the gene isAhHPPD, its sequence as shown in SEQ ID NO.1, Amino acid sequence is shown in SEQ ID NO.2.

Peanut of the inventionAhHPPDGene is set by the basis of the sequencing data of peanut 454 and gene microarray analysis result Meter primer, using the RACE technologies of improvement, clone and separate peanutHPPDThe cDNA sequence of gene, and do relevant biological information credit Analysis.Cloned by RACE technologies and obtain full length gene cDNA as shown in figure 1, Cloning of full length gene nucleotide series such as SEQ ID Shown in No.1.

With the method for qRT-PCR to it in peanut different tissues and peanut embryonic development different times（20, 40, 60DAP） In expression identified and analyzed.WithAhHPPDExpression quantity of the gene in leaf is reference, the expression in fruit pin Amount it is high 14.5 times compared with leaf, the expression quantity in cotyledon, embryo, flower and stem be respectively leaf in 1.4,2.2,2.3 and 2.5 times. The expression quantity planted in skin and root is relatively low, and the expression quantity in leaf is respectively their 3 times and 25 times, as shown in Figure 2.QRT-PCR points AnalysisAhHPPDThe relative expression quantity of mRNA shows in 20 days, 40 days and 60 days embryos of peanut, with gradually reaching maturity for embryo,AhHPPDExpression quantity gradually reduce, the expression quantity in 60 days embryos less than 1/10th in 20 days embryos, as shown in Fig. 2.Explanation The characteristics of there is the gene tissue and space and time difference to express.

The construction method of the overexpression vector of the gene is to connect to replace plant expression vector pBI121- by digestion Gus gene in GUSA, builds CaMV 35S promoters and drivesAhRRS5Gene plant expression vector pBI121-AhHPPD- OE, By its Transformed E HA105 Agrobacteriums, in being conducted into dark green No. one and Ben's tobacco by leaf disk method, tested through PCR and RT-PCR CardAhHPPDGene has been incorporated into tobacco gene group, and being capable of effectively expressing.

Transgene tobacco positive plant VE contents are detected through RP-HPLC, is positive to turn 35S by Molecular Identification::AhHPPD Ten plants of dark green (CB-1) tobacco and Ben's tobacco (N.benthamiana) four plants, cigarette is extracted using direct solvent extraction Alpha-tocopherol in careless plant leaf, every plant of transgene tobacco repeats to extract three times, with the dark green of non-transgenic wild type (WT) No. one is control with Ben's tobacco, and wild-type tobacco extracts three plants.HPLC detect α in transgenosis and non-transgenic tobacco blade- The changes of contents situation of tocopherol, each sample duplicate detection three times, the results are shown in Table 1.Can illustrateAhHPPDSuccessfully turn people Dark green No. one and Ben's tobacco plant are simultaneously expressed.Turn 35S::AhHPPDThe content of alpha-tocopherol is improved in tobacco plant, table The bright clone's from peanutAhHPPDTool functional p _ Hydroxyphenyl pyruvic acid dioxygenase can be encoded.

Peanut of the inventionAhHPPDGene can encode tool functional p _ Hydroxyphenyl pyruvic acid dioxygenase, can urge Change p _ Hydroxyphenyl pyruvic acid (HPP) generation alcapton, and then generate alpha-tocopherol, overexpressing the gene can substantially increase dimension life The content of plain E, this will lay the foundation to improve the content of peanut vitamin E with the storage stability of activity and grease.To prolong The shelf life of peanut long has important practice significance.

Brief description of the drawings

Fig. 1 RACE technologies clone obtainAhHPPDThe electrophoretogram of the full-length cDNA of gene；A：5 ' RACE purpose fragments；B： 3 ' RACE purpose fragments；C：CDNA purpose fragments.

Fig. 2 isAhHPPDPhylogenetic analysis.

Fig. 3 is analyzed for qPCRAhHPPDGene existsAhHPPDGene is respectively organized and interim mRNA during embryonic development three in peanut Relative expression quantity.

