CN104404063B - One cultivate peanut vitamin E synthesis key gene and application - Google Patents
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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
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
<110>University Of Agriculture and Forestry In Fujian
<120>One cultivate peanut vitamin E synthesis key gene and application
<130> 18
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 1575
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<213>Artificial sequence
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gcaccttcca tcacattccc tccacacagt catggttacc caaacgcaac agaacgctcc 60
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cccgaaatcg gaccgcttca aggtaatccg gttccaccat gtggagttct ggtgcaccga 180
tgcaaccaac accgccagcc gcttctcttg gggcctcgga atgccgatcg tagcgaagtc 240
cgacctctcc accggaaacg cagttcatgc ctcctacctc ctccgctccg gcgacctcaa 300
tttcctcttc tccgctcctt attccgcctc aatcgccggc gatggtgcct ccgcttccgt 360
tccctccttc tctgcctctg attgccactc cttcgcagct aaacacggcc tcgccgtccg 420
cgccgtcgcc atcgaggtgg aagacgcctc cgccgccttc tccgccagcg tcgccaacgg 480
cgcgaaacct gtatctccgg cggttctcct cgacaacagc gtcggattcg ccgaggttca 540
tctctacggc gacgtcgttc tccgctacat cagctactcc gccccgaccc gagaatctga 600
cctgaatcct agcttacggt tcctccctgg attcgaagcc gtggaggctg attcgtcgtt 660
cccggagcta gattacggta ttcggcgact cgaccacgcc gtcgggaacg tgccggagct 720
ggcgccggca gtgaattact tgaagaaatt caccggattc cacgagttcg cagagttcac 780
ggcggaggac gttggaacga gtgagtcagg gctgaactcg gtggttcttg cgagcaacga 840
tgagatggtt cttcttccgt tgaacgaacc ggtgtacgga acaaagagga agagccaaat 900
tgagacgtat cttgagcaca acgaaggtgc agggttgcag cacttggcgt tggtctccga 960
agacatattc aggactctga gggagatgag gaagagaagc accatcggcg gttttcagtt 1020
catgccgtct ccgccgccaa cgtattaccg gaacttgaag aagcgtgccg gtgatgtgct 1080
gagtgatgaa cagattaagg agtgtgagga gcttggaatc cttgttgata gggacgatca 1140
gggcactctg cttcagattt tcaccaaacc tgttggtgat aggccaacca tttttataga 1200
gataattcag agaattggat gcatgctcaa ggatgaagaa ggaaaggtgt accaaaaagg 1260
tggatgtggg ggttttggaa aaggcaactt ctctgagctt tttaaatcca ttgaagaata 1320
tgagaagact ttggaaagta gaagaactgc ataatgggat tcggtccctt gcaatttccc 1380
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gctcatagat aaagcaacac aattacacag tcactcaggc aatgctcctt tcttcttgta 1500
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Met Val Thr Gln Thr Gln Gln Asn Ala Pro Ser Ser Ser Pro Glu Pro
1 5 10 15
Ala Phe Lys Leu Val Gly Phe Lys Asn Phe Val Arg Thr Asn Pro Lys
20 25 30
Ser Asp Arg Phe Lys Val Ile Arg Phe His His Val Glu Phe Trp Cys
35 40 45
Thr Asp Ala Thr Asn Thr Ala Ser Arg Phe Ser Trp Gly Leu Gly Met
50 55 60
Pro Ile Val Ala Lys Ser Asp Leu Ser Thr Gly Asn Ala Val His Ala
65 70 75 80
Ser Tyr Leu Leu Arg Ser Gly Asp Leu Asn Phe Leu Phe Ser Ala Pro
85 90 95
Tyr Ser Ala Ser Ile Ala Gly Asp Gly Ala Ser Ala Ser Val Pro Ser
100 105 110
Phe Ser Ala Ser Asp Cys His Ser Phe Ala Ala Lys His Gly Leu Ala
115 120 125
Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Ser Ala Ala Phe Ser
130 135 140
Ala Ser Val Ala Asn Gly Ala Lys Pro Val Ser Pro Ala Val Leu Leu
145 150 155 160
Asp Asn Ser Val Gly Phe Ala Glu Val His Leu Tyr Gly Asp Val Val
165 170 175
