CN102321649B - Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application - Google Patents
Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application Download PDFInfo
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Abstract
The invention discloses a lycium chinense miller lycopene beta-cyclase gene, a recombinant vector containing the gene, a host cell and application. Total RNA (Ribonucleic Acid) of fresh lycium chinense miller leaf is extracted, and lycopene beta-cyclase gene LmLycB is cloned by using 3' RACE (Rapid Amplification of cDNA Ends) technology to obtain a complete gene sequence of 1,506bp. An Escherichia coli expression vector pMON-LmLycB is constructed; an escherichia coli heterogenous expression system is applied; the activity of cloned LmLycB gene-coded enzyme is identified; and two beta-lonone rings can be added at the tail end of lycopene by LmLycB to produce beta-carotene. A maize inbred line immature embryo genetic transformation system is optimized; a plant expression vector pCAMBIA2300-LmLycB-Bar containing a target gene is transformed into maize callus to finally obtain a transgenic positive plant; through HPLC (High Performance Liquid Chromatography) detection, the total carotinoid content of transgenic maize leaf is 165.70mug/g FW and is 12.76 percent greater than that of a wild maize; and beta-carotene content is 80.17mug/g FW and is 54.83 percent greater than that of the wild maize.
Description
Technical field
The present invention relates to a kind of matrimony vine (Lycium chinense Miller) lycopene beta cyclase gene and comprise recombinant vectors and host cell and the application of this gene.
Background technology
In the Carotenoid in Plants biosynthetic pathway, the cyclization of Lyeopene is most important tapping point wherein, and symmetrical cyclisation forms two alpha, beta-lonone rings at the two ends of linear Lyeopene, produces β-carotenoid.Lycopene beta cyclase (Lycopene β-cyclase, LycB) be rate-limiting enzyme important in Carotenoid biosynthetic pathway, the catalysis Lyeopene transforms [Bartley GE to β-carotene, Scolnik PA, Plant carotenoids:pigments for photoprotection, visual attraction, and human health, Plant Cell, 1995,7:1027-1038].So far, be separated to the LycB gene from the plants such as soybean, tomato, capsicum, Arabidopis thaliana, corn, daffodil, Nicotiana gossei.
Carotenoid is the hydrocarbon polymer of C40, extensively be present in the fat-soluble pigment in plant, some photosynthetic bacteriums and algae, mainly comprise carotene (carotenes) and the large class of their oxidized derivatives xenthophylls (xanthophylls) two.Carotenoid plays vital effect in photosynthesis of plant, they are photosynthetic antenna and the indispensable constituent of photoresponse central complex, can also protect chlorophyll to avoid the photoxidation destruction that high light causes.Carotenoid is the precursor of vitamin A, has and strengthens function immune, anti-oxidant, that enhancement cell gap junction exchanges, prevents, delays and treat cancer.Aluru etc. are cascaded bacterium crtB (pds) and crtI gene, specific expressed in corn embryosperm, in endosperm, carotenoid content improves 34 times, and has accumulated the precursor of a large amount of vitamin A: β-carotene [Aluru M, Xu Y, Guo R, et al, Generation of transgenic maize with enhanced provitamin A content, J Exp Bot, 2008,59 (13): 3551-3562].
In case after the free radical with damage ability that in biosystem, height of formation is active, will carry out strong destruction to cytogenetics material and cytolemma, cause cell function to descend, the generation of body aging and disease.Carotenoid, especially β-carotene can suppress, remove interior free yl, can delay senility and the diseases 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 the 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.It is reported, except β-carotene, Lyeopene etc. also have the function that increases immunizing power.
Along with the continuous discovery of the mankind to carotenoid pharmaceutical use and medical care effect, to the demand of the kind of carotenoid and output also with increasing, yet carotenoid is difficult to chemical process synthetic.The development of modern molecular biology research means, the gene that makes a series of key enzymes in Carotenoid biosynthetic pathway is by isolation identification successively, opened up road for by DNA recombinant technology and genetic engineering regulation and control, producing carotenoid, particularly by the carotenoid genetically engineered, obtain " golden paddy rice " and " polishes dish ", greatly strengthened people and carried out the engineered confidence of Carotenoid in Plants.
Summary of the invention
The object of the present invention is to provide a kind of matrimony vine lycopene beta cyclase gene.