Fig. 4 is to turn AhHPPD genetic tobacco Molecular Identifications.Fig. 4 .a transgenic tobacco plants PCR checkings, hole 1-19：19 plants Transgene tobacco genomic DNA amplification product；M：Maker2000；ck1、ck2：Negative control（Non-transgenic tobacco genome DNA cloning result）；ck3：Positive control（Plasmid p35S::AhHPPD amplified productions）；ck4：Water is compareed；Fig. 4 .b transgenosis cigarettes Careless plant RT-PCR checkings, hole 1-13：13 plants of transgene tobacco cDNA amplified productions；M：Maker2000；ck1、ck2：It is negative right According to（Non-transgenic tobacco cDNA amplifications）；ck3：Positive control（Plasmid p35S::AhHPPD amplified productions）；ck4：Water pair According to.

Specific embodiment

【Embodiment 1】Cloned using electronic cloning technologyAhCYP707AGene open reading frame

The candidate gene fragment that the transcript profile data being sequenced according to peanut 454 are obtained, design primer AhHPPD-F (5 '- TCCACGAGTTCGCTGAGTTC-3 ') and AhHPPD-R（5 '-CAAGTGCTGCAACCCTGCACCTTCGTTG -3 '), checking Fujian spends whether No. 6 each tissue mixing cDNA libraries have the genetic fragment, then in conjunction with the LibF- designed according to carrier library 45（5 '-GATTCTGTGGATAACCGTATTACCGCCTTACGCGTGTAAAACGAC-3 ') and LibR-39（5’- ACCAGGATCTCCTAGGGAAACAGCTATGACCATGTTCAC-3 ') primer, carry out RACE reactions.RACE reaction conditions：With AhHPPD-R and LibF-45 carries out 5 ' RACE reactions, PCR amplification conditions for primer：94℃ 5min；94 DEG C of 30s, 70 DEG C 2min, 10 circulations；94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 2min, 25 circulations；72℃ 10min.With AhHPPD-F and LibR-39 carries out 3 ' RACE reactions, PCR amplification conditions for primer：94℃ 5min；94 DEG C of 30s, 70 DEG C of 2min, 10 are followed Ring；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 25 circulations；72℃ 10min.

The size of the purpose band gene according to Bioinformatics Prediction is autotelic to RACE-PCR products to reclaim connection T-A clones are carried out, positive colony serves extra large Hua Da sequencing.5 ' and 3 ' unknown nucleotide sequences for obtaining and candidate gene fragment are carried out Splicing designs total length primer AhHPPD-FL-F to obtain complete cDNA sequence from sequence two ends （GCACCTTCCATCACATTCCCTCCACACAG）And AhHPPD-FL-R（CATTCATGCACAACTATATGGTAAATAATAC） , with cDNA library as template, amplification obtains AhHPPD full length cDNA sequences.Purpose band is purified, connection, conversion, after identification Sequencing.

After through sequencing, pcr amplification reaction is carried out by primer of AhHPPD-R and LibF-45, obtain the 5 ' of 1,200bp or so RACE fragments（Fig. 1）, pcr amplification reaction is carried out by primer of AhHPPD-F and LibR-39, obtain the 3 ' RACE of 900bp or so Fragment（Fig. 1）, compared with candidate gene fragment through sequencing, confirm as same gene.By BioEdit softwares by sequence assembly, One overall length of acquisition is the cDNA integration sequences of 1,575 bp（Fig. 1）, its sequence such as Seq1 temporarily namesAhHPPD.In cDNA sequence Total length primer AhHPPD-FL-F is designed in both sides and AhHPPD-FL-R, PCR amplification obtain the band that size is about 1,500 bp（Figure 1）, sequencing result show its with splicing cDNA sequence it is basically identical.Its full length cDNA sequence is passed through as shown in SEQ ID No.1 440 amino acid of the gene code are analyzed, 5 ' non-translational regions about 31bp long, the 3 ' end a length of 224bp of non-translational region include Poly (A) tail of 27bp.The other plant registered in the peanut AhHPPD amino acid sequences and ncbi database that will derive HPPD protein sequences carry out homologous comparison, and the systematic evolution tree of p _ Hydroxyphenyl pyruvic acid dioxygenase is built using MEGA6.0（Figure 2）, as seen from the figure, peanut AhHPPD protein sequences are all a small branch with the GmHPPD protein sequences of soybean.

