CN103205456A - Plant binary expression vector - Google Patents
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Abstract
The present invention discloses a plant binary expression vector. The plant binary expression vector is obtained according to the follow method: a fluorescent protein reporter gene expression cassette and an exogenous gene expression cassette are constructed in a starting vector pBI121, wherein the exogenous gene expression cassette is composed of a promoter, an intron and a terminator connected together in sequence, a plurality of restriction enzyme cutting sites are arranged between the promoter and the intron, and a plurality of restriction enzyme cutting sites are arranged between the intron and the terminator; and all restriction enzyme cutting sites of other positions in the constructed plant binary expression vector are different from the restriction enzyme cutting sites of both sides of the intron. The constructed plant binary expression vector in the present invention can be used for plant transgenic overexpression studies and RNAi studies; the plant binary expression vector has an EGFP reporter gene, so detection for transgenetic seedlings can be easily performed; the plant binary expression vector has a kanamycin resistance gene which is relatively safe, so efficiently screening and production applications can be performed.
Description
Technical field
The present invention relates to a kind of plant binary expression vector.
Background technology
Plant expression vector is to carry out in the transgenosis process plant, with exogenous gene transfered plant cell and be integrated in DNA medium on the plant chromosome.Most widely used on plant genetic engineering is binary expression vector, contains the T-DNA sequence in agrobacterium tumefaciens source, can realize the integration of foreign gene and plant chromosome.At present, existing a series of plant expression vector is applied, as the pCambia serial carrier.These binary expression vectors generally contain marker gene, reporter gene and multiple clone site.But employed reporter gene mostly is GUS greatly.Relative GUS, the detection of reporter gene GFP is easier, only needs fluorescent microscope or excitation light source, need not reaction substrate or other cofactor material; GFP need not pre-treatment when detecting, and can carry out somatoscopy.Marker gene commonly used on the plant expression vector is neomycin phosphotransferase (neomycin phosphotransfe-rase, NPT II) gene, streptomycin phosphotransferase (streptomycin phosphotransferase, SPT) gene, hygromix phosphotransferase (hygromycin phosphotransferase, HPT) gene and anti-herbicide gene etc.Comparatively speaking, the NPTII gene of kalamycin resistance uses kantlex to screen, and is relatively safe a kind of marker gene.
RNA interference technique (RNA interference, RNAi) be a kind of specific gene interrupter technique that development in recent years is got up, be with double-stranded RNA (double strained RNA, dsRNA) cause a kind of cell response process that the specific target gene mRNA is degraded in the transfered cell, it is gene silencing (PTGS) phenomenon after a kind of the transcribing.In plant, the main method that triggers RNAi be make up can be transcribed into hairpin RNA (hairpin RNA, plant expression vector hpRNA) transforms plant and produces RNAi, therefore making up efficiently, the RNAi expression vector is the key of utilizing transgenosis RNAi.Studies show that: inserting the reorganization binary vector of reverse repeated fragment in the promotor downstream, is the basic structure that produces RNAi.(its reticent efficient is higher for intron-contain-ing hairpin RNA, the ihpRNA) plant of structure representation dsRNA and with the hairpin RNA that contains intron.By making up and optimizing various RNAi carriers, can make things convenient for the RNA of plant to interfere research greatly.
Summary of the invention
The purpose of this invention is to provide a kind of plant binary expression vector.
Plant binary expression vector provided by the invention is to obtain as follows: make up fluorescent protein report gene expression cassette and exogenous gene expression box in the carrier pBI121 that sets out; Described exogenous gene expression box is connected in sequence by promotor, intron and terminator, has several restriction enzyme sites between described promotor and the described intron, has several restriction enzyme sites between described intron and the described terminator; The restriction enzyme site of the restriction enzyme site of other position and described intron both sides is all inequality in the plant binary expression vector that structure obtains.
In the above-mentioned plant binary expression vector, several restriction enzyme sites between described promotor and described intron are: XbaI, Age I, Kpn I, Avr II and BamH I, several restriction enzyme sites between described intron and the described terminator are: EcoR I, Sac I, Sal I and SnaB I.
In above-mentioned arbitrary described plant binary expression vector, the nucleotide sequence of described exogenous gene expression box is shown in sequence 4.
In above-mentioned arbitrary described plant binary expression vector, the method of the expression cassette of described structure fluorescent protein report gene obtains the expression cassette of described fluorescent protein report gene for replace the gus gene in the gus gene expression cassette among the described carrier pBI121 that sets out with fluorescent protein report gene.
In above-mentioned arbitrary described plant binary expression vector, described fluorescent protein report gene be the EGFP reporter gene.
