CN103757034A - Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene - Google Patents

Abstract

The invention relates to a Lycium chinense Miller geranylgeranyl pyrophosphate synthetase (GGPS) gene and a recombinant vector comprising the gene, and specifically relates to cloning of GGPS gene (i) Lm (/ i) (i) GGPS1 (/ i) in Lycium chinense Miller. The total RNA is extracted from fresh Lycium chinense Miller leaves, LmGGPS1 in Lycium chinense Miller is cloned to obtain a complete gene sequence 1134bp; an Escherichia coli expression vector pET28a-LmGGPS1 and a binary plant expression vector pCAMBIA2300-LmGGPS1 are constructed; and the vectors are transformed into Agrobacterium C58 cells by an electroporation method; and the cells are used for tobacco transformation, so as to obtain transgenic tobacco. Through the tests, the transgenic tobacco gains greatly improved content of plant lycopene, and increased content of carotene. The invention can be used for the preparation of transgenic maize, soybean, rice, peanut, sunflower, potato, cotton, millet, barley, and flower and vegetable plants.

Description

For improving the matrimony vine GGPS1 gene of stress resistance of plant and the recombinant vectors of this gene

Technical field

The present invention relates to a kind of matrimony vine ( lycium chinense Miller) yak base geranylpyrophosphate synthase gene (Geranylgeranyl pyrophosphate synthase, gGPS) and comprise and the recombinant vectors of this gene be specially yak base geranylpyrophosphate synthase gene in matrimony vine lmGGPS1clone.

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 Lyeopene of generation not only gives tomato bright-coloured color and luster, and has important healthy nutritive value.Medical research shows, in serum, the Lyeopene of high-content can reduce the sickness rate of various cancers significantly, the occurrence probability of the kinds cancers such as the content of lycopene in blood and prostate cancer, digestive tract cancer, mammary cancer, lung cancer, bladder cancer, skin carcinoma is negative correlation, and research shows that high lycopene content in human body can reduce the occurrence probability of prostate cancer significantly.The effect of Lyeopene protection cardiovascular and cerebrovascular is mainly by its antioxygenation; reduce the peroxidation of serum lipid and low-density lipoprotein; thereby the sickness rate that reduces arteriosclerosis and coronary heart disease, the absorption of high hycopene can reduce the incidence probability of cardiovascular and cerebrovascular diseases significantly.Lyeopene also has the effect such as improve immunizing power, delay senility.

Lyeopene can quenching singlet oxygen, remove free radical, prevents that protein and DNA are subject to the effects such as destruction of oxygen.The ability that Lyeopene is removed singlet oxygen is to commonly use at present 10 times of antioxidant vitamin E, 2 times of β-carotene.The quantity of the conjugated double bond that Lyeopene 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 Lyeopene 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 most of terpenoid compound of 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 phytoene by phytoene synthetase (PSY) catalyzed polymerization.Phytoene generates the former Lyeopene (prolycopene) of cis-structure through continuous dehydrogenation reaction, former Lyeopene isomery under the effect of carotene isomerase (CRTISO) becomes transconfiguration Lyeopene.Under the Lyeopene 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 Lyeopene and carotenoid pharmaceutical use and medical care effect, to the demand of the kind of Lyeopene and carotenoid and output also by increasing, yet Lyeopene 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 Lyeopene and Carotenoid biosynthetic pathway is by isolation identification successively, for producing Lyeopene by DNA recombinant technology and genetic engineering regulation and control and carotenoid has been opened up road, particularly by carotenoid genetically engineered, obtain " golden paddy rice " and " polishes dish ", greatly strengthened people and carried out the engineered confidence of Carotenoid in Plants.

Terpenoid is the maximum compounds of quantity in plant metabolites, by isoprene, is that structural unit forms.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, IPP) and isomers---dimethylallylpyrophosphate ester (Dimethylallyl pyrophosphate, DMAPP).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 lmGGPS1, 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 lmGGPS1the 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 lmGGPS1recombinant cloning vector pMD19-T- lmGGPS1.

Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene lmGGPS1recombinant vectors, these recombinant vectorss comprise plasmid.

Described plasmid expression vector coli expression carrier pET28a- lmGGPS1.

Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene lmGGPS1recombinant vectors, these recombinant vectorss comprise plasmid.

Described plasmid expression vector coli expression carrier pET28a-LmGGPS1.

Described plasmid expression vector double base plant expression vector pCAMBIA2300-LmGGPS1.