【Embodiment 2】The extraction of peanut different tissues and different development stage embryo total serum IgE

No. 6 are spent as material with the peanut improved seeds Fujian of oil plant institute of University Of Agriculture and Forestry In Fujian seed selection, take its different development stage Root, stem, leaf, flower, fruit pin, pericarp, the embryo of planting skin, the tissue of embryo 8 and different development stage be to take 20 after its fruit pin buries My god, 40 days, the fresh embryo of 60 days, store in -80 DEG C it is standby.By 1.7mLCTAB extracts（65℃）Preheated with 80 μ l mercaptoethanols To 65 DEG C；Add ground material, vortex 2min, 65 DEG C of warm bath 15-30min to overturn mix 3-4 times therebetween, add 1/10 The absolute ethyl alcohol of volume, the 3MKAc of 1/10 volume, isometric phenol/chloroform/isoamyl alcohol (25：24：1), overturn at room temperature and mix 10min.4 DEG C, 12000r/min centrifugations 15min；Supernatant is taken, isometric chloroform/isoamyl alcohol (24 is added：1), run at room temperature Mix 10 minutes.4 DEG C, 12000r/min centrifugations 15min.Supernatant adds the 10mol L-1 LiC1 of 1/3 volume, -80 DEG C of mistakes Night is precipitated；4 DEG C, 12000r/min centrifugation 15min abandon supernatant, are washed with 75% ethanol 1 time, and drying at room temperature or vacuum drying are heavy Form sediment；Add TE（10 mmol•I Tris•HCl pH8.0.1 mmol/L EDTA）Dissolution precipitation, adds isometric water saturation Phenol：Chloroform：Isoamyl alcohol（25：24：1）, mixing, 12000 r/min are centrifuged 15 min, move supernatant to new centrifuge tube；Supernatant adds The 10mol L of 1/3 volume^-1LiC1, -80 DEG C of precipitation 1h；4 DEG C, 12000r/min centrifugation 15min abandon supernatant, are washed with 75% ethanol Wash 2 times.It is stored in water after drying at room temperature, or vacuum drying, -80 DEG C save backup.

【Embodiment 3】QRT-PCR is analyzedAhHPPDExpression pattern

Peanut different tissues and different development stage embryo total serum IgE are extracted respectively using above-mentioned modified CTAB method, and 1 μ g are taken respectively With PrimeScript Reverse Transcriptase reverse transcriptases（Purchased from TAKARA companies）Carry out reverse transcription.Will be single Chain cDNA takes 2 μ L for template after diluting 10 times, grasped according to TAKARA companies SYBR Premix Ex Taq kit protocols Make, use Mastercylcer ep realplex (Eppendorf) with special primer AhHPPD_RT_F（5’- AGAAGAACTGCATAATGGGATTCGGTC-3’)；AhHPPD_RT_R（5’-GAAAGGAGCATTGCCCAAGTGACTGTG- 3’）；Ahactin F（5’-GAGGAGAAGCAGAAGCAAGTTG-3’）, Ahactin R（5’- AGACAGCATATCGGCACTCATC-3’）Carry out qRT-PCR.Gene expression feelings in the sample are analyzed according to Δ Δ CT methods Condition.Analyzed using qRT-PCRAhHPPDGene peanut respectively organize and during embryonic development three interim mRNA relative expression quantity, knot Fruit sees Fig. 4.As seen from the figure,AhHPPDRespectively organized in peanut and embryonic development different times have expression, and expression has significantly Spatio-temporal difference.WithAhHPPDExpression quantity of the gene in leaf is reference, expression quantity in fruit pin compared with leaf in it is high 14.5 times, Expression quantity in cotyledon, embryo, flower and stem is respectively 1.4,2.2,2.3 and 2.5 times in leaf.Expression quantity in kind of skin and root compared with Low, the expression quantity in leaf is respectively their 3 times and 25 times.QRT-PCR is analyzedAhHPPDIn peanut 20 days, 40 days and 60 days The relative expression quantity of mRNA shows in embryo, and with gradually reaching maturity for embryo, the expression quantity of AhHPPD is gradually reduced, in 60 days embryos Expression quantity less than 1/10th in 20 days embryos.Illustrate the characteristics of there is the gene tissue and space and time difference to express.

【Embodiment 4】AhHPPDOverexpression vector builds

By AhHPPD-Spe1-F（5’-AGGAACTAGTCTCCACACAGTCATGGTTACC-3’）And AhHPPD- Asc1-R（5’-AGGAAGGCGCGCCCCATTATGCAGTTCTTCTAC-3’）This is to primer from the plasmid with complete reading frame Middle amplification is obtainedAhHPPDGene cDNA encoding area, the end of 5 ' end 3 ' is respectively provided with Spe1 and Asc1 restriction enzyme sites, to this experiment What the pBI121-GUSA driven by 2 × CaMV 35S promoters and amplification that room builds were obtainedAhHPPDPurpose fragment is used simultaneously Spe1（Purchased from NEB companies）And Asc1（Purchased from NEB companies）Double digestion, E. coli DH5 is transformed into after reclaiming connection In α, the plasmid of positive colony is extracted, Agrobacterium EHA105 is converted by freeze-thaw method, for converting dark green No. one and Ben's tobacco.