In above-mentioned arbitrary described plant binary expression vector, the expression direction of described exogenous gene expression box is opposite with the expression direction of the expression cassette of described fluorescent protein report gene.
Above-mentioned arbitrary described plant binary expression vector also belongs to protection scope of the present invention in the application that goal gene is imported in the plant that sets out.
Application in the goal gene afunction of above-mentioned arbitrary described plant binary expression vector in making the plant that sets out also belongs to protection scope of the present invention.
In above-mentioned arbitrary described application, described goal gene afunction in the plant that sets out disturb to be realized by RNA.
In above-mentioned arbitrary described application, the described plant that sets out is dicotyledons or monocotyledons; Described dicotyledons is specially tobacco or cotton; Described monocotyledons is specially onion.
The constructed plant binary expression vector of the present invention can be used for plant transgene and crosses expression study and RNAi research, TUB intron gene on the described carrier can be replaced to goal gene, be transformed into the plant expression vector that contains goal gene, also can on the multiple clone site of TUB intron gene fragment both sides, add forward and reverse fragment of target gene fragment respectively, be transformed into purpose RNAi carrier.Described plant binary expression vector has the GFP reporter gene, can carry out the detection of transfer-gen plant easily; Described plant binary expression vector has kalamycin resistance gene, and kalamycin resistance gene is safer, the application that can screen efficiently and produce.
Description of drawings
Fig. 1 is the restriction enzyme site checking result schematic diagram of carrier pCRI1210.
Fig. 2 is the collection of illustrative plates synoptic diagram of carrier pCRI1210.
Fig. 3 is plasmid vector pCRI1210 and pBI121 double digestion result schematic diagram.
Fig. 4 is that the specific detection primer of gus gene carries out the result schematic diagram that PCR detects amplification.
Fig. 5 is the double digestion detected result synoptic diagram of gus gene.
Fig. 6 is the result schematic diagram that the transient expression examining report gene GFP of carrier in onion epidermis expresses.
Fig. 7 is that the transient expression in cotton healing tissue of carrier detects the result schematic diagram that GUS expresses.
Fig. 8 transforms v tobacco young shoot GUS coloration result synoptic diagram.
Fig. 9 is transformation of tobacco blade GUS coloration result synoptic diagram.
Figure 10 is transformation of tobacco root GUS coloration result synoptic diagram.
Figure 11 is the expression of results synoptic diagram with converting cotton callus examining report gene EGFP.
Figure 12 is transformation of tobacco gus gene group DNA specific detection primer PCR detected result synoptic diagram.
Figure 13 transforms and non-transformation of tobacco growth of seedling state comparison diagram.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, makes up the interference expression cassette
Be template with the pBI121 carrier (GenBank:AF485783.1) that contains the CaMV 35S promoter, having obtained two ends with primer 35S1/35S2 and 35S1/35S3 by the twice PCR amplification has respectively added the CaMV35S promoter fragment of restriction enzyme site (its nucleotide sequence is shown in sequence in the sequence table 1, the restriction enzyme site that 5 ' end adds is Hind III, SmaI and XmaI, the restriction enzyme site that 3 ' end adds is BglII, KpnI, XbaI and XhoI), and (this carrier is available from Dalian TAKARA company to be connected to pMD19-T simple carrier, catalog number is: D104A), obtain intermediate carrier pMD19T-35S.Primer sequence is as follows:
35S1：5’-AAgCTTCCCgggTATTggCTAgAgCAgCTT-3’，
35S2：5’-TACCggTgATCTAgAAgCTCgAgAgAgATAgAT-3’，
35S3：5’-CCgAgATCTATAggTACCTgACCggTgATCTAgAAgC-3’。
Be template with the pCADS1341 carrier that contains CaMV 35SpolyA terminator, having obtained two ends with primer 35SpolyA1/35SpolyA2 and 35SpolyA1/35SpolyA3 by the twice PCR amplification has added the CaMV 35SpolyA terminator of restriction enzyme site (its nucleotide sequence is shown in sequence in the sequence table 2, the restriction enzyme site that 5 ' end adds is SacI, Sal I and SnaBI, the restriction enzyme site that 3 ' end adds is BglII) and be connected on the pMD19-Tsimple carrier, intermediate carrier pMD19T-polyA obtained.Primer sequence is as follows:
35SpolyA1：5’-TTgCTAgATCTCCgTACTgAATTAACgCCgAAT-3’，
35SpolyA2：5’-gTCgACTCTACgTATCTgTCgATCgACAAgCT-3’，
35SpolyA3：5’-ATgAgCTCTCTTgTCgACTCTACgTATCTgT-3’。
From the genomic dna of upland cotton, obtain TUB intron intron fragment with primer AF1/AF3 by pcr amplification, (its nucleotide sequence is shown in sequence in the sequence table 3 to add restriction enzyme site with primer AF2/AF4 at TUB intron fragment two ends by pcr amplification, the restriction enzyme site that 5 ' end adds is KpnI, AvrII and BamHI, the restriction enzyme site that 3 ' end adds is BglII, SacI and EcoRI), T/A is cloned among the pMD19-T simple, obtains intermediate carrier pMD19T-AF.Primer sequence is as follows:
AF1:5 '-CCTAggTATggATCCACTCCAAgTTgTAAgTAT-3 ' and
AF3：5’-gAgCTCACTgAATTCTgAACATCAAACATTACA-3’；
AF2:5 '-TAggTACCAATTACCTAggTTATggATCCACT-3 ' and
AF4：5’-AgATCTTgAgAgCTCACTgAATTCTgAACAT-3’。
Kpn I/BglII is the above-mentioned intermediate carrier pMD19T-35S of double digestion and pMD19T-AF respectively, reclaim the fragment of 3.5kb and 590bp respectively, the intermediate carrier that obtains after the connection carries out Sac I/BglII double digestion again, reclaim the 4.1kb fragment, the fragment that reclaims is connected with the fragment of the 240bp that Sac I/BglII double digestion carrier pMD19T-polyA obtains, obtains containing the intermediate carrier pMD19T-35SAP that interferes expression cassette.Carrier pMD19T-35SAP is carried out sequence verification, the result contains the interference expression cassette between the HindIII of pMD19-T and BglII restriction enzyme site, interfere the expression cassette structure as follows: to be followed successively by to the downstream from the upstream: CaMV 35S promoter (sequence 1 in the sequence table), TUB intron intron (sequence 3 in the sequence table) and CaMV 35SpolyA terminator (sequence 2 in the sequence table), and contain multiple clone site at TUB intron intron two ends, 5 ' end at TUB intron has 5 restriction enzyme sites: Xba I, Age I, Kpn I, AvrII, BanH I has 4 restriction enzyme sites at 3 ' end: EcoR I, Sac I, Sal I, SnaB I.
Two, modify the pBI121 carrier
With Sac I/BamH I double digestion pBI121 carrier, the carrier segments that obtains and reporter gene EGFP (GenBank:AF323988.1, its sequence is seen the sequence 5 in the sequence table) connect, and obtain the pBI121-EGFP expression vector.The pBI121-EGFP carrier is put down the endization back and is connected with EcoR I single endonuclease digestion, removes the EcoR I restriction enzyme site on the carrier.With the method same with removing EcoR I restriction enzyme site, remove Sac I, BamH I and Xba I restriction enzyme site on the pBI121-EGFP carrier respectively, obtain intermediate carrier pBI121-GFPQ.EcoR I, Sac I, BamH I and Xba I restriction enzyme site are arranged in the both sides of pBI121 carrier gus gene.
Checking: by Sac I/BamH I double digestion pBI121-EGFP expression vector, agarose gel electrophoresis detects, and can access the band of about 720bp, illustrates that EGFP correctly is connected in the pBI121-EGFP expression vector.
Adopt Sac I/BglII double digestion plasmid, do not do contrast and verify that pBI121 goes up the removal effect of corresponding restriction enzyme site to remove plasmid before the restriction enzyme site.Owing on the pBI121 carrier 2 Bgl II restriction enzyme sites are arranged, and 4 restriction enzyme sites removing are all between these 2 Bgl II sites.After if restriction enzyme site is removed, double digestion can only cut 2 Bgl II sites, obtains two bands of 5.6k and 8.0k.If restriction enzyme site is not removed, then can cut 3 bands.According to the checking result, EcoR I, Sac I, BamH I and four restriction enzyme sites of Xba I are all removed.
Three, make up the pCRI1210 carrier
With HindIII single endonuclease digestion carrier pBI121-GFPQ, reclaim carrier segments, put down the reaction of endization reaction and dephosphorylation; With HindIII/BglII double digestion carrier pMD19T-35SAP, reclaim the 1.7kb fragment, put down the endization reaction after, be connected with the carrier segments that reclaims behind the above-mentioned single endonuclease digestion carrier pBI121-GFPQ, obtain binary vector pCRI1210 (Fig. 2).Utilize the multiple clone site of carrier pCRI1210 both sides to carry out enzyme and cut checking and sequence verification.
Enzyme is cut and is combined as A:Avr II/EcoR I; B:Age I/EcoR I; C:Kpn I/Sal I; D:Xba I/Sac I; E:SnaB I/BamH I, enzyme is cut effect and is seen that (Fig. 1: swimming lane A is that the enzyme of Avr II/EcoR I is cut the checking result to Fig. 1, B is that Age I/EcoR I enzyme is cut the checking result, C Kpn I/Sal I enzyme is cut the checking result, D Xba I/Sac enzyme is cut the checking result, E SnaB I/BamH I enzyme is cut the checking result), each cuts the endonuclease bamhi that combination has all obtained 550bp to enzyme, show in the carrier of structure and only about intron, have outside the above polyclone restriction enzyme site, all the other positions do not have above polyclone restriction enzyme site, illustrate that this carrier has obtained correct connection, the multiple clone site on it also is available.And the sequence of sending order-checking company, result to interfere expression cassette is shown in the sequence 4, verifies that further this expression vector is correct.
The functional verification of embodiment 2, plant binary expression vector
One, the structure of expression vector pCRI1210-GUS
With the gus gene fragment among the BamHI/SacI double digestion plasmid pBI121 and the TUB intron part among the plasmid pCRI1210, enzyme is cut product and is carried out electrophoresis, (Fig. 3: No. 1 swimming lane is MakerIII to electrophoresis result as shown in Figure 3, the BamHI/SacI double digestion collection of illustrative plates that the BamHI/SacI double digestion collection of illustrative plates that No. 2 swimming lanes are plasmid pBI121, No. 3 swimming lanes are plasmid pCRI1210.), at about 1.9kb and 15kb place amplified band has appearred respectively.The 1.9kb that is recovered to and two portions dna fragmentation of 15kb are coupled together, obtain containing the carrier pCRI1210-GUS of GUS and GFP gene.Wherein, the resistance screening gene of this carrier is kalamycin resistance gene.
Join in the centrifuge tube that the bacillus coli DH 5 alpha competent cell (available from Tiangen company) that has just melted is housed connecting product pCRI 1210-GUS, flick mixing, ice bath 30 minutes; This centrifuge tube is placed 42 ℃ of water-bath heat shocks 90 seconds, after the taking-up ice bath 2-3 minute immediately, during do not rock centrifuge tube; In centrifuge tube, add the LB liquid nutrient medium that 250-500ul does not contain kantlex, 150rpm, 37 ℃ of concussions were cultivated 45 minutes; With the bacterium liquid mixing in the centrifuge tube, draw 100ul bacterium liquid and join on the LB solid medium that contains kantlex, gently bacterium liquid is coated with out with aseptic elbow glass stick, treat the planar surface drying after, be inverted flat board; Cultivate after 12-16 hour for 37 ℃, picking list bacterium colony shook bacterium about 12 hours in the LB liquid nutrient medium that contains kantlex; Simultaneously, design the intestinal bacteria (positive control) of unconverted intestinal bacteria (blank) and conversion pBI121 carrier; Utilize TIANprep Mini Plasmid Kit (available from Tiangen company) to extract plasmid, with the specific detection primer of gus gene the plasmid that extracts is carried out the PCR detection, specificity GUS primer sequence is:
Upstream primer GUSF1:5 '-CTACACCACGCCGAACACCT-3 ' and
Downstream primer GUSR1:5 '-CACGCTTGGGTGGTTTTTGTC-3 '.
The PCR detected result shows, the intestinal bacteria of conversion pCRI1210-GUS carrier No. 1 and No. 2 mono-clonals amplified band occurred with the intestinal bacteria (positive control) that transform the pBI121 carrier, and (Fig. 4: No. 1 swimming lane is MakerIII at the 690bp place, No. 2 swimming lanes are unconverted intestinal bacteria (blanks), No. 3 swimming lanes are No. 1 mono-clonal, No. 4 swimming lanes are No. 2 mono-clonals, No. 5 swimming lanes show that for transforming the intestinal bacteria (positive control) of pBI121 carrier goal gene GUS successfully is building up among the carrier pCRI1210.Simultaneously, the plasmid that extracts is carried out enzyme cut detection, plasmid with the extraction of BamHI/SacI double digestion, carrying out agarose gel electrophoresis then detects, the result shows No. 1 and No. 2 mono-clonals amplified band occurred (Fig. 5: No. 1 swimming lane is MakerIII at the 1.9kb place, No. 2 swimming lanes are No. 1 mono-clonal, and No. 3 swimming lanes are No. 2 mono-clonals), show that the gus gene has been connected on the pCRