CN102816790A - Plant expression vector pCamE and application thereof in gene transformation - Google Patents
Plant expression vector pCamE and application thereof in gene transformation Download PDFInfo
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Abstract
The invention provides a plant expression vector containing a promoter-multiple cloning site-terminator sequence, which is constructed by sequentially cloning a CaMV 35S promoter, a multiple cloning site of a plasmid vector and an NOS terminator through a PCR reaction, restriction enzyme digestion and connection technology and inserting the multiple cloning site and the NOS terminator of a binary vector into the multiple cloning site of the binary vector. The plant expression vector pCamE provided by the invention can completely and independently drive the expression of an inserted gene, 8, 4 and 3 restriction enzyme cutting sites are respectively provided at the multiple cloning site, the promoter upstream and the terminator downstream of the vector, and 15 restriction enzyme cutting sites are provided in total, so that the vector can be conveniently used. The pCamE vector has the characteristics of high copy number in escherichia coli, stable heredity and expression in a plant host material, and convenience, rapidness, economy and practicability in the using process.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to a kind of plant expression vector and the application in gene transformation thereof.
Background technology
Day by day routinize, generalize in that the scientific experiment of biological field is indoor for the research of gene and gene transformation, and in this research process, the smooth structure of conversion carrier is to concern one of experiment key of success factor.The normal expression of a gene needs a complete expression assembly, comprising promotor and terminator.In carrier, often need MCS and insert wherein, accomplish the expression vector establishment of a goal gene to make things convenient for foreign gene to be inserted in the expression vector.Wherein promotor is a critical elements that drives genetic transcription, and terminator is the element that instructs genetic transcription to stop.Expressing assembly is a prerequisite assembly of expression vector, and the insertion of a reporter gene (like gfp, gus) is often arranged in the expression assembly of the existing plant expression vector that uses, and the restriction endonuclease sites that can provide seldom.
The expression vector that makes up goal gene at present is generally through two approach; 1. the MCS at binary vector carriers such as (like pBIN19 (GI:1256363), pGreen (GI:4204727), pEGFP1 (GI:1377908), pPZP (GI:506651) and pCAMBIA (GI:7638064)) inserts goal gene; But this needs to insert at the goal gene upper reaches simultaneously promotor; Terminator is inserted in downstream, therefore, makes the building process complex steps of plant expression vector.2. (like pBI121 (GI:19569229), pEGAD (GI:7453571), pCAMBIA1304 (GI:7638083) and pBin-GFP) go up and insert goal gene having the expression vector of reporter gene; Keep (or excision) reporter gene (like the gus gene of pBI121 carrier, the GFP gene of pEGAD carrier) simultaneously, but owing to the fusion of reporter gene and the limited amount of restriction enzyme site make the use of carrier have certain limitation.
Therefore, press for a kind of complete expression assembly that has, do not contain reporter gene, can driven independently insert expression of gene and contain the plant expression vector that enriches restriction enzyme site and be applied in the gene transformation research work.
Summary of the invention
The object of the present invention is to provide a kind of plant expression vector that contains the expressed intact assembly.
The plant expression vector that contains promotor-MCS-terminator sequence provided by the invention is that the promotor-MCS-MCS of terminator sequence insertion binary vector is made up and obtains.
Wherein, said binary vector is pCAMBIA1300.
Wherein, said promotor is the CaMV 35S promoter, and its nucleotide sequence is shown in SEQ ID NO.1.
Wherein, said terminator is the NOS terminator, and its nucleotide sequence is shown in SEQ ID NO.2.
Wherein, said MCS is the MCS of pUC19, and its nucleotide sequence is shown in SEQ ID NO.3.
Further, plant expression vector provided by the invention is pCamE, and its nucleotide sequence is shown in SEQ ID NO.4.The collection of illustrative plates of pCamE plant expression vector is as shown in Figure 1.
Plant expression vector provided by the invention is that its MCS of pCamE, the promotor upper reaches and terminator downstream have 8,4 and 3 restriction enzyme digestion sites respectively.