Second purpose 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 lycopene beta cyclase gene LmLycB, the nucleotide sequence as shown in SEQ ID NO.1 in sequence table forms.
The invention provides a kind of protein of above-mentioned matrimony vine lycopene beta cyclase gene LmLycB 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 lycopene beta cyclase gene LmLycB recombinant cloning vector pBS-T-LmLycB.
Contain the recombinant vectors of above-mentioned matrimony vine lycopene beta cyclase gene LmLycB, these recombinant vectorss comprise plasmid and plant expression vector.
Described plasmid expression vector coli expression carrier pMON-LmLycB.
Described recombinant plant expression vector pCAMBIA2300-LmLycB-Bar.
Contain the host cell of the complete coding reading frame sequence of above-mentioned matrimony vine lycopene beta cyclase gene LmLycB, 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 maize cell.
The invention provides two kinds and contain the LmLycB genetic engineering bacterium.
The application of above-mentioned matrimony vine lycopene beta cyclase gene LmLycB comprises the application of albumen in intestinal bacteria and plant of this LmLycB genes encoding; Use described recombinant vectors, as plant expression vector maize transformation cell; Perhaps, with described Agrobacterium and the co-culture of cells such as corn, soybean, paddy rice, peanut, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetables that contain this gene, obtain genetically modified regeneration plant; Perhaps with described matrimony vine lycopene beta cyclase gene LmLyc genetic transformation, obtain above-mentioned species transfer-gen plant.
Technical scheme of the present invention specifically is summarized as follows:
A kind of matrimony vine lycopene beta cyclase gene LmLycB, nucleotide sequence as shown in SEQ ID NO.1 in sequence table, also comprise that the nucleotide sequence shown in SEQ ID No.1 add, replace, insert or delete 70% above homologous sequence or its allelotrope and the derivative nucleotide sequence thereof of one or more Nucleotide.
A kind of bacillus coli DH 5 alpha that contains the recombinant vectors pMON-LmLycB of lycopene beta cyclase gene LmLycB.
A kind of Agrobacterium C58 that contains the recombinant vectors pCAMBIA2300-LmLycB-Bar of lycopene beta cyclase gene LmLycB.
A kind of albumen of lycopene beta cyclase gene LmLycB coding, aminoacid sequence as shown in SEQ ID NO.2 in sequence table.
A kind of Expression and Application of albumen in intestinal bacteria of lycopene beta cyclase gene LmLycB coding.
A kind of application of albumen in corn of lycopene beta cyclase gene LmLycB coding.
Described matrimony vine lycopene beta cyclase gene also is applied to prepare the plant such as genetically engineered soybean, paddy rice, peanut, Sunflower Receptacle, potato, cotton, millet, barley and flowers and vegetables.
Cloning process of the present invention is comprised of following step:
Extract total RNA from the matrimony vine blade, the aminoacid sequence design degenerated primer P1 according to the lycopene beta cyclase in other plant in GenBank, is characterized in that having the sequence shown in SEQ ID NO.3 in sequence table.Then utilize the amplification of 3 ' RACE method to obtain complete gene order and be 1506bp.
The present invention builds coli expression carrier pMON-LmLycB and the plant expression vector pCAMBIA2300-LmLycB-Bar that contains lycopene beta cyclase gene LmLycB, following step, consists of:
1) build the intermediate carrier pBS-T-LmLycB that contains lycopene beta cyclase gene LmLycB:
Design is by the upstream primer P1 shown in SEQ ID NO.3, with the downstream primer P2 by shown in SEQ ID NO.4, take the cDNA of matrimony vine lycopene beta cyclase gene as template, carry out pcr amplification, pcr amplification product is connected in the pBS-T carrier, obtains to contain the intermediate carrier pBS-T-LmLycB of the LmLycB gene shown in SEQ ID NO.1 in ordered list.
2) build coli expression carrier pMON-LmLycB:
Design is by the upstream primer P3 shown in SEQ ID NO.5, with the downstream primer P4 by shown in SEQ ID NO.6, take plasmid pBS-T-LmLycB as template, carry out pcr amplification, with pcr amplification product after BamHI and SalI enzyme are cut, coli expression carrier pMON-LmLycB is cut through BamHI and SalI enzyme, both carry out ligation, obtain coli expression carrier pMON-LmLycB.