Contain above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene lmGGPS1the 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 LmGGPS1 of containing gene.

Above-mentioned matrimony vine yak base geranylpyrophosphate synthase gene lmGGPS1the 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 Agrobacterium and the co-culture of cells such as corn, soybean, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetables, obtain genetically modified regeneration plant; Or obtain above-mentioned species transfer-gen plant with described LmGGPS1 genetic transformation.

Technical scheme of the present invention is specifically summarized as follows:

Cloning process of the present invention is comprised of following step:

From the fresh blade of matrimony vine, extract total RNA, according to transcribing matrimony vine yak base geranylpyrophosphate synthase gene in group Unigene sequence lmGGPS1nucleotide sequence design upstream primer LmGGPS1-F1:5 ' TAGGTGGCGGGAACGAAGAT-3 ', then utilize 3 ' RACE method to expand and obtain 3 ' end sequence, design degenerate primer LmGGPS1-F2:5 ' ATGAGATCTATGAAYS TTGTYGATTCATGGG and GGPS1-R:5 '-TCCCATAAGTTTCGGATACG-3 ', pcr amplification obtains 5 ' end sequence of this gene, splices above-mentioned obtained three sections lmGGPS1gene order, the full length sequence of splicing is 1224bp.Design primer GGPS1-F3:5 '-ATGAGATCTATGAATGTTGTTGATTC-3 '; GGPS1-R-FULL:5 '-CAAGATTCAAATCACAAGCCTG-3 '; Pcr amplification obtains gGPS1full length sequence.

The present invention builds coli expression carrier pET28a-LmGGPS1 and the plant expression vector pCAMBIA2300-LmGGPS1 that contains yak base geranylpyrophosphate synthase gene LmGGPS1, following step, consists of:

1) build the intermediate carrier pMD19-T-LmGGPS1 that contains yak base geranylpyrophosphate synthase gene.

Take GGPS1-F3/GGPS1-R-FULL as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD19-T carrier, obtain the intermediate carrier pMD19-T-LmGGPS1 containing the LmGGPS1 gene shown in SEQ ID NO.1 in ordered list.

2) build coli expression carrier pET28a-LmGGPS1

Take GGPS1-F3/GGPS1-R-FULL as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD19-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-LmGGPS1.

3) build plant expression vector pCAMBIA2300-LmGGPS1

Take GGPS1-F3/GGPS1-R-FULL as primer, take matrimony vine cDNA as template, carry out pcr amplification, pcr amplification product is connected in to pMD19-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-LmGGPS1.

The invention provides a kind of for improving the matrimony vine of stress resistance of plant gGPS1the recombinant vectors of gene and this gene is isolated the global cDNA of coding yak base geranylpyrophosphate synthase gene first from matrimony vine, is connected on coli expression carrier, utilizes the expressing protein of heterogenous expression system verification matrimony vine LmGGPS1 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 lmgGPS1 is 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. be 3 ' RACE PCR electrophorogram.

Fig. 2. be the electrophorogram of 5 ' end PCR product.

Fig. 3. be LmGGPS1 total length pcr amplification electrophorogram.

Fig. 4. be pMD19-T-LmGGPS1 carrier schematic diagram.

Fig. 5. be pET-28a-LmGGPS1 carrier schematic diagram.

Fig. 6. be pCAMBIA2300-LmGGPS1 carrier schematic diagram.

Fig. 7. be transgene tobacco Genomic PCR.

Fig. 8. be transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, millet, wheat, barley, cotton gene group PCR.

Fig. 9. be transgenosis Chinese rose, Lisianthus, An Zuhua, butterfly orchid Genomic PCR.

Figure 10. be transgenic poplar, fragrant flower Chinese scholar tree Genomic PCR.

Embodiment

The experimental technique of unreceipted actual conditions in embodiment, conventionally according to the condition described in normal condition and handbook, or the condition of advising according to manufacturer.

Embodiment 1

The clone of yak base geranylpyrophosphate synthase gene LmGGPS1 in matrimony vine

gGPS13 ' end clone:

Utilize Trizol reagent, from the fresh matrimony vine blade of 100mg, extract totalRNA, according to the Unigene sequences Design upstream primer of transcribing group, in matrimony vine, the upstream primer of yak base geranylpyrophosphate synthase gene LmGGPS1 is LmGGPS1-F1:5 ' TAGGTGGCGGGAACGAAGAT-3 ', utilize 3 ' FULLRACE Core Set Ver.2.0 (TaKaRa, Japan) test kit amplification to obtain complete gene order.Concrete steps: 1. take totalRNA as template, utilize 3 ' RACE Adaptor primer to carry out reverse transcription reaction, synthetic 1st Strand cDNA, reaction system is as follows:

RNA	2μL.
		3‘ RACE Adaptor	1μL
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.