The invention provides the host cell that contains said plant expression vector.
The invention provides the application of above-mentioned plant expression vector in gene transformation.
The invention provides the application of above-mentioned plant expression vector in the preparation transgenic plant.
The present invention also provide the construction process of plant expression vector pCamE, comprising:
(1) clone CaMV 35S promoter gene pro, the primer that is used for amplification gene pro is: proF:5'-CAAGCTTAATTAAGAGCTCGCATGCCCTTTCAG-3 '; ProR:5'-CGTCGACGACCGGTAGCGCTAGAGTCCCCG-3 '; The goal gene pro that pcr amplification is obtained is connected with the pGM-T carrier, obtains carrier pGMpro;
(2) clone NOS terminator gene Ter, the primer that is used for amplification gene Ter is: TerF:5'-CGGTACCGGGCCCTGATCTCGAGGAGCTAGCTCGAATTTCCCC-3 '; TerR:5'-CGAATTCGAATTAATTCCCGATCTAGTAACATAGATG-3'; The goal gene Ter that pcr amplification is obtained is connected with the pGM-T carrier, obtains carrier pGMTer;
(3) with being connected with behind HindIII and the Sal I double digestion respectively of carrier pGMpro and carrier pUC19, obtain carrier pUCpro
(4) plasmid of carrier pGMTer and carrier pUCpro is connected with behind Kpn I and the EcoR I double digestion respectively, obtains carrier pUCpro-MCS-Ter;
(5) plasmid of intermediate carrier pUCpro-MCS-Ter and binary vector pCAMBIA1300 is connected with behind HindIII and the EcoR I double digestion respectively, transforms, cultivate, extract plasmid, obtain plant expression vector pCamE.
Wherein, above-mentioned steps (5) is that the connection product is adopted thermal shock method transformed into escherichia coli competent cell.Overnight cultures in picking positive colony to the 3ml LB nutrient solution, alkaline lysis method of extracting plasmid are extracted and are dissolved in the 50 μ l TE damping fluids, and enzyme is cut detection, acquisition plant expression vector pCamE.Can know that through calculating the copy number of plant expression vector pCamE in bacillus coli DH 5 alpha is 237.This carrier copy number belongs to relaxed type carrier (copy number is the relaxed type carrier greater than 10～15) greater than 10, and this vector plasmid size is 10131bp, and less than 15000bp, separate easily purifying relatively in intestinal bacteria is high copy number plasmid.
Plant expression vector pCamE provided by the invention has the complete expression assembly that is made up of promotor-MCS-terminator sequence, and it can completely drive independently and insert expression of gene.Provide 8,4 and 3 respectively in MCS, the promotor upper reaches and the terminator downstream of this carrier, amounted to 15 restriction enzyme sites, the MCS that only foreign gene is directly inserted this place and provides when using this carrier gets final product.PCamE carrier of the present invention itself does not contain reporter gene, can avoid well because the fusion of reporter gene, causes carrier property to descend and then has limited the use of carrier.Green fluorescence protein gene gfp is inserted the Apa I restriction endonuclease sites of this plant expression vector pCamE; Made up expression vector pCamGFP; And pCamGFP onion epidermis cell and Arabidopis thaliana plant have been transformed respectively; The result shows that the entrained green fluorescence protein gene gfp of pCamGFP carrier can genetic stability and normal expression in vegetable material; Explain that the plant expression vector pCamE among the present invention can be used for transforming the plant host material, and integrate the back stably express with plant chromosome.It is high that the pCamE carrier has in intestinal bacteria a copy number, and in use have convenient, fast, economy and practical characteristics.
Description of drawings
Fig. 1 is the collection of illustrative plates of plant expression vector pCamE; Runic TATAA is an eukaryotic promoter TATA frame, and runic AATAAA is Eukaryotic tailing signal, and TGA and TAG are translation stop codon in the frame.
The expression of Fig. 2 plasmid pCamGFP in onion entocuticle cell; A figure is the expression of plasmid pCamGFP in onion entocuticle cell under the excited by visible light, and B figure is the blue-light excited expression of plasmid pCamGFP in onion entocuticle cell down.
