CN103820475A - Geranylgeranyl pyrophosphate synthetase gene in Lycium chinense Miller, and encoded protein and application thereof - Google Patents
Geranylgeranyl pyrophosphate synthetase gene in Lycium chinense Miller, and encoded protein and application thereof Download PDFInfo
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Abstract
The invention relates to a geranylgeranyl pyrophosphate synthetase gene in Lycium chinense Miller, and encoded protein and application thereof, in particular to a clone of the geranylgeranyl pyrophosphate synthetase gene LmGGPS2 in Lycium chinense Miller. Total RNA in fresh Lycium chinense Miller leaves is extracted, the geranylgeranyl pyrophosphate synthetase gene LmGGPS2 in Lycium chinense Miller is cloned, and the complete sequence of the obtained gene is 1246bp; an escherichia coli expression vector pET28a-LmGGPS2 is constructed, and LmGGPS2 expression protein is obtained through an escherichia coli heterologous expression system; a binary plant expression vector pCAMBIA2300-LmGGPS2 is constructed, the vector is transformed to agrobacterium C58 cells through an electric shock method, and tobaccos are transformed through the cells to obtained transgenic tobaccos. Tests find that the transgenic tobaccos greatly increase the content of ycopene of plant, and the content of carotene is also increased.
Description
Technical field
The present invention relates to protein and the application of a kind of matrimony vine yak base geranylpyrophosphate synthase gene and coding thereof, be specially matrimony vine (
lycium chinense Miller) middle yak base geranylpyrophosphate synthase gene
lmGGPS2clone.
Background technology
Yak base geranylpyrophosphate (GGPP) is the ubiquitous important mesostate of organic sphere. in plant, GGPP participates in the synthetic of the products such as chlorophyll, carotenoid, Plant hormones regulators,gibberellins, plastoquinone, vitamin-E, monoterpene and benzoquinones, to photosynthesis of plant, grow and product quality etc. has material impact.This product is is directly catalyzed and synthesized by GGPS.GGPP is not only carotenoid the most direct biosynthetic precursor substance, or the precursor substance such as plant materials inner gibberellin, chlorophyll and plastoquinone.GGPP synthetic is the speed limit process in carotenogenesis.By a series of enzymatic reaction, the lycopene of generation is not only given the color and luster that tomato is bright-coloured, and has important healthy nutritive value.Medical research shows, in serum, the lycopene of high-content can show the sickness rate of the various cancers of reduction that land, the occurrence probability of the kinds cancers such as the lycopene content in blood and prostate cancer, digestive tract cancer, mammary cancer, lung cancer, bladder cancer, skin carcinoma is negative correlation, and research shows the occurrence probability of the reduction prostate cancer that in human body, high lycopene content can show.The effect of lycopene protection cardiovascular and cerebrovascular is mainly by its antioxygenation; reduce the peroxidation of serum lipid and low-density lipoprotein; thereby reduce the sickness rate of arteriosclerosis and coronary heart disease, the incidence probability of the reduction cardiovascular and cerebrovascular diseases that the absorption of high lycopene can show.Lycopene also has the immunizing power of raising, the effect such as delay senility.
Lycopene can quenching singlet oxygen, remove free radical, prevents that protein and DNA are subject to the effects such as the destruction of oxygen.It is to commonly use at present 10 times of antioxidant vitamin E that lycopene is removed the ability of singlet oxygen, 2 times of β-carotene.The quantity of the conjugated double bond that lycopene molecule contains in all carotenoid is maximum, and its singlet-oxygen quenching rate constant is 100 times of vitamin-E, is the carotenoid that resistance of oxidation is the strongest.The chemical structure of lycopene has determined that it has the ability of very strong removal reactive oxygen species and free radicals, is a kind of good antioxidant.And carotenoid, especially β-carotene can suppress, remove interior free yl, can delay senility and the disease such as prophylaxis of tumours, thrombus, atherosclerosis.Carotenoid can increase the vigor of B cell in immunity system, the pathogenic bacteria of elimination external source invasion, can improve the vigor of lymph helper cell, assists B cell to produce antibody, and improves the activity of other immune component; Can also increase the number of natural killer cell, to eliminate infected cell or cancer cells in body.
In plant, under GGPS gene and other enzyme acting in conjunction, generate diterpene, tetraterpene and polyterpene compound.GGPS is the key enzyme of synthetic diterpene, tetraterpene and polyterpene.Tobacco terpenoid and tabacco fragrance are closely related, and they are not only present in tobacco leaf as important tobacco aroma precursor, and in flue gas, have also found the most of terpenoid compound having in tobacco leaf.If diterpene is the chief component composition of blade face body of gland colloid secretory product, its main component is western cypress three enediols, and its degraded product solanone and derivative thereof are important fragrance matters; Tetraterpene compounds carotene is the important perfumery precursor that causes in tobacco leaf, its degraded product as trans-Damascenone, jononeionone, Megastigmatrienone etc. be important aroma component in tobacco leaf.In China's leaf tobacco production, increase tobacco leaf terpenoid content, particularly with the closely-related fragrance precursor substance of flavouring essence quality as the accumulation in tobacco leaf such as diterpene, carotenoid, can increase tobacco incense tolerance, improve quality of tabacco fragrance.Therefore, research transgenosis GGPS tobacco is significant.
The synthetic step of Carotenoid in Plants is as follows: 3-methyl-3, 5-dihydroxy-acid (MVA) is synthetic isopentenylpyrophosphate (IPP) under the katalysis of enzyme, isopentenylpyrophosphate (IPP) first generates the isomers dimethyl allene tetra-sodium (DMAPP) with propenyl structure under the effect of isopentenylpyrophosphate isomerase (IPI), it is precursor substance essential in isopentene route of synthesis, DMAPP and IPP generate yak base tetra-sodium (GPS) successively through continuous condensation, farnesyl pyrophosphate (FPP), yak base yak base tetra-sodium (GGPP).Bimolecular GGPP becomes octahydro lycopene by octahydro lycopene synthetic enzyme (PSY) catalyzed polymerization.Octahydro lycopene generates the former lycopene (prolycopene) of cis-structure through continuous dehydrogenation reaction, former lycopene isomery under the effect of carotene isomerase (CRTISO) becomes transconfiguration lycopene.Under the lycopene catalysis of corresponding cyclase subsequently, generate a, β-carotene, and further generate the pigment (Tanaka et al., 2008) of other type.
Along with the continuous discovery of the mankind to lycopene and carotenoid pharmaceutical use and medical care effect, kind to lycopene and carotenoid and the demand of output are also by increasing, but lycopene carotenoid is difficult to by chemical process synthetic.The development of modern molecular biology research means, the gene that makes a series of key enzymes in lycopene and Carotenoid biosynthetic pathway is by isolation identification successively, for producing lycopene by DNA recombinant technology and genetic engineering regulation and control and carotenoid has been opened up road, particularly obtain " golden paddy rice " and " polishes dish " by carotenoid genetically engineered, greatly strengthened people and carried out the engineered confidence of Carotenoid in Plants.
Terpenoid is the maximum compounds of quantity in plant metabolites, is that structural unit forms by isoprene.At respiration, the photosynthesis of plant materials, and play an important role in growth, growth, breeding, signal transduction and defence.Some terpenoid has important economic worth, and also some can be used as natural perfume and perfume compound or anti-tumor chemotherapeutic medicine.In plant, the biosynthesizing of terpenoid occurs in tenuigenin and plastid, its precursor substance is isopentenyl pyrophosphate (the Isopentenyl pyrophosphate of C5 structure, and isomers---dimethylallylpyrophosphate ester (Dimethylallyl pyrophosphate, DMAPP) IPP).Via mevalonic acid (Mevalonic acid, MVA) the synthetic IPP of approach is synthetic farnesyl pyrophosphate (Farnesyl pyrophosphate, FPP, C15) precursor, FPP finally synthesizes sesquiterpene, triterpene and sterol in tenuigenin.In plastid, synthetic IPP and the DMAPP of methyl erythritol phosphoric acid (methyl-erythritol-phosphate, MEP) approach is the precursor that generates geranyl tetra-sodium (Geranyl pyrophosphate, GPP, C10).GPP generates monoterpene under the effect of monoterpene synthetic enzyme; At geranyl geranylpyrophosphate synthetic enzyme (Geranylgeranyl pyrophosphate Synthase, GGPPS) lower geranyl geranylpyrophosphate (the Geranylgeranyl pyrophosphate that generates of effect, GGPP,, and then under the effect of enzyme, generate diterpene, tetraterpene and polyterpene compound C20).
Summary of the invention
The object of the present invention is to provide a kind of matrimony vine yak base geranylpyrophosphate synthase gene.
Second object of the present invention is to provide the protein of this genes encoding.
The present invention also aims to provide the recombinant vectors and the host cell that contain this gene.
Another object of the present invention is to provide the purposes of this gene.
The invention provides a kind of matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2, the nucleotide sequence as shown in SEQ ID NO.1 in sequence table forms.
The invention provides a kind of above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2the protein of coding, the protein of the aminoacid sequence as shown in SEQ ID NO.2 in sequence table.
The invention provides a kind of above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2recombinant cloning vector pMD18-T-
lmGGPS2.
Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2recombinant vectors, these recombinant vectorss comprise plasmid.
Described plasmid expression vector coli expression carrier pET28a-
lmGGPS2.
Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2recombinant vectors, these recombinant vectorss comprise plasmid.
Described plasmid expression vector coli expression carrier pET28a-LmGGPS2.
Described plasmid expression vector double base plant expression vector pCAMBIA2300-LmGGPS2.
Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2the host cell of complete coding reading frame sequence, as the host cell that contains above-mentioned recombinant vectors also belongs to protection scope of the present invention.
Described host cell is selected from Bacillus coli cells, agrobatcerium cell or tobacco cell.
The invention provides a kind of engineering bacteria of the LmGGPS2 of containing gene.
Above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene
lmGGPS2the application application of albumen in plant that comprise this genes encoding; With described recombinant vectors, as plant expression vector maize transformation cell; Or with described in contain this gene the co-culture of cells such as Agrobacterium and corn, soybean, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetables, obtain genetically modified regeneration plant; Or with described
lmGGPS2genetic transformation obtains above-mentioned species transfer-gen plant.
Technical scheme of the present invention is specifically summarized as follows:
Cloning process of the present invention comprises the steps:
From the fresh blade of matrimony vine, extract total RNA, according to transcribing matrimony vine yak base geranylpyrophosphate synthase gene in group Unigene sequence
lmGGPS2nucleotide sequence design upstream primer
lmGGPS2-F1:5 '-GTCATGTCTATGCTAATAGGTGT-3 ', LmGGPS-R1:5 '-GGTCA
GTTTCTGATGGAGAAATT-3 ', pcr amplification obtains this full length gene sequence 1246bp.
The present invention builds and contains yak base geranylpyrophosphate synthase gene
lmGGPS2coli expression carrier pET28a-
lmGGPS2with plant expression vector pCAMBIA2300-
lmGGPS2, formed by following step:
1) build the intermediate carrier pMD18-T-that contains yak base geranylpyrophosphate synthase gene
lmGGPS2.
Take LmGGPS-F1/ LmGGPS-R1 as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD18-T carrier, obtain containing shown in SEQ ID NO.1 in ordered list
lmGGPS2the intermediate carrier pMD18-T-LmGGPS2 of gene.
2) build coli expression carrier pET28a-LmGGPS2
Take LmGGPS-F1/ LmGGPS-R1 as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD18-T carrier, and be transformed in competence intestinal bacteria TOP10, after bacterium colony PCR checking, extract plasmid, with BamHI and this plasmid of SalI double digestion, by BamHI and SalI double digestion for pET28a empty carrier, after purifying, connect respectively, obtain coli expression carrier pET28a-LmGGPS2.
3) build plant expression vector pCAMBIA2300-LmGGPS2
Take LmGGPS-F1/ LmGGPS-R1 as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD18-T carrier, and be transformed in competence intestinal bacteria TOP10, after bacterium colony PCR checking, extract plasmid, with BamHI and this plasmid of SalI double digestion, by BamHI and SalI double digestion for pCAMBIA2300-35S-OCS empty carrier, after purifying, connect respectively, obtain plant expression vector pCAMBIA2300-LmGGPS2.
The invention provides protein and the application of a kind of matrimony vine yak base geranylpyrophosphate synthase gene and coding thereof.The global cDNA of isolating first coding yak base geranylpyrophosphate synthase gene from matrimony vine, is connected on coli expression carrier, utilizes heterogenous expression system verification matrimony vine
lmGGPS2the expressing protein of gene; Then be connected on plant expression vector, utilize Agrobacterium infestation method transformation of tobacco, obtain transfer-gen plant, show that the carotenoid content of transfer-gen plant increases by research, i.e. the present invention has widespread use at aspects such as strengthening Carotenoid in Plants content.
Matrimony vine yak base geranylpyrophosphate synthase gene of the present invention
lmGGPS2be expected for the preparation of transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetable plant strain.
Accompanying drawing explanation
Fig. 1. for
lmGGPS2total length pcr amplification electrophorogram.
Fig. 2. be pMD18-T-LmGGPS2 carrier schematic diagram.
Fig. 3. be pET-28a-LmGGPS2 carrier schematic diagram.
Fig. 4. be pCAMBIA2300-LmGGPS2 carrier schematic diagram.
Fig. 5. be transgene tobacco Genomic PCR.
Fig. 6. be transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, millet, wheat, cotton gene group PCR.
Fig. 7. be transgenosis Chinese rose, Lisianthus, An Zuhua, butterfly orchid Genomic PCR.
Fig. 8. be transgenic poplar Genomic PCR.
Embodiment
Yak base geranylpyrophosphate synthase gene in matrimony vine
lmGGPS2clone
From the fresh blade of matrimony vine, extract total RNA, utilize 3 ' FULLRACE Core Set Ver.2.0 (TaKaRa, Japan) test kit by synthetic RNA reverse transcription cDNA, concrete steps are with reference to specification sheets, and reaction system is as follows:
| RNA | 2μL. |
| OligdT | |
| 5×M-MLV Buffer | 2μL |
| dNTP Mixture | 1μL |
| Rnase Inhibitor | 0.25μL |
| Reverse Transcriptase M- MLV | 0.25μL |
| Rnase Free ddH 2O | 3.5μL |
Reaction conditions: 42 ℃, 60min; 70 ℃, 15min.
According to transcribing matrimony vine yak base geranylpyrophosphate synthase gene in group Unigene sequence
lmGGPS2nucleotide sequence design upstream primer LmGGPS-F1:5 '-GTCATGTCTATGCTAATAGGTGT-3 ', LmGGPS-R1:5 '-GGTCAGTTTCTGATGGAGAAATT-3 ', take the cDNA that synthesizes as template, PCR reaction system is as follows:
| 1 st Strand cDNA | 1μL |
| LmGGPS2-F1 | 0.5μL |
| LmGGPS2-R1 | 0.5μL |
| 2.5mM dNTP Mixture | 2μL |
| 10×LA Taq PCR buffer | 2.5μL |
| TaKaRa LA Taq archaeal dna polymerase | 0.25μL |
| ddH 2O | 18.25μL |
| Total volume | 25μL |
Configure altogether 4 tube reaction liquid, reaction conditions is as follows: 94 ℃, and 4min; 94 ℃, 30Sec; 56 ℃, 30Sec; 72 ℃, lmin20 Sec; 72 ℃, 10min; 30 circulations.After reaction, utilize Tian Gen company common DNA product purification test kit to carry out purifying to PCR reaction product, obtain the LmGGPS PCR fragment of purifying, as Fig. 1, the operation of test kit specification sheets.
The building process of cloning vector pMD18-T-LmGGPS2
By shown in sequence table
lmGGPS2gene is connected with pMD19-T carrier, and reaction system is as follows:
| LmGGPS2Total length reclaims product | 4μL |
| PMD18-T carrier | 1μL |
| SolutionI | 5μL |
lmGGPS2pCR fragment is recovery in embodiment 1
gGPSfull length product.
Reaction conditions: 16 ℃, 30min.Connect product transformed competence colibacillus E-Coli.TOP10, be coated on the LB Agar Plating of IPTG, 100mg/L Amp of the X-Gal, the 16ml50mg/ml that contain 40ml 25mg/ml and cultivate, form single bacterium colony.Select white colony, bacterium colony PCR method is confirmed the length scale of Insert Fragment in T carrier, as Fig. 2, consistent with expection, this carrier is sent to the order-checking of Hua Da genome company, and we have obtained this gene base sequence of 1074bp, carry out blast at NCBI, high with Solanaceae homology, be indicated as this gene clone success.
The building process of coli expression carrier pET28a-LmGGPS2
The intestinal bacteria that contain pMD19-T-LmGGPS2 plasmid that obtain in enlarged culturing experimental example 2, extract plasmid, with BamHI and this plasmid of SalI double digestion, by BamHI and SalI double digestion for pET28a empty carrier, after purifying, connect respectively, obtain coli expression carrier pET28a-LmGGPS2, the enzyme of the two is cut to product and connect.
Linked system is as follows:
| PET28a empty carrier fragment | 2μL | |
| PMD19-T-LmGGPS2 | 5μL | |
| 5×T4 DNA ligase buffer | 2μL | |
| T4 DNA ligase | 1μL |
Connect product transformed competence colibacillus e. coli bl21.Coat containing on the LB flat board of 100mg/L kana resistance 37 ℃ of cultivations.After 12h, picking list bacterium colony carries out bacterium colony PCR checking, as Fig. 3, bacterium colony PCR is verified to positive bacterium, shakes bacterium and extracts plasmid, and enzyme is cut evaluation and obtained object band, finally send the order-checking of Hua Da gene sequencing company, and result shows carrier pET28a-
lmGGPS2build correct.
The structure of double base plant expression vector pCAMBIA2300-LmGGPS2
The intestinal bacteria that contain pMD19-T-LmGGPS2 plasmid that obtain in enlarged culturing experimental example 2, extract plasmid, with BamHI and this plasmid of SalI double digestion, pCAMBIA2300 empty carrier plasmid is also used to BamHI and SalI double digestion, the enzyme of the two is cut to product and connect, linked system is as follows:
| PCAMBIA2300 empty carrier fragment | 2μL | |
| PMD19-T-LmGGPS2 | 5μL | |
| 5×T4 DNA ligase buffer | 2μL | |
| T4 DNA ligase | 1μL |
Connect product Transformed E-Coli.DH5 α, coat on the LB flat board containing 100mg/L concentration kana resistance.37 ℃ of cultivations, after 12h, picking list bacterium colony carries out bacterium colony PCR checking, and bacterium colony PCR is verified to positive bacterium, shakes bacterium and extracts plasmid, and enzyme is cut evaluation and is obtained object band, as Fig. 4.
Be used for the structure of the Agrobacterium engineering strain C58:pCAMBIA2300-LmGGPS2 of plant transgene.
The preparation of Agrobacterium competent cell
By mono-Agrobacterium C58 colony inoculation in 5mLYEP liquid nutrient medium, 28 ℃, 180r/min shaking culture.
2. above-mentioned bacterium liquid is proceeded in l00mLYEP liquid nutrient medium, 28 ℃, 180r/min, shaking culture is to (OD
600value is about 0.5).
3. after ice bath 30min, 4 ℃, the centrifugal l0min of 4000r/min, collects thalline, is resuspended in the H of 20mL precooling
2in O.
4. 4 ℃, the centrifugal 10min of 4000r/min, collect thalline, be resuspended in 10% glycerine of precooling, every pipe 200 μ L quick-frozens, be stored in-80 ℃ for subsequent use.
The electric shock of plant expression vector transforms
1. the C58 competent cell of-80 ℃ of taking-ups is placed on ice, it is slowly melted;
2. add 4 μ L plasmids, mix ice bath 5min;
3. be transferred in electric shock cup;
4. electric shock conversion instrument parameter: 1500V is set, 0.2s, electric shock transforms;
5. room temperature adds 500 μ LYEB liquid nutrient mediums after leaving standstill 2min, and 28 ℃, 180r/min shaking culture 3h;
6. the centrifugal 10min of room temperature 4000r/min, sucking-off 400 μ L supernatant liquors, mix remaining bacterium liquid, coat on the YEB flat board containing 100mg/L kantlex and 100mg/L rifampicin resistance, be inverted flat board, 28 ℃ of cultivations, 48h, until see single bacterium colony clearly.
7. picking list bacterium colony, bacterium colony PCR checking.
Agriculture bacillus mediated tobacco genetic transformation
The sterile culture of tobacco seedling: select full, healthy tobacco seed, with 75% alcohol immersion lmin, 25% peace tiformin (available chlorine 2.5%) aqueous solution sterilizing 8min, rinsed with sterile water three times, seed is placed in MS substratum, and 25 ℃ of light are cultivated, the 16h/8h photoperiod.
The substratum that this experiment is used is as shown in the table
The Agrobacterium-mediated Transformation of tobacco
Infect the preparation of bacterium liquid
1. the single bacterium colony of the positive Agrobacterium of picking, is inoculated into 5ml containing in the YEP liquid nutrient medium of 100mg/L kantlex, incubated overnight on 28 ℃, the shaking table of 200r/min.
2. next day, get 3ml bacterium liquid, be inoculated in the 50mlYEP liquid nutrient medium containing 100mg/L kantlex, when bacterium liquid is in vigorous period (OD
600=0.6-0.9) time, pour bacterium liquid into 50ml centrifuge tube, at 3500r/min, centrifugal 10min at 4 ℃, abandons supernatant liquor, collects thalline.Resuspended with the MS liquid nutrient medium of equivalent, make OD
600=0.9-1.
Explant is contaminated
1. tobacco leaf is removed to master pulse and limb edge, then blade is cut into 0.5cm × 0.5cm size, the Agrobacterium bacterium liquid that immersion prepares, soak 15-20min, shake 2-3 time therebetween, make blade fully contact bacterium liquid, take out blade, with the bacterium liquid that it is unnecessary that aseptic filter paper exhausts, blade face down, blade back is inoculated in common training substratum upward, 25 ℃ of dark cultivations 2 days in left and right.
2. blade is transferred in screening culture medium, changes a subculture about 20 days, induction of resistance bud produces, and when resistant buds grows to 1cm left and right from callus, cuts resistant buds from callus, is inoculated in resistance seedling rooting substratum.
Transgenic seedling is transplanted
The tobacco tissue cultured seedling good root growth, vitality is vigorous is taken out from tissue culture bottle, rinse substratum (as far as possible reduce root system damage) with tap water, be planted in frog stone and compost humous, coating film heat and moisture preserving 15 days, then open film, regularly water, apply fertilizer, make it normal growth in greenhouse.
The mensuration of transgene tobacco Molecular Detection and carotene carotene content
The PCR of transgene tobacco genomic dna detects: 1.CTAB method is extracted the total DNA of tobacco; 2. detect take genomic dna as template performing PCR, primer is GGPS-F3 and GGPS-R-FULL, reaction conditions:: 94 ℃, 4min; 94 ℃, 30Sec; 54 ℃, 30Sec; 72 ℃, lmin10 Sec; 72 ℃, 10min; 30 circulations.Get above-mentioned PCR product 5 μ l and carry out electrophoresis detection, as Fig. 5, explanation
lmGGPS2gene successfully proceeds to tobacco.