Downstream primer 3 ' RACE out the primer:5 '-TACCGTCGTTCCACTAGTGATTT-3 ' providing according to the upstream primer of gene and test kit, the 1 st Strand cDNA of take is template, carries out PCR reaction, reaction system is as follows:

1 st Strand cDNA	1μL
		LmGGPS1 upstream primer	1μL
3’RACE out primer	1μL
		2.5mM dNTP Mixture	2μL
10×LA Taq PCR buffer	2.5μL
		TaKaRa LA Taq archaeal dna polymerase	0.25μL
ddH 2O	17.25μL
		Total volume	25μL

Configure altogether 4 tube reaction liquid, reaction conditions is as follows: 94 ℃, and 4min; 94 ℃, 30Sec; 52 ℃, 30Sec; 72 ℃, lmin10 Sec; 72 ℃, 10min; 34 circulations.Reaction product, by 1% agarose gel electrophoresis detection validation, as Fig. 1, is utilized the sepharose DNA of Tian Gen company to reclaim test kit the object fragment in PCR reaction product is reclaimed after checking, according to test kit specification sheets, operate.

gGPS15 ' end clone

Design degenerate primer LmGGPS1-F2:5 ' ATGAGATCTATGAAYSTTGTYGATTCA TGGG and GGPS1-R:5 '-TCCCATAAGTTTCGGATACG-3 ', the 1 st Strand cDNA of take is template, pcr amplification obtains 5 ' end sequence of this gene, and reaction system is as follows:

1 st Strand cDNA	1μL
		LmGGPS1-F2	1μL
GGPS1-R	1μL
		2.5mM dNTP Mixture	2μL
10×LA Taq PCR buffer	2.5μL
		TaKaRa LA Taq archaeal dna polymerase	0.25μL
ddH 2O	17.25μL
		Total volume	25μL

Configure altogether 4 tube reaction liquid, reaction conditions is as follows: 94 ℃, and 4min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, lmin; 72 ℃, 10min; 34 circulations.As Fig. 2, as aforesaid method reclaims object fragment.

gGPS1full length sequence amplification

According to gGPS1gene 3 ' end sequence, 5 ' end sequence design primer GGPS1-F3:5 '-ATGAGATCTATGAATGTTGTTGATTC-3 '; GGPS1-R-FULL:5 '-CAAGATTCAAATCACAAGCCTG-3 '; Pcr amplification obtains gGPS1full length sequence is 1224bp, as Fig. 3.

Embodiment 2

The building process of cloning vector pMD19-T-LmGGPS1

LmGGPS1 gene shown in sequence table is connected with pMD19-T carrier, and reaction system is as follows:

LmGGPS1 total length reclaims product	4μL
		PMD19-T carrier	1μL
SolutionI	5μL

LmGGPS1 PCR fragment is recovery in embodiment 1 gGPS1full length product.

Reaction conditions: 16 ℃, 30min.Connect product transformed competence colibacillus E-Coli.TOP10, be coated on the IPTG of the X-Gal, the 16ml50mg/ml that contain 40ml 25mg/ml, cultivate on the LB Agar Plating of 100mg/L Amp, form single bacterium colony.Select white colony, bacterium colony PCR method is confirmed the length scale of Insert Fragment in T carrier, 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, at NCBI, carry out blast, high with Solanaceae homology, be indicated as this gene clone success.

Embodiment 3

The building process of coli expression carrier pET28a-LmGGPS1

The intestinal bacteria that contain pMD19-T-LmGGPS1 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-LmGGPS1, the enzyme of the two is cut to product and connect.

Linked system is as follows:

PET28a empty carrier fragment	2μL
		PMD19-T-LmGGPS1 endonuclease bamhi	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, and 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 it is correct that result shows that carrier pET28a-LmGGPS1 builds.

Embodiment 4

The structure of double base plant expression vector pCAMBIA2300-LmGGPS1

The intestinal bacteria that contain pMD19-T-LmGGPS1 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-LmGGPS1 endonuclease bamhi	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.

Embodiment 5

The structure that is used for the Agrobacterium engineering strain C58:pCAMBIA2300-LmGGPS1 of plant transgene.