The expression of Fig. 3 plasmid pCamGFP in transgenic arabidopsis; A figure is the expression of plasmid pCamGFP in Arabidopis thaliana under the excited by visible light, and B is the blue-light excited expression of plasmid pCamGFP in Arabidopis thaliana down.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize; Enzyme reagent such as restriction endonuclease used among the embodiment are all available from precious biotechnology (Dalian) ltd; Primer is by the synthetic completion of Beijing three rich polygala root biotechnology Ltds; Examining order is accomplished by Sangon Biotech (Shanghai) Co., Ltd., and non-the indicating outward especially of other biochemical reagents is conventional commercial reagent, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
(1) alkaline lysis method of extracting plasmid
Picking contains the e.colistraindh5 of plasmid pEGAD, is inoculated in 1.5ml LB liquid nutrient medium and (contains Tryptones 10g in 1 liter of substratum, yeast extract 5g; NaCl 10g; PH7.0) in, add penbritin to final concentration 0.1mg/ml, 37 ℃ of shaking culture are spent the night; The centrifugal 30s of 12000rpm abandons supernatant.The solution I [50mM TrisHCl (pH7.5), 10mM EDTA (pH8.0)] that adds 100 μ l precoolings, thermal agitation disperses deposition, and fully room temperature is placed 5min behind the mixing.(0.2M NaOH 1%SDS), puts upside down mixing rapidly 5 times, leaves standstill 5min on ice to add 200 μ l solution II.Add 150 μ l solution III (the 1.32M Potassium ethanoate, pH4.8), gentle mixing 4-5 time is placed 5min on ice, the centrifugal 10min of 12000rpm shifts supernatant to another centrifuge tube, adds 3 μ lRNase, 37 ℃ of water-bath 15min behind the gentle mixing.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing after, the centrifugal 10min of 12000rpm gets supernatant to another centrifuge tube, adds 2 times of cold absolute ethyl alcohols of volume, behind the mixing ,-20 ℃ leave standstill 20min; The centrifugal 10min of 12000rpm abandons supernatant.With 70% washing with alcohol deposition, be dissolved in after the drying at room temperature among the 10 μ l TE.
(2) clone and the order-checking of CaMV 35S promoter gene pro
The sequence information of the plasmid pEGAD that provides according to NCBI website (http://www.ncbi.nlm.nih.gov/) DateBase DB, the PCR primer of designs C aMV 35S promoter gene pro:
proF：5'-CAAGCTTAATTAAGAGCTCGCATGCCCTTTCAG-3'
proR：5'-CGTCGACGACCGGTAGCGCTAGAGTCCCCG-3'
With plasmid pEGAD is template, utilizes PCR primer proF and proR to obtain 35S promoter gene pro through the round pcr amplification.The PCR reaction system is following:
PCR reaction amplification program is following:
95 ℃ 5 minutes; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.
The purpose fragment of the PCR product of 35S promoter gene pro after reclaiming after the agarose electrophoresis is connected in the pGM-T carrier, and reaction system is following:
4 ℃ of connections behind the above-mentioned mixed solution mixing are spent the night.
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.Use the picking positive colony, shake alkaline lysis method of extracting plasmid and order-checking behind the bacterium, obtain the sequence information of 35S promoter gene pro.
The sequence alignment analysis of sequencing result and carrier pEGAD confirms that the cloned sequence of gained 35S promoter gene pro is correct.CaMV 35S promoter gene pro sequence is shown in SEQ ID NO.1.
(3) PCR of NOS terminator gene Ter clone and order-checking
The sequence information of the plasmid pEGAD that provides according to NCBI website DateBase DB, the PCR primer of design NOS terminator gene Ter:
TerF：
5'-CGGTACCGGGCCCTGATCTCGAGGAGCTAGCTCGAATTTCCCC-3'
TerR：5'-CGAATTCGAATTAATTCCCGATCTAGTAACATAGATG-3'。
With plasmid pEGAD is template, utilizes PCR primer TerF and TerR to obtain NOS terminator gene Ter through the round pcr amplification.The PCR reaction system is following:
PCR reaction amplification program is following:
95 ℃ 5 minutes; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.