3) build plant expression vector pCAMBIA2300-LmLycB-Bar:
Design is by the upstream primer P5 shown in SEQ ID NO.7, with the downstream primer P6 by shown in SEQ ID NO.8, take plasmid pBS-T-LmLycB as template, carry out pcr amplification, with pcr amplification product after BamHI and SalI enzyme are cut, plant expression vector pCAMBIA2300-35S-OCS is cut through BamHI and SalI enzyme, both carry out ligation, obtain plant expression vector pCAMBIA2300-LmLycB.
Then utilize the XhoI restriction enzyme site, the NPTII gene on the pCAMBIA2300-LmLycB carrier is removed, from 5 ' inserted the Bar gene of anti-glufosinates to 3 ' direction, obtain the pCAMBIA2300-LmLycB-Bar plant expression vector.
The invention provides a kind of matrimony vine lycopene beta cyclase gene and comprise recombinant vectors and host cell and the application of this gene, isolate first the global cDNA of coding for tomato red pigment beta cyclase from matrimony vine, be connected on coli expression carrier, utilize heterogenous expression system verification matrimony vine LmLycB gene to have the activity of enzyme at the product of protein expression.Then be connected on plant expression vector, utilize Agrobacterium infestation method maize transformation, the transfer-gen plant that obtains carries out HPLC and detects, result shows that transgenic corns blade total carotinoid content is 165.70 μ g/g FW, improved 12.76% than wild-type corn, wherein content beta-carotene is 80.17 μ g/g FW, than wild-type corn, has improved 54.83%.In Table 2.
The present invention isolates the gene LmLycB of coding for tomato red pigment beta cyclase from matrimony vine, the overexpression of this gene can cause the raising of β-carotene biosynthesizing amount in transgenic corns.Change pathways metabolism by the genetic engineering breeding method, improve β-carotene output, to reach better, more accurately the purpose of corps nutrient breeding, or by utilizing microorganism to carry out the biosynthesizing β-carotene of being correlated with as bio-reactor, to meet people's nutritional need.
Description of drawings
Fig. 1 pMON-LmLycB carrier schematic diagram.
Fig. 2 pMON-LmLycB PCR, enzyme are cut the result.
Fig. 3 pCAMBIA2300-LmLycB-Bar carrier schematic diagram.
Fig. 4 pCAMBIA2300-LmLycB-Bar PCR, enzyme are cut the result.
The expression of Fig. 5 LmLycB in intestinal bacteria.
Fig. 6 expresses the intestinal bacteria HPLC detected result of LmLycB.
Fig. 7 transgenic corns Genomic PCR checking electrophoresis result.
Fig. 8 transgenic corns RT-PCR verifies electrophoresis result.
The HPLC detected result of Fig. 9 wild-type corn and transgenic corns Carotenoid in Leaves.
Embodiment
The experimental technique of unreceipted actual conditions in embodiment, usually according to the condition described in normal condition and handbook, or the condition of advising according to manufacturer.
The clone of matrimony vine lycopene beta cyclase gene LmLycB:
With RNeasy Plant Mini Kit (QIAGEN, German) test kit, extract Total RNA from the fresh matrimony vine blade of 100mg, according to the aminoacid sequence design degenerated primer P1 of the lycopene beta cyclase in the species such as corn, tobacco, Arabidopis thaliana, Radix Dauci Sativae, green pepper in GenBank, the sequence as shown in SEQ ID NO.3 in sequence table.Utilize 3 '-amplification of FULL RACE Core Set Ver.2.0 (TaKaRa, Japan) test kit obtains complete gene order.Concrete steps:, 1. take Total RNA as template, use 3 ' RACE Adaptor primer to carry out reverse transcription reaction, synthetic 1st Strand cDNA, reaction system is as follows:
RNA： 2μl
3′RACEAdaptor： 1μl
5×M-MLV Buffer： 2μl
dNTPMixture： 1μl
RNase Inhibitor： 0.25μl
Reverse Transcriptase M-MLV： 0.25μl
RNase Free dH
2O： 3.5μl
Reaction conditions: 42 ℃, 60min; 70 ℃, 15min.
2. by the upstream primer P1 shown in SEQ ID NO.3, with by the downstream primer P2 shown in SEQ ID NO.4,, take 1st Strand cDNA as template, carry out the PCR reaction, reaction system is as follows:
1st PCR product: 2 μ l
dNTP Mixture： 8μl
P1： 2μl
P2： 2μl
10×LA PCR BufferII： 4μl
MgCl
2： 3μl
TaKaRa LATaq： 0.25μl
dH
2O： 28.75μl
Reaction conditions: 94 ℃, 3min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min30sec; 72 ℃, 10min, 30 circulations.