The preparation of Agrobacterium competent cell

By the mono-colony inoculation of Agrobacterium C58 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, collects thalline, be resuspended in 10% glycerine of precooling, every pipe 200 μ L quick-frozens, be stored in-80 ℃ standby.

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. after the standing 2min of room temperature, add 500 μ LYEB liquid nutrient mediums, 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.

Embodiment 6

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, from callus, cuts resistant buds, is inoculated in resistance seedling rooting substratum.

Transgenic seedling is transplanted

Root growth is good, vitality is vigorous tobacco tissue cultured seedling takes out from tissue culture bottle, with tap water, rinse substratum (as far as possible reducing root system damage), 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.

Embodiment 7

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. take genomic dna as the detection of template performing PCR, primer is GGPS1-F3 and GGPS1-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. 7, explanation lmGGPS1gene successfully proceeds to tobacco.

Extract the carotenoid of transgene tobacco, by HPLC, measure transgene tobacco carotenoid content apparently higher than wild-type tobacco, illustrate that this gene has vital role at raising carotenoid content.For transgene tobacco Genomic PCR.

With reference to above-mentioned condition, carry out same experiment:

Embodiment 8

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 ( lmGGPS1) obtain transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, millet, barley, wheat, cotton plants, Genomic PCR electrophorogram, as Fig. 8, B: negative control, 1-9 is followed successively by 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 Lyeopene and carotenoid, and HPLC measures transgenosis group seedling Lyeopene 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 utilizes Agrobacterium infestation method to carry out transgenosis (LmGGPS) to flowers such as Chinese rose, Lisianthus, An Zuhua, butterfly orchides, obtain transgenosis Chinese rose, Lisianthus, An Zuhua, butterfly orchid plant, Genomic PCR electrophorogram, as Fig. 9, B: negative control, 1-6 is followed successively by from left to right Chinese rose, Lisianthus, peace ancestral and spends a, peace ancestral to spend the Genomic PCR of b, butterfly orchid a and butterfly orchid b.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 utilizes Agrobacterium infestation method to carry out transgenosis (LmGGPS) to arbor trees such as willows, obtains transgenic poplar plant, and Genomic PCR electrophorogram, as Figure 10., B: negative control, 1-2 is willow Genomic PCR.

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 Lyeopene and carotenoid content obviously increase than wild-type plant.

Experimental example 11

At transgene tobacco seedling and the wild-type contrast seedling of greenhouse normal growth, water respectively the water containing the NaCI of 300mmol/L and 500mmol/L concentration, in greenhouse, keep 85% humidity.Detect and find, under the growth conditions of 300mmol/LNaCI, wild-type plant strain growth is relatively slow.And transgenosis sapling can normal growth.Under the condition of the NaCI of 500mmol/L concentration, grow after one week, the jaundice of wild-type seedling leaf, wilts, and tissue necrosis, can not normal growth.Although the growth of transgene tobacco seedling is also subject to inhibition to a certain extent, obvious unlike wild-type seedling, substantially can normal growth.

Sequence table

<110> University Of Tianjin

The matrimony vine of <120> for improving stress resistance of plant gGPS1the recombinant vectors of gene and this gene

<130> 20131024

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1224

<212> DNA

<213> is series manually

<220>

<221> gene

<222> (1)..(1224)