Extract the carotenoid of transgene tobacco, measure transgene tobacco carotenoid content apparently higher than wild-type tobacco by HPLC, illustrate that this gene has vital role at raising carotenoid content.
This laboratory by vegetative point infestation method to the farm crop mature embryo transgenosiss such as corn, soybean, paddy rice, peanut, Sunflower Receptacle, millet, barley, wheat, cotton (
lmGGPS2), success obtains transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, millet, barley, wheat, cotton plants, Genomic PCR electrophorogram, as Fig. 6, B: negative control, 1-8 is respectively the Genomic PCR of corn, soybean, paddy rice, peanut, Sunflower Receptacle, millet, barley, wheat, cotton.
At greenhouse normal growth, transgenosis seedling and wild-type contrast seedling, extract respectively lycopene and carotenoid, and HPLC measures transgenosis group seedling lycopene and carotenoid content apparently higher than wild-type group.Moreover, transgenosis group growth of seedling is thick and strong, and wild-type growth phase is to slowly, and biomass is relatively less.
Embodiment 9
This laboratory utilize Agrobacterium infestation method to the flowers such as Chinese rose, Lisianthus, An Zuhua, butterfly orchid carry out transgenosis (
lmGGPS1), obtain transgenosis Chinese rose, Lisianthus, An Zuhua, butterfly orchid plant, Genomic PCR electrophorogram, as Fig. 7, B: negative control, 1-4 is respectively the Genomic PCR of Chinese rose, Lisianthus, An Zuhua, butterfly orchid.Transgenic flower seedling at greenhouse normal growth is more thick and strong than wild-type growth, and pattern is bright-coloured, and petal carotenoid content increases.
Embodiment 10
This laboratory utilize Agrobacterium infestation method to the arbor trees such as willow carry out transgenosis (
lmGGPS2), obtaining transgenic poplar plant, Genomic PCR electrophorogram, for willow Genomic PCR, as Fig. 8.
At transgenosis sapling and the wild-type contrast seedling of greenhouse normal growth, transfer-gen plant is better than wild-type plant strain growth, and transfer-gen plant lycopene and carotenoid content obviously increase than wild-type plant.
Sequence table
<110> University Of Tianjin
Protein and the application of <120> matrimony vine yak base geranylpyrophosphate synthase gene and coding thereof
<130> 20131025
<160> 2
<170> PatentIn version 3.3
<210> 1
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<220>
<221> gene
<222> (1)..(1246)
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Thr Phe Arg Gln Ala Gly Val Phe Asp Phe Lys Ser Tyr Met Leu Gln
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Lys Ile Lys Ser Val Asn Thr Ala Leu Asp Ala Ala Val Pro Ile Arg
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Glu Pro Ile Lys Phe His Glu Ala Met Arg Tyr Ser Leu Leu Ser Glu
100 105 110
Gly Lys Arg Val Cys Pro Val Leu Cys Ile Ala Thr Cys Glu Leu Val
115 120 125
Gly Gly Gln Glu Ser Thr Ala Met Pro Ala Ala Cys Gly Met Glu Met
130 135 140
Ile His Ala Met Cys Met Met His Asp Asp Leu Pro Cys Met Asp Asn
145 150 155 160
Asp Asp Leu Arg Arg Gly Lys Leu Ser His His Lys Val Tyr Gly Glu
165 170 175
Asn Val Thr Val Leu Ala Gly Tyr Ser Leu Val Ala Leu Ala Phe Glu
180 185 190
His Ile Ala Ile Ala Thr Lys Gly Val His Pro Lys Thr Met Val Arg
195 200 205
Ala Val Gly Glu Leu Ala Arg Leu Ile Gly Pro Glu Gly Ala Thr Ala
210 215 220
Gly Gln Val Val Asp Leu Leu Cys Gly Gly Lys Ser Gly Thr Arg Leu
225 230 235 240
Glu Glu Leu Glu Tyr Ile His Arg His Lys Thr Ala Asp Phe Ala Glu
245 250 255
Ala Ala Ser Ile Val Gly Ala Ile Leu Gly Gly Ala Ser Glu Asp Glu
260 265 270
Ile Asn Arg Leu Arg Lys Phe Ser Gln Cys Ile Gly Leu Leu Phe Gln
275 280 285
Val Val Asp Asp Ile Leu Asp Val Thr Lys Ser Ser Glu Gln Leu Gly
290 295 300
Lys Thr Ala Gly Lys Asp Leu Leu Ala Asn Lys Leu Thr Tyr Pro Lys
305 310 315 320
Met Ile Gly Ile Asp Lys Ser Lys Lys Tyr Ala Gln Lys Leu Ser Lys
325 330 335
Glu Ala Lys Glu Gln Leu Val Gly Phe Asp Pro Glu Lys Ala Ala Pro
340 345 350
Leu Leu Ser Met Ala Asp Phe Val Leu His Arg Gln Lys
355 360 365
。
Claims (8)
1. a matrimony vine yak base geranylpyrophosphate synthase gene, is characterized in that this gene is the nucleotide sequence shown in SEQ ID NO.1.
2. the protein of matrimony vine yak base geranylpyrophosphate synthase gene coding claimed in claim 1, is characterized in that described protein is the aminoacid sequence shown in SEQ ID NO.2.
3. a recombinant vectors, is characterized in that the complete sequence or the Partial Fragment that contain matrimony vine yak base geranylpyrophosphate synthase gene claimed in claim 1.
4. a recombinant vectors claimed in claim 3, is characterized in that it is plasmid expression vector coli expression carrier pET28a-LmGGPS.
5. a recombinant vectors claimed in claim 3, is characterized in that it is recombinant plant expression vector pCAMBIA2300-LmGGPS.
6. a host cell, is characterized in that containing yak base geranylpyrophosphate synthase gene complete sequence claimed in claim 1 or Partial Fragment.
7. a host cell claimed in claim 6, is characterized in that it is agrobatcerium cell, tobacco cell.
8. a matrimony vine yak base geranylpyrophosphate synthase gene claimed in claim 1 is applied to and prepares transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetable plant strain.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235044A (en) * | 2019-12-31 | 2020-06-05 | 天津大学 | Recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, construction method and application |
| CN112391327A (en) * | 2019-08-14 | 2021-02-23 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN114574507A (en) * | 2022-03-09 | 2022-06-03 | 中国科学院西北高原生物研究所 | Key gene for regulating biosynthesis of zeaxanthin palmitate and application thereof |
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| CN103153043A (en) * | 2010-10-08 | 2013-06-12 | 韩国生命工学研究院 | Ggps gene for promotimg higher growth or biomass of plant and use thereof |
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Patent Citations (1)
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| CN103153043A (en) * | 2010-10-08 | 2013-06-12 | 韩国生命工学研究院 | Ggps gene for promotimg higher growth or biomass of plant and use thereof |
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| LI F ET AL.: "GenBank Accession:AFC88470", 《GENBANK》 * |
| P A SCOLNIK ET AL.: "Nucleotide sequence of an arabidopsis cDNA for geranylgeranyl pyrophosphate synthase.", 《PLANT PHYSIOL》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391327A (en) * | 2019-08-14 | 2021-02-23 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN112391327B (en) * | 2019-08-14 | 2022-08-05 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN111235044A (en) * | 2019-12-31 | 2020-06-05 | 天津大学 | Recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, construction method and application |
| CN111235044B (en) * | 2019-12-31 | 2022-01-04 | 天津大学 | Recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, construction method and application |
| CN114574507A (en) * | 2022-03-09 | 2022-06-03 | 中国科学院西北高原生物研究所 | Key gene for regulating biosynthesis of zeaxanthin palmitate and application thereof |
| CN114574507B (en) * | 2022-03-09 | 2023-10-03 | 中国科学院西北高原生物研究所 | Key gene for regulating biosynthesis of zeaxanthin palmitate and application thereof |
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| CN103820475B (en) | 2016-06-22 |
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