The building process of intermediate carrier pBS-T-LmLycB
LmLycB gene shown in SEQ ID NO.1 is connected with the pBS-T carrier, and reaction system is as follows:
Purpose PCR fragment: 3 μ l
PBS-T carrier: 1 μ 1
2×T4DNA Rapid Ligation Buffer： 5μl
T4DNA Ligase： 1μl
Reaction conditions: 23 ℃, 10min.Connect product Transformed E-Coli.TOP10, coat the LB that contains the ammonia benzyl dull and stereotyped.Carry out take goal gene as primer PCR (reaction conditions: 94 ℃, 3min; 94 ℃, 30sec; 54 ℃, 30sec; 72 ℃, 2min30sec; 72 ℃, 10min, 30 circulations.) obtain the 1800bp product, extract plasmid respectively through BamHI and PstI double digestion: 37 ℃, 16hrs, enzyme is cut (15 μ l plasmid DNA, 3 μ l R buffer, 0.5 μ lBamHI excision enzyme, 0.5 μ l PstI excision enzyme, 13 μ l ddH2O) obtain the purpose band, send finally the order-checking of the large gene sequencing company of China, result shows that vector construction is correct.
The building process of coli expression carrier pMON-LmLycB
At first, take pBS-T-LmLycB as masterplate, P3 and P4 are respectively upstream and downstream primer amplification LmLycB fragment, and its reaction conditions is 94 ℃, 3min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min30sec; 72 ℃, 10min, 30 circulations.Introduce BamHI restriction enzyme site (GGATCC) and a base A in P3, introduce SalI restriction enzyme site (GTCGAC) in P4.Then, PCR product and pMON38201 plasmid are respectively through BamHI and SalI double digestion, and both enzyme is cut the product connection: 16 ℃, 16hrs, connect (2 μ l 10 * T4buffer, 0.5 μ l T4DNA ligase enzyme, 5 μ l carrier DNAs, 7.5 μ l foreign DNAs, 5 μ l ddH
2O).Connect product Transformed E coli.DH5 α, coat the LB that contains Amp dull and stereotyped, as shown in Figure 1, be constructed pMON-LmLycB carrier schematic diagram.Carry out PCR take goal gene as primer and obtain the 1500bp product, enzyme is cut and is identified and to obtain the purpose band, send finally the order-checking of the large gene sequencing company of China, and it is correct that result shows that carrier pMON-LmLycB builds, as Fig. 2, for pMON-LmLycBPCR and enzyme are cut the result.
The plant expression vector pMON-LmLycB that builds is transformed bacillus coli DH 5 alpha:
The building process of plant expression vector pCAMBIA2300-LmLycB-Bar
At first, take pBS-T-LmLycB as masterplate, P5 and P6 are respectively upstream and downstream primer amplification LmLycB fragment, and its reaction conditions is 94 ℃, 3min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min30sec; 72 ℃, 10min, 30 circulations.Introduce BamHI restriction enzyme site (GGATCC) in P5, introduce SalI restriction enzyme site (GTCGAC) in P6.Then, PCR product and pCAMBIA2300-35S-OCS plasmid are respectively through BamHI and SalI double digestion, and both enzyme is cut the product connection: 16 ℃, 16hrs, connect (2 μ l10 * T4buffer, 0.5 μ l T4DNA ligase enzyme, 5 μ l carrier DNAs, 7.5 μ l foreign DNAs, 5 μ l ddH
2O).Connect product Transformed E-Coli.TOP10, coat the LB that contains kantlex dull and stereotyped.Carry out PCR take goal gene as primer and obtain the 1500bp product, enzyme is cut evaluation and is obtained the purpose band, send finally the order-checking of the large gene sequencing company of China, and result shows that carrier pCAMBIA2300-LmLycB builds correctly, as shown in Figure 3, cut the result for pCAMBIA2300-LmLycB PCR and enzyme.
Utilize XhoI (CTCGAG) restriction enzyme site, the NPTII gene on the pCAMBIA2300-LmLycB carrier is removed, from 5 ' inserted the Bar gene of anti-glufosinates to 3 ' direction, obtain the pCAMBIA2300-LmLycB-Bar plant expression vector, as Fig. 4.
The plant expression vector pCAMBIA2300-LmLycB-Bar that builds is transformed Agrobacterium C58, and concrete steps are: 1. the C58 competent cell of-80 ℃ of taking-ups is placed on ice, makes its slow thawing; 2. add 2 μ L plasmids, mix; 3. be transferred in the electric shock cup, above operation is all carried out on ice; 4. electric shock conversion instrument parameter is set: 25 μ F, 400olm, 1500V, 5ms, electric shock transforms; 5. add 1mL YEB liquid nutrient medium after the standing 2min of room temperature, 28 ℃, 180r/min shaking culture 4h; 6. get 50 μ L bacterium liquid and coat on the YEB flat board that contains 100mg/L Ka resistance, be inverted flat board, cultivate 48h for 28 ℃, until see single bacterium colony clearly.
The functional verification of LmLycB gene in intestinal bacteria
Cotransformation plasmid pACCRT-EBI in intestinal bacteria
Eu(chlorampenicol resistant) and expression vector pMON-LmLycB (ammonia benzyl mycin resistance), concrete steps are: 1. add each 1 μ L of two kinds of plasmid DNA in E.coli (DH5 α) competent cell, mix, place 30min on ice; 2. 42 ℃ of heat shock 90s, then place 2-3min on ice; 3. add 800 μ L, the LB liquid nutrient medium of 37 ℃ of preheatings (not containing microbiotic), 37 ℃, 150r/min shaking culture 45min; 4. draw the 50 above-mentioned nutrient solutions of μ L in containing on corresponding antibiotic LB solid medium, coated plate; 5. be inverted flat board, 37 ℃, cultivate 12hrs.The enzyme of matrimony vine LmLycB coding can be converted into the Lyeopene of redness yellow β-carotene, shows as the intestinal bacteria colony colour and changes yellow into by redness, as Fig. 5.The LB liquid nutrient medium consists of: 10g/L peptone, 10g/LNaCl, 5g/L yeast extract.
The extraction of intestinal bacteria carotenoid: 1. centrifugal collection thalline, the thalline quality is no more than 0.8g; 2. add 20ml methyl alcohol in thalline, resuspended; 3. add 2ml, 60%KOH, resuspended; 4. 60 ℃ of temperature are bathed 20min, then sample is cooled to room temperature; 5. the sherwood oil (bp.40-60 ℃) that sample and 20ml is contained 10% ether is transferred in separating funnel, fully mixes extraction; , if 6. sample and ether/sherwood oil layered effect is bad, can add appropriate saturated NaCl solution and a small amount of ethanol; 7. collect upper organic phase; 8. logical nitrogen, dry up sample.
The HPLC of intestinal bacteria carotenoid detects: sample is dissolved in 40 μ L acetone, uses nucleosil 100-3c250 * 4.6mm (MN, Germany) chromatographic column, blue rich (import SSI) quaternary gradient pump.According to Sander (Lane C.Sander, Katherine Epler Sharpless, et al, Development of Engineered Stationary Phases for the Seperation of Carotenoid Isomers, Anal.Chem, 1994,66:1667-1674) etc. the method for (1994) is carried out efficient liquid phase chromatographic analysis.Moving phase is acetonitrile: methyl alcohol: Virahol=85: 10: 5, flow velocity is 1.0mL/min, use simultaneously Thermo diode array (diode-array detector, DAD) detector, full wavelength scanner carotenoid spectrogram, as Fig. 6, for expressing the intestinal bacteria HPLC detected result of LmLycB.
Agriculture bacillus mediated maize genetic transforms
1. the acquisition of corn II type callus: maize immature embryos used is from this laboratory test Tanaka's corn inbred line 7922, after pollination 10-15d, get young female fringe, peel off bract, first use 75% ethanol disinfection surface in super clean bench, use again 0.1% mercury chloride soaking disinfection 15min, sterile water wash 3 times.The complete rataria of the female fringe of the picking big or small approximately 1-2mm in middle part, scultellum is seeded in respectively on 4 kinds of calli induction medias (medium component is shown in Table 1) up, and dark the cultivation, induce II type callus 3-4 week.The tissue block that the II type callus that grows fine that induces is divided into 2-3mm, upper dark cultivation of subculture medium (medium component is shown in Table 1), approximately 2-3 week subculture once, carries out the amplification of II type callus.
2. the cultivation of Agrobacterium: the Agrobacterium C58 that will contain plant expression vector pCAMBIA2300-LmLycB-Bar draws flat board, separates single bacterium colony; Picking list bacterium colony, in containing the 5mL YEB liquid nutrient medium of 100mg/L Ka, 28 ℃, 180r/min cultivates 12hrs; Above-mentioned bacterium liquid is inoculated in the fresh 30mL YEB liquid nutrient medium that contains 100mg/L Ka in 1: 100 ratio, 28 ℃, 180r/min, shaking culture to OD600 value is about the 0.6-0.8 left and right; Collect bacterium liquid, the centrifugal 10min of 4000r/min with the 50mL centrifuge tube; With infect liquid resuspended, clean thalline 2 times, adding Syringylethanone to final concentration is 100 μ mol/L, infects standby.
3. the genetic transformation of corn: the II type callus of the corn inbred line 7922 that will grow fine is cut into the 3-5mm fritter, be placed in culture dish, to adding appropriate resuspended liquid (N6 is a large amount of+B5 trace+molysite+N6 is organic+1.5mg/L2,4-D+0.7g/L L-Pro+36g/L glucose+68.4g/L sucrose) in culture dish.Prepare bacterium liquid in step 2 and pour in culture dish, bacterium liquid is outwelled after infecting end, callus is placed on filter paper, blots unnecessary bacterium liquid.
The rataria callus shield that will infect faces up, and is inoculated on common culture medium (medium component is shown in Table 1), 28 ℃, secretly cultivates 3-4d.Be inoculated on (medium component is shown in Table 1) through the rataria callus after cultivating altogether, 28 ℃, secretly cultivate 7d.When taking off bacterium, the foreign vector that just changes in plant is given full expression in vegetable cell.
After postponing to cultivate, change on the screening culture medium (medium component is shown in Table 1) that contains PPT 5mg/L, 25 ℃, secretly cultivated for 2 weeks, then subculture is on the screening culture medium that has improved screening pressure, and 2 all subcultures are once.After 6 weeks, just have new callus to produce, simultaneously most of immature embryo brownings are dead.These new callus can be considered transformant, continue subculture.
The picking embryo callus is inoculated on regeneration culture medium I (medium component is shown in Table 1), 25 ℃, the dark cultivation 2-3 week, in this process, most of somatic embryo swelling, bleach, some has formed coleoptile, the part of the long root of these callus is cut away, it is inoculated into regeneration culture medium II upper (medium component is shown in Table 1), 25 ℃ of light are cultivated (intensity of illumination is 5000Lux, and the photoperiod is 16h/8h) again.Within one week, the body embryo just can come into leaves, and 10d just can transplant left and right.
When the corn seedling grows to the 5cm size, the culturing bottle lid is opened hardening 3-5d in culturing room.Then carefully seedling is taken out from bottle with tweezers, clear water rinses out agar, avoids injuring root and blade in this process.Plant is moved into small flower, fill compost, water permeablely, move into greenhouse.
Table 1 plant culturing based component
aThe substratum pH value is 5.8;
bThe substratum pH value is 5.2; CH: casein food grade; MES:2-(N-morpholine) ethyl sulfonic acid
The Molecular Detection of transgenic corns and HPLC detect
The PCR of transgenic corns genomic dna detects: 1. the CTAB method is extracted total corn DNA; 2., take genomic dna as template, carry out PCR and detect, primer is P5, P6, reaction conditions: 94 ℃, and 3min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min30sec; 72 ℃, 10min, 30 circulations, electrophoresis result is as shown in Figure 7.
The RT-PCR of transgenic corns detects: 1. extract the total RNA of maize leaf; 2., take the total RNA of maize leaf as template, use Reverse Transcription System (Promega) test kit to carry out reverse transcription, synthetic cDNA the first chain, reaction conditions: 42 ℃, 50min; 95 ℃, 5min, 4 ℃, 5min; 3. getting 1 μ L reaction solution is template, carries out the PCR reaction, and primer is P7, P8, reaction conditions: 94 ℃, and 3min; 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 45s; 72 ℃, 10min, 30 circulations, electrophoresis result is as shown in Figure 8.
The extraction of maize leaf carotenoid: 1. get the fresh maize leaf in 50mg left and right (second, top blade), be placed in mortar, be milled to Powderedly with liquid nitrogen, change in the 50ml centrifuge tube; 2. add 20ml methyl alcohol in centrifuge tube, mix; 3. add 2ml, 60%KOH, mix; 4. 60 ℃ of temperature are bathed 20min, then sample is cooled to room temperature; 5. the sherwood oil (bp.40-60 ℃) that sample and 15ml is contained 50% ether is transferred in separating funnel, fully mixes extraction; , if 6. sample and ether/sherwood oil layered effect is bad, can add appropriate saturated NaCl solution and a small amount of ethanol; 7. collect upper organic phase; 8. logical nitrogen, dry up sample.
Transgenic corns total carotinoid assay: the carotenoid that will extract is dissolved in 10mL acetone, and ultraviolet spectrophotometer is measured the absorbancy at 450nm place,, as Fig. 9, is the HPLC detected result of wild-type corn and transgenic corns Carotenoid in Leaves.And calculate as follows maize leaf total carotinoid content:
X(μg/g)＝(A×1×V×l0
6)/(ε×100×W)
In formula: X is carotenoid content (μ g), and A is the absorbancy OD value that sample is measured at the 450nm place, and V is the volume (10mL) of extracting solution, and ε is carotenoid molecule mean absorption coefficient 2100, and W is the weight (g) of maize leaf.
Transgenic corns blade HPLC detects: under the 450nm wavelength, with calibration curve method working sample content beta-carotene, formula is as follows:
X(μg/g FW)＝(C×V)/(A×W)
In formula: X is content beta-carotene, the content beta-carotene (μ g) of C for from typical curve, trying to achieve; V is extracting solution cumulative volume (40 μ L); Volume used (20 μ L) when A is mensuration; W is sample heavy (g).The results are shown in Table 2.
Table 2
Claims (7)
1. a matrimony vine lycopene beta cyclase gene, is characterized in that this gene is the nucleotide sequence shown in SEQ ID No.1.
2. the protein of matrimony vine lycopene beta cyclase genes encoding 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 containing matrimony vine lycopene beta cyclase gene complete sequence claimed in claim 1.
4. a recombinant vectors claimed in claim 3, is characterized in that it is coli expression carrier pMON-LmLycB or recombinant plant expression vector pCAMBIA2300-LmLycB-Bar.
5. a host cell, is characterized in that containing matrimony vine lycopene beta cyclase gene complete sequence claimed in claim 1.
6. host cell according to claim 5, is characterized in that it is Bacillus coli cells or agrobatcerium cell.
7. the application of matrimony vine lycopene beta cyclase gene claimed in claim 1, is characterized in that it is for the preparation of transgenic corns.
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| ES2558953B1 (en) * | 2015-11-23 | 2016-11-18 | Universitat De Lleida | Corn enriched in antioxidants to improve the nutritional quality of the egg |
| CN105255926A (en) * | 2015-11-25 | 2016-01-20 | 天津大学 | Wolfberry lycopene epsilon-cyclase gene and recombinant vector comprising gene |
| CN105368849A (en) * | 2015-11-25 | 2016-03-02 | 天津大学 | Lycium 9-cis-epoxy-carotenoid dioxygenase enzyme gene with function of stress resistance and application of lycium 9-cis-epoxy-carotenoid dioxygenase enzyme gene with function of stress resistance |
| CN106119234B (en) * | 2016-07-07 | 2019-08-23 | 中国科学院华南植物园 | A kind of genetic engineering bacterium producing alpha-carotene and its construction method and application |
| CN106367410B (en) * | 2016-08-29 | 2019-08-20 | 中国科学院华南植物园 | A kind of genetic engineering bacterium producing compound carotenoid and its construction method and application |
| CN108004258A (en) * | 2017-11-01 | 2018-05-08 | 江西中医药大学 | The protein of cape jasmine lycopene beta cyclase b2 genes and its coding, the gene of optimization and their application |
| CN110423766B (en) * | 2019-08-29 | 2021-06-29 | 南京大学 | Lycopene beta-cyclase gene and coding protein and application thereof |
| CN114703225B (en) * | 2022-04-09 | 2023-05-16 | 吉林省农业科学院 | Method for improving carotenoid content of corn kernels by using gene editing technology |
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