<400> 1

atgagatcta tgaatgttgt tgattcatgg gctcaagctt gtttagtttt caatcaaacc 60

ttaccttata aatccttcaa taatggattc atgaaaatcc ctctcagaaa tcccaaaatc 120

aaaccccaaa caagacccat aagcatttca gctatagcta ccaaagaaga tgaaaaagtt 180

gttaccgagc agttcaattt caaactgtac gtagcagaaa aggctattct tgtaaacaaa 240

gcattagatg aggctattat aataaaagac ccacctgtta tccacgaatc aatgcgttac 300

tcccttctcg ccggcggtaa acgtgtccgg ccaatgcttt gcctcgccgc cgccgaactc 360

gtcggcggcg accagcgcgc cgccgtgccg gcagcttgcg ccgtcgagat gatacatacg 420

atgtcgttaa ttcatgatga tttgccttgt atggataatg atgatctccg tcgtggaaaa 480

ccgacgaatc ataaagtgta cggtgaggat gtggcggtgc ttgctggaga ttcgttactt 540

gcgtttgcgt ttgagtatct tgctacggcg acgacgggag tttcgccggc gaggatagtt 600

gctgccgttg ctgaattggc gaaatctatt ggaattgaag ggttagtagc tggacaagtg 660

gcggatatag cttgtacagg taatccgaat gtgggattag acacgcttga attcatccac 720

acacacaaaa cagcagcatt actagaagct tcagtagtac taggagcaat cctaggtggc 780

gggaacgaag atgacgtgga caagttaagg agatttgcta gatgtattgg actattattt 840

caagtagttg atgatatact tgacgttaca aagtcatctg aagagctagg aaaaactgct 900

ggaaaagatt tggcagtgga taaaacgacg tatccgaaac ttatgggatt agaaaaggct 960

aaggaatttg cggcagagct taacagggaa gctaaagaac agttggttga atttgatcca 1020

cataaagctg ctcctttgat tgctttggct gattacattg ctcatcgtga aaattaggtg 1080

ggggggaaga ttatttgtaa tactttattt ttcaggcttg tgatttgaat cttgatttaa 1140

caagtgaaaa tgaaaggtga tttgttactt aaccaatttc tattgttaga aattggatgt 1200

taaaaaaaaa aaaaaaaaaa aaaa 1224

<210> 2

<211> 358

<212> PRT

<213> MUTAGEN

<220>

<221> MUTAGEN

<222> (1)..(358)

<400> 2

Met Arg Ser Met Asn Val Val Asp Ser Trp Ala Gln Ala Cys Leu Val

1 5 10 15

Phe Asn Gln Thr Leu Pro Tyr Lys Ser Phe Asn Asn Gly Phe Met Lys

20 25 30

Ile Pro Leu Arg Asn Pro Lys Ile Lys Pro Gln Thr Arg Pro Ile Ser

35 40 45

Ile Ser Ala Ile Ala Thr Lys Glu Asp Glu Lys Val Val Thr Glu Gln

50 55 60

Phe Asn Phe Lys Leu Tyr Val Ala Glu Lys Ala Ile Leu Val Asn Lys

65 70 75 80

Ala Leu Asp Glu Ala Ile Ile Ile Lys Asp Pro Pro Val Ile His Glu

85 90 95

Ser Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg Pro Met

100 105 110

Leu Cys Leu Ala Ala Ala Glu Leu Val Gly Gly Asp Gln Arg Ala Ala

115 120 125

Val Pro Ala Ala Cys Ala Val Glu Met Ile His Thr Met Ser Leu Ile

130 135 140

His Asp Asp Leu Pro Cys Met Asp Asn Asp Asp Leu Arg Arg Gly Lys

145 150 155 160

Pro Thr Asn His Lys Val Tyr Gly Glu Asp Val Ala Val Leu Ala Gly

165 170 175

Asp Ser Leu Leu Ala Phe Ala Phe Glu Tyr Leu Ala Thr Ala Thr Thr

180 185 190

Gly Val Ser Pro Ala Arg Ile Val Ala Ala Val Ala Glu Leu Ala Lys

195 200 205

Ser Ile Gly Ile Glu Gly Leu Val Ala Gly Gln Val Ala Asp Ile Ala

210 215 220

Cys Thr Gly Asn Pro Asn Val Gly Leu Asp Thr Leu Glu Phe Ile His

225 230 235 240

Thr His Lys Thr Ala Ala Leu Leu Glu Ala Ser Val Val Leu Gly Ala

245 250 255

Ile Leu Gly Gly Gly Asn Glu Asp Asp Val Asp Lys Leu Arg Arg Phe

260 265 270

Ala Arg Cys Ile Gly Leu Leu Phe Gln Val Val Asp Asp Ile Leu Asp

275 280 285

Val Thr Lys Ser Ser Glu Glu Leu Gly Lys Thr Ala Gly Lys Asp Leu

290 295 300

Ala Val Asp Lys Thr Thr Tyr Pro Lys Leu Met Gly Leu Glu Lys Ala

305 310 315 320

Lys Glu Phe Ala Ala Glu Leu Asn Arg Glu Ala Lys Glu Gln Leu Val

325 330 335

Glu Phe Asp Pro His Lys Ala Ala Pro Leu Ile Ala Leu Ala Asp Tyr

340 345 350

Ile Ala His Arg Glu Asn

355

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-LmGGPS1.

5. a recombinant vectors claimed in claim 3, is characterized in that it is recombinant plant expression vector pCAMBIA2300-LmGGPS1.

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 yak base geranylpyrophosphate synthase gene claimed in claim 1 is applied to prepare transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetable plant strain.

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