【Embodiment 5】The genetic transformation of tobacco and the Molecular Identification of transgene tobacco

Agrobacterium tumefaciens mediated leaf disk method transformation of tobacco is taken, being respectively provided with plasmid p35S::The super tables of AhHPPD-OE Agrobacterium up to carrier converts Ben's tobacco and dark green No. by leaf disk method（CB-1）, use 100mg/L kanamycins（Kan）Sieve Leaf bud and root growth are selected, with 500 mg/L cephalosporins in incubation（Cef）To suppress the growth of Agrobacterium, bar is cultivated 25 DEG C ± 2 DEG C of part temperature, illumination daily 14h daytimes/10h nights.Co-culture culture medium：MS culture medium+0.1mg/L NAA（a- Methyl α-naphthyl acetate）+ 1mg/L 6-BA（6-benzyl aminopurine）, induce screening and culturing medium：MS culture medium+0.1mg/L NAA+1mg/L Mg/L kan+500mg/L the Cef of 6-BA+ 100, root media：MS culture medium+50mg/L Kam+500mg/L Cef. NAA, 6-BA, kan and Cef are purchased from Sigma companies.

Transgenosis and the non-DNA for turning base tobacco leaf are extracted using CTAB methods, with the DNA as template, by CaMV 35S Promoter anti-sense primer Trans id 35s fwd (5 '-TGATGTGATATCTCCACTGACGTAAG-3 ') and downstream of gene draw Thing AhHPPD-R（5’-TCTGTTCATCACTCAGCACATC-3’）Carry out pcr amplification reaction.PCR amplification conditions：94℃ 5min；94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 2min, 35 circulations；72℃10min.

Transgenosis and the non-RNA for turning base tobacco leaf are extracted using Trizol methods, according to PrimeScript Reverse Transcript kits（Purchased from TAKARA）The synthesizing single-stranded cDNA of reverse transcription.As template after single-stranded cDNA is diluted into 3 ~ 5 times, By after CaMV 35S promoter transcription initiation sites design primer Trans id 35s fwd1 (5 '- GAAGTTCATTTCATTTGGAGAGAACAC-3 ') and downstream of gene primer AhHPPD-R（5’- TCTGTTCATCACTCAGCACATC-3’）Carry out RT-PCR amplified reactions.PCR reaction systems are：The μ l of 10 × buffer 2.0, The 0.5 μ l of μ l, 35S-F of dNTP 1.5,AhHPPDThe μ l of-R 0.5, Taq enzyme 0.1 μ l, ddH2O 14.4 μ l, the μ l of template DNA 1.0, Cumulative volume is 20 μ l.PCR amplification conditions：94℃ 5min；94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 2min, 35 circulations；72℃ 10min。

Extract DNA and enter performing PCR the result such as Fig. 4 a（Only display portion result）, verify with 35S promoterAhHPPD Whether gene order is incorporated into tobacco gene group.Transgenosis and the non-RNA for turning base tobacco leaf are extracted, reverse transcription is synthesizing single-stranded cDNA.It is template with the cDNA for diluting 3 ~ 5 times, with Trans id 35s fwd1 and AhHPPD-R as primer, carries out RT-PCR Whether amplified reaction, checking 35S promoter drivesAhHPPDExpressed in tobacco, as a result such as Fig. 4 b（Only display portion result）. ShowAhHPPDGene has successfully been incorporated into the genome of tobacco plant and has been expressed.