The PCR product of NOS terminator gene Ter is connected in the pGM-T carrier after reclaiming after the agarose electrophoresis, reaction system is following:
4 ℃ of connections behind the above-mentioned mixed solution mixing are spent the night.
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.Use the picking positive colony, shake alkaline lysis method of extracting plasmid and order-checking behind the bacterium, obtain sequence information and the intermediate carrier pGMTer of 35S promoter gene pro.
The sequence alignment analysis of sequencing result and carrier pEGAD confirms that the cloned sequence of gained NOS terminator gene Ter is correct.NOS terminator gene Ter gene order is seen shown in the SEQ ID NO.2.
(4) structure of carrier pUCpro
The plasmid of carrier pGMpro and cloning vector pUC19 was used HindIII and Sal I double digestion respectively 30 minutes.
PGMpro endonuclease reaction system is following:
PUC19 endonuclease reaction system is following:
Enzyme is cut product behind 1% agarose gel electrophoresis, uses gel recovery test kit recovery cloning vector also to be connected with goal gene pro fragment and spends the night, and reaction system is following:
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.The picking positive colony, the alkaline lysis method of extracting plasmid extracts and enzyme is cut detection, obtains carrier pUCpro.
(5) structure of carrier pUCpro-MCS-Ter
The plasmid of intermediate carrier pGMTer and cloning vector pUCpro was used Kpn I and EcoR I double digestion respectively 30 minutes.
PGMTer endonuclease reaction system is following:
PUCpro endonuclease reaction system is following:
Enzyme is cut product behind 1% agarose gel electrophoresis, uses gel recovery test kit recovery cloning vector also to be connected with goal gene Ter fragment and spends the night, and reaction system is following:
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.Picking positive colony, alkaline lysis method of extracting plasmid, enzyme are cut and are detected and order-checking.Sequencing result shows that constructed expression assembly is made up of MCS MCS (shown in the SEQ ID NO.3) and NOS terminator gene Ter three parts of 35S promoter gene pro, plasmid pUC19.Confirm to express the structure completion of assembly (promotor-MCS-terminator), obtain intermediate carrier pUCpro-MCS-ter thus.
The structure of embodiment 2 plant expression vector pCamE
(1) structure of plant expression vector pCamE
Intermediate carrier pUCpro-MCS-ter that embodiment 1 is obtained and the plasmid of binary vector pCAMBIA1300 were used HindIII and EcoR I double digestion respectively 30 minutes.
PUCpro-MCS-ter endonuclease reaction system is following:
PCAMBIA1300 endonuclease reaction system is following:
Enzyme is cut product behind 1% agarose gel electrophoresis, uses gel recovery test kit recovery binary vector also to be connected with purpose fragment pro-MCS-ter and spends the night, and reaction system is following:
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.Overnight cultures in picking positive colony to the 3mlLB nutrient solution, alkaline lysis method of extracting plasmid are extracted and are dissolved in the 50ul TE damping fluid, and enzyme is cut detection, acquisition plant expression vector pCamE.
(2) plant expression vector pCamE copy number calculates
Get the TE damping fluid that 1.2ul is dissolved with plasmid pCamE and detect through micro-spectrophotometer, its plasmid concentration is 300ng/ul, and can to get the contained plasmid copy number of 3ml bacterium liquid be 300ng/ul * 10 through calculating
-9* 6.02 * 10
23* 50ul/10131bp/660dalton/bp=1.35 * 10
12
The LB bacterium liquid dilution 10 of the plasmid pCamE that extracts
6Utilizing blood counting chamber to calculate e. coli concentration doubly is 1.9/microlitre, and can get the contained bacterial number of 3ml bacterium liquid through calculating is 1.9 * 10
6* 10
3* 3ml=5.70 * 10
9
The copy number of plant expression vector pCamE in bacillus coli DH 5 alpha is 1.35 * 10
12/ 5.70 * 10
9=237.This carrier copy number belongs to relaxed type carrier (copy number is the relaxed type carrier greater than 10～15) greater than 10, and this vector plasmid size is 10131bp, and less than 15000bp, separate easily purifying relatively in intestinal bacteria is high copy number plasmid.