Leu Arg Tyr Ile Ser Tyr Ser Ala Pro Thr Arg Glu Ser Asp Leu Asn
180 185 190
Pro Ser Leu Arg Phe Leu Pro Gly Phe Glu Ala Val Glu Ala Asp Ser
195 200 205
Ser Phe Pro Glu Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val
210 215 220
Gly Asn Val Pro Glu Leu Ala Pro Ala Val Asn Tyr Leu Lys Lys Phe
225 230 235 240
Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr
245 250 255
Ser Glu Ser Gly Leu Asn Ser Val Val Leu Ala Ser Asn Asp Glu Met
260 265 270
Val Leu Leu Pro Leu Asn Glu Pro Val Tyr Gly Thr Lys Arg Lys Ser
275 280 285
Gln Ile Glu Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gln His
290 295 300
Leu Ala Leu Val Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg
305 310 315 320
Lys Arg Ser Thr Ile Gly Gly Phe Gln Phe Met Pro Ser Pro Pro Pro
325 330 335
Thr Tyr Tyr Arg Asn Leu Lys Lys Arg Ala Gly Asp Val Leu Ser Asp
340 345 350
Glu Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp
355 360 365
Asp Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg
370 375 380
Pro Thr Ile Phe Ile Glu Ile Ile Gln Arg Ile Gly Cys Met Leu Lys
385 390 395 400
Asp Glu Glu Gly Lys Val Tyr Gln Lys Gly Gly Cys Gly Gly Phe Gly
405 410 415
Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys
420 425 430
Thr Leu Glu Ser Arg Arg Thr Ala
435 440
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tccacgagtt cgctgagttc 20
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence
<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.
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CN201410744726.6A CN104404063B (en) | 2014-12-09 | 2014-12-09 | One cultivate peanut vitamin E synthesis key gene and application |
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CN104404063B true CN104404063B (en) | 2017-07-07 |
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Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104711275B (en) * | 2015-03-30 | 2017-07-28 | 青岛农业大学 | Peanut vitamin E synthesis related gene AhPK and its application in plant vitamin E contents and salt tolerance is improved |
CN106636098B (en) * | 2017-01-22 | 2018-09-18 | 中国农业科学院北京畜牧兽医研究所 | A kind of promoter and its preparation method and application of alfalfa HPPD genes |
CN107373568B (en) * | 2017-08-04 | 2020-04-28 | 华南理工大学 | Method for increasing α -tocopherol content in peanuts |
CN107794263B (en) * | 2017-09-30 | 2019-10-11 | 河北大学 | It is a kind of improve melanin yield gene and its application |
CN108728448B (en) * | 2018-06-04 | 2020-06-09 | 青岛农业大学 | Peanut oil synthesis related gene and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101265295A (en) * | 2008-05-13 | 2008-09-17 | 中国农业科学院生物技术研究所 | Tocopherol synthesis correlated albumen, coding gene and application thereof |
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101265295A (en) * | 2008-05-13 | 2008-09-17 | 中国农业科学院生物技术研究所 | Tocopherol synthesis correlated albumen, coding gene and application thereof |
Non-Patent Citations (3)
Title |
---|
改良花生维生素E的表达载体的构建及转化;庄伟建 等;《全国"植物生物技术及其产业化"研讨会论文摘要集》;20071101;164页 * |
花生维生素E合成关键酶基因的克隆、序列分析及γ-TMT基因的转化研究;李拴柱;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130215(第02期);D047-157 * |
花生预苯胺酸脱氢酶基因AhADH的克隆及分子特性分析;张福婷 等;《分子植物育种》;20140528;第12卷(第3期);478-484页 * |
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