【Embodiment 6】Transgene tobacco vitamin E is extracted and high performance liquid chromatography detection

It is positive to turn 35S by Molecular Identification::AhHPPDTen plants of dark green (CB-1) tobacco and Ben's tobacco (N.benthamiana) four plants of alpha-tocopherols extracted using direct solvent extraction in tobacco leaf.Concrete operations are as follows：It is accurate Fresh tobacco leaves 1g is really weighed, liquid feeding nitrogen is fully ground in powdered, is quickly transferred to the 10ml centrifuge tubes containing 3mL n-hexanes In, vortex 30s is placed in water-bath 20min in 60 DEG C of water-baths, and water-bath is vortexed twice therebetween；4,000 rpm are centrifuged 15min, take It is placed in clearly in new centrifuge tube；Precipitation is resuspended with the n-hexane of 3mL, water-bath 20min in 60 DEG C of water-baths, 4,000rpm centrifugations 15min, takes supernatant, merges supernatant；Supernatant is placed in after being evaporated in vacuum desiccator and is substantially soluble in 1mL methyl alcohol, crosses 0.45 filter membrane, Filtrate sealing is kept in dark place and determined for HPLC.

The chromatographic condition that vitamin E is determined：Chromatographic column：Shimadzu C18 (4.6 × 250mm, 5 μm)；Mobile phase：Methanol-water (98: 2, V/V)；Ultraviolet detection wavelength：294nm；The μ L of sample size 10；Flow velocity：1.0mL/min；Column temperature：30℃.

It is control with dark green No. one of non-transgenic wild type (WT) and Ben's tobacco, wild-type tobacco extracts three plants. HPLC detects the changes of contents situation of alpha-tocopherol in transgenosis and non-transgenic tobacco blade, each sample duplicate detection three It is secondary, the results are shown in Table 5.Can illustrateAhHPPDDark green No. one of people and Ben's tobacco plant are successfully turned and have been expressed.Turn 35S::AhHPPDThe content of alpha-tocopherol is improved in tobacco plant, shows what is cloned from peanutAhHPPDTool functional can be encoded P _ Hydroxyphenyl pyruvic acid dioxygenase.

The content of alpha-tocopherol in the transgenosis of table 5 and non-transgenic tobacco blade

The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with Modification, should all belong to covering scope of the invention.

SEQUENCE LISTING

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<400> 4

caagtgctgc aaccctgcac cttcgttg 28

<210> 5

<211> 45

<212> DNA

<213>Artificial sequence

<400> 5

gattctgtgg ataaccgtat taccgcctta cgcgtgtaaa acgac 45

<210> 6

<211> 39

<212> DNA

<213>Artificial sequence

<400> 6

accaggatct cctagggaaa cagctatgac catgttcac 39

<210> 7

<211> 29

<212> DNA

<213>Artificial sequence

<400> 7

gcaccttcca tcacattccc tccacacag 29

<210> 8

<211> 31

<212> DNA

<213>Artificial sequence

<400> 8

cattcatgca caactatatg gtaaataata c 31

<210> 9

<211> 27

<212> DNA

<213>Artificial sequence

<400> 9

agaagaactg cataatggga ttcggtc 27

<210> 10

<211> 27

<212> DNA

<213>Artificial sequence

<400> 10

gaaaggagca ttgcccaagt gactgtg 27

<210> 11

<211> 22

<212> DNA

<213>Artificial sequence

<400> 11

gaggagaagc agaagcaagt tg 22

<210> 12

<211> 22

<212> DNA

<213>Artificial sequence

<400> 12

agacagcata tcggcactca tc 22

<210> 13

<211> 31

<212> DNA

<213>Artificial sequence

<400> 13

aggaactagt ctccacacag tcatggttac c 31

<210> 14

<211> 33

<212> DNA

<213>Artificial sequence

<400> 14

aggaaggcgc gccccattat gcagttcttc tac 33

<210> 15

<211> 26

<212> DNA

<213>Artificial sequence

<400> 15

tgatgtgata tctccactga cgtaag 26

<210> 16

<211> 22

<212> DNA

<213>Artificial sequence

<400> 16

tctgttcatc actcagcaca tc 22

<210> 17

<211> 27

<212> DNA

<213>Artificial sequence

<400> 17

gaagttcatt tcatttggag agaacac 27

<210> 18

<211> 22

<212> DNA

<213>Artificial sequence

<400> 18

tctgttcatc actcagcaca tc 22

Claims (2)

1. one cultivate peanut vitamin E synthesis key gene build overexpression vector tobacco vitamin E synthesis in effect, institute Stating gene isAhHPPD, its sequence is as shown in SEQ ID NO.1.

2. application according to claim 1, it is characterised in that：The gene is from peanut transcript profile data and chip analysis Screened in result and obtained, its full length cDNA sequence is obtained by RACE technologies.