(3) sequence information of plant expression vector pCamE obtains
PCamE checks order to the gained plant expression vector, confirms the vector construction completion.Its sequence is shown in SEQ ID NO.4.
(1) the basic module information analysis of plant expression vector pCamE
According to the sequence information of plant expression vector pCamE, this carrier of blast compare of analysis possesses the basic module of plant expression vector through the NCBI website.Analytical results information such as table 1.
The basic module of table 1 plant expression vector pCamE
(2) the mutational site information analysis of plant expression vector pCamE
According to the sequence information of plant expression vector pCamE, find that through NCBI blast compare of analysis there are 8 mutational sites in the sequence of this carrier sequence and plasmid pCAMBIA1300, pEGAD and pUC19.
Mutational site information analysis and the explanation of table 2 plant expression vector pCamE
(3) the restriction endonuclease sites information analysis of plant expression vector pCamE
According to the sequence information of plant expression vector pCamE, to 117 restriction endonuclease sites analyses, analytical results shows that 94 restriction enzymes have 323 restriction enzyme sites (table 3) in this carrier with the sequence of this carrier of DNAMAN software.23 restriction enzymes do not have restriction enzyme site in this carrier.Wherein in plant expression vector, do not have 23 restriction enzymes of restriction enzyme site following:
AscⅠ，AvrⅡ，BlⅡ，Bsp1407Ⅰ，BstEⅡ，Bsu36Ⅰ，Cvn?Ⅰ，Eam1105Ⅰ，Eco72Ⅰ，Hpa?Ⅰ，I-PpoⅠ，MluⅠ，Mst?Ⅱ，Nru?Ⅰ，PmaC?Ⅰ，PstⅠ，Sau?Ⅰ，SnaB?Ⅰ，Spe?Ⅰ，Spo?Ⅰ，SrfⅠ，StuⅠ，Swa?Ⅰ。
323 sites of 94 restriction enzymes are analyzed among the table 3 plant expression vector pCamE
(4) plant expression vector pCamE collection of illustrative plates is drawn
According to sequence information and the bioinformatic analysis of gained plant expression vector pCamE, use DNAMAN software to draw out this carrier collection of illustrative plates (Fig. 1).
The structure of embodiment 4pCamGFP expression vector
In order to study and verify the practical and stable of carrier pCamE of the present invention, be the Apa I site that material is connected to this carrier with green fluorescence protein gene (egfp), be built into expression vector pCamGFP.Concrete steps are following:
(1) clone of egfp gene
According to the sequence information that NCBI website DateBase DB provides, the PCR primer of design egfp gene:
eGFPF：5'-GGGCCCATGGTGAGCAAGGGCGAGGAGCTGTTC-3'
eGFPR：5'-GGGCCCTTACGCAGCTCCGGACTTGTACAGCTCGTCC-3'
With plasmid pEGAD is template, utilizes PCR primer eGFPF and eGFPR to obtain the egfp gene order through pcr amplification, makes these sequence two ends all have Apa I restriction enzyme site simultaneously.The PCR reaction system is following:
PCR reaction amplification program is following:
95 ℃ 5 minutes; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of 30 circulations in 1 minute; 72 ℃ 10 minutes.
Wgfp gene PCR product is connected in the pGM-T carrier after reclaiming after the agarose electrophoresis, reaction system is following:
4 ℃ of connections behind the above-mentioned mixed solution mixing are spent the night.
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.Use the picking positive colony, shake alkaline lysis method of extracting plasmid and order-checking behind the bacterium, obtain the sequence information and the expression vector pGMGFP of egfp gene.
The sequence alignment analysis of sequencing result and carrier pEGAD confirms that the cloned sequence of gained egfp gene is correct, and the egfp gene order is shown in SEQ ID NO.5.
(2) structure of expression vector pCamGFP and order-checking
The plasmid of carrier pGMGFP and plant expression vector pCamE was cut 30 minutes with Apa I enzyme respectively.
PGMGFP endonuclease reaction system is following:
PCamE endonuclease reaction system is following:
Enzyme is cut product behind 1% agarose gel electrophoresis, uses gel recovery test kit recovery plant expression vector pCamE also to be connected with goal gene egfp fragment and spends the night, and reaction system is following:
Connect product and adopt thermal shock method transformed into escherichia coli competent cell.The picking positive colony, the alkaline lysis method of extracting plasmid extracts and enzyme is cut detection and order-checking, obtains expression vector pCamGFP.The sequence alignment analysis of the sequence of gdp gene and carrier pCamE confirms that the insertion site of gfp gene among the gained expression vector pCamGFP is restriction endonuclease Apa I site among sequencing result and the carrier pEGAD, and cloned sequence is correct.
The expression study of embodiment 5 expression vector pCamGFP in onion epidermis cell
(1) particle gun bombardment onion epidermis cell
Alkaline lysis method of extracting plasmid pCamGFP uses particle gun (PDS-1000/He system) bombardment onion epidermis cell.Get 50 μ L (3mg) bronze suspension-s to the 1.5ml centrifuge tube, add 5ug pCamGFP DNA, 25ul 5M CaCl successively
2With 5ul 0.4M spermidine, add water to 45 μ L at last; Limit edged vibration continues vibration 2-3min, and room temperature is placed 1min, and instantaneous centrifugal 2s abandons supernatant; Add 140ul 70% ethanol and wash deposition 1 time, abandon supernatant.Add the 140ul absolute ethyl alcohol and wash deposition 1 time, abandon deposition.Deposition is suspended in the 48 μ L absolute ethyl alcohols again, can supply 6 rifles (every rifle 8ul) to use.Get 8 μ L suspension-s and evenly be applied to loading film (micro mist slide glass) central authorities rapidly; In case micro mist caking; Dry environment dries in short-term, can split film and be soaked in the 3-5min that sterilizes in 70% Virahol in short-term, and taking-up can be split film and be installed on the tin hat rapidly; Be screwed to clockwise on the gas accelerator, screw with tool bar.Installation stops net, contains exsiccant loading film set collar successively in stopping the net bracket, and the cap that is tightened inserts bore and shuts up the door carefully.Open the main valve of helium tank, the registration of helium pressure list index is higher than splits film pressure 200psi.Open the particle gun switch, put Vac/Vent/Hold Switch key, start vacuum pump, treat vacuum tightness to 27～29inHg, streak the vent shelves rapidly, then pin emission key, and keep motionless, till percussion to the Hold shelves in the Vac shelves.After treating that by the ventilation key vacuum meter returns zero, take out sample.
(2) fluorescence microscopy of expression vector pCamGFP in onion epidermis cell observed
Use fluorescent microscope Olympus BX51 significant egfp expression (Fig. 2) to be arranged in onion epidermis cell at the blue-light excited pCamGFP of observation down.The result show the GFP of pCamGFP can be in onion epidermis cell normal expression, explain that the green fluorescence protein gene that inserts pCamE carrier A pa I site can be expressed by the 35S promoter normal transcription of new structure.
The expression study of embodiment 6 expression vector pCamGFP in Arabidopis thaliana
(1) expression vector pCamGFP transforms Agrobacterium GV3101
In 50 μ l Agrobacterium GV3101 competent cells, add pCamGFP DNA 1 μ g, ice bath 30min; Put into liquid nitrogen 1min, put into 37 ℃ of water-bath water-bath 5min then immediately; Take out centrifuge tube, add 0.5ml LB, 28 ℃, 220rpm shaking culture 3～5hr; Take out bacterium liquid in containing coated plate on the corresponding antibiotic LB flat board, in 28 ℃ of incubators, be inverted and cultivated about 2 days, occur up to bacterium colony.
(2) the reorganization Agrobacterium is identified
Picking list bacterium colony is inoculated in and contains in the corresponding antibiotic LB liquid nutrient medium, and 28 ℃ of shaking culture are spent the night.Alkaline lysis method of extracting plasmid DNA and enzyme are cut evaluation.
(3) arabidopsis thaliana transformation
Matrix (vermiculite and nutrition soil 3:1 mixes) is inserted small-sized flowerpot and is placed in the plastic culture box.Pour the Horgland nutritive medium in the plastic culture box into.Make nutritive medium slowly soak into matrix, and make retained part nutritive medium in the plastic culture box by the small flower bottom.With (the suitable humidity of vernalization in right amount; Place 2d for 4 ℃) the Columbia type Arabidopis thaliana planting seed preserved of this chamber on above-mentioned matrix top layer (8-10 grain seed/basin), and spray nutrient solution so that seed tightly invests the leech surface, move to the illumination cultivation chamber after secretly being cultured to germination; Normal illumination (16h light cultivation/8h secretly cultivates); Intensity of illumination is 5000lx, and temperature 20-24 ℃, humidity is 80%.Water the Horgland nutritive medium weekly 1 time.Arabidopis thaliana is grown on above-mentioned matrix about 6-7 week, grows to 10-15cm its main tongue, and when growing up to 1-2 angle fruit, promptly can be used for conversion.Also can be when its main tongue grows to 5-6cm; Cut whole inflorescence at the inflorescence base portion; Remove its apical dominance, 1 week back grows 4-6 newborn side tongue at the axillalry bud position, treats that its side tongue inflorescence forms bud and part and blooms or forms 1-2 angle really the time; Promptly can be used for transforming, need cut off flower of having bloomed and the angle that has grown up to before transforming really.
The picking activatory contains the positive Agrobacterium GV3101 of the mono-clonal bacterial strain of pCamGFP to the fresh YEB liquid nutrient medium of 20ml (containing kantlex and Rifampin), 28 ℃ of shaking culture 24h.Get above-mentioned bacterium liquid 5ml and be connected in the fresh YEB liquid nutrient medium of 500ml, 28 ℃ of 250rpm cultivate 18-24h, and the OD value is reached about 0.8.The centrifugal 20min of room temperature 5500g; Supernatant discarded; Reach about 0.8 with the resuspended OD of the being precipitated to value of conversion medium (1/2MS is a large amount of, 1 * molysite, 1 * trace, 1 * organic, 5%sucrose, 0.01 μ g/ml benzyladenine (BAP), 0.03%silwetL-77, KOH adjust pH to 5.7).With Arabidopis thaliana plant (T to be transformed
0Generation) is inverted, over-ground part is soaked in the above-mentioned conversion medium, contaminate 5min.Get rid of immersion liquid gently plant kept flat, and cover the Arabidopis thaliana over-ground part with plastics bag and preserve moisture, secretly cultivate 16-24h after, carefully remove plastics bag, be positioned in the plastic culture box, and water sufficient Horgland nutritive medium, recover normal illumination (16h/8h).Normal management, results mature seed (T1).
Transform 5-6 after week, can water nutritive medium in right amount less for quickening the Arabidopis thaliana maturation.Treat the indivedual angles of Arabidopis thaliana really begin withered and yellow after, can its angle really be cut and be put in the petridish inner drying.Arabidopis thaliana angle fruit most of withered and yellow after, can collect in the EP pipe that whole seeds are stored in 1.5ml (pricking an aperture so that drying covering).Behind the seed complete drying, be put in 4 ℃ of short-terms preservations in the new EP pipe of 1.5ml, can be placed on prolonged preservation in-20 ℃ of refrigerators like needs.
(4) screening of transgenic positive plant and evaluation
Get the T of about 50 μ l volumes
1Seed is put in the aseptic EP pipe of 2.0ml.Add the 1ml sterilized water, surface infiltration 1min, the centrifugal 30s of 5000rpm, supernatant discarded.Add 1.5ml 2.5% Youxiaolin, resuspended seed, sterilization 10min, the centrifugal 30s of 5000rpm carefully discards Youxiaolin.With the aseptic resuspended seed of 0.01% agar-agar soln, move to the MS that contains 50 μ g/ml Kan with the rifle head and select on the substratum (not containing sugar), evenly tiling.Diameter is that the petridish of 6cm is planted 800～1500 Arabidopis thaliana seeds approximately, 4 ℃ of vernalization 2d.Move to culturing room and carry out the normal illumination cultivation, culture condition is 23 ℃ of temperature, intensity of illumination 5000lx, light/dark incubation time 16h/8h.Behind the about 7-10d of screening on the screening culture medium; When the positive seedling (T1 plant) of success importing goal gene grows to 4 true leaves; Be transplanted in the matrix of soaking into fully with the Horgland nutritive medium, on cover preservative film or the sheet glass 3-5d that preserves moisture, normal illumination is cultivated.After the positive Arabidopis thaliana plant angle fruit maturation of treating to filter out through kantlex, the T of individual plant results Arabidopis thaliana
2Seed, dry back 4 ℃ of short-terms are preserved or-20 ℃ of prolonged preservation.
With results transgenic positive plant seed kind on the MS screening culture medium that contains kantlex; After 10 days; Add up its become to live strain number and dead strain number; It separates and to meet the plant strain system that 3:1 Mendelian separates than rule than (ratio of become to live strain number and dead strain number) and be heterozygote, and it is to be homozygote that its become to live strain number and the ratio of planting the plant number meet the plant strain that 1:1 separates than rule.It separates the ratio experiment and separates than rule than still meeting Mendelian in testing in later separation, explains that the pCamGFP carrier that has the gfp gene also can genetic stability with its chromosomal integration in Arabidopis thaliana.
(5) fluorescence microscopy of expression vector pCamGFP in transgenic arabidopsis observed
Use fluorescent microscope Olympus BX51 to observe the expression of expression vector pCamGFP in transgenic arabidopsis down blue-light excited, the result show the GFP of pCamGFP can be in transgenic arabidopsis normal expression.Explain that the GFP albumen that pCamGFP expresses can genetic stability and expression (Fig. 3) in transgenic arabidopsis.Normally functionating is set up in the expression that the pCamE vector construction is described, can be applicable to the Study on Transformation of plant host material.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. contain the plant expression vector of promotor-MCS-terminator sequence, it is characterized in that, the MCS structure that promotor-MCS-terminator sequence is inserted binary vector obtains.
2. plant expression vector as claimed in claim 1 is characterized in that, said binary vector is pCAMBIA1300.
3. plant expression vector as claimed in claim 1 is characterized in that, said promotor is the CaMV 35S promoter, and its nucleotide sequence is shown in SEQ ID NO.1.
4. plant expression vector as claimed in claim 1 is characterized in that, said terminator is the NOS terminator, and its nucleotide sequence is shown in SEQ ID NO.2.
5. plant expression vector as claimed in claim 1 is characterized in that, said MCS is the MCS of pUC19, and its nucleotide sequence is shown in SEQ ID NO.3.
6. plant expression vector as claimed in claim 1, it is pCamE, its nucleotide sequence is shown in SEQ ID NO.4.
7. plant expression vector as claimed in claim 6 is characterized in that, its MCS, the promotor upper reaches and terminator downstream have 8,4 and 3 restriction enzyme digestion sites respectively.
8. the host cell that contains each said plant expression vector of claim 1 ~ 7.
9. each described plant expression vector application in gene transformation of claim 1 ~ 7.
10. the application of each described plant expression vector of claim 1 ~ 7 in the preparation transgenic plant.
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| CN109929874A (en) * | 2019-03-27 | 2019-06-25 | 吉林大学 | The construction method of Agrobacterium Ti plasmid carrier PCHF1302 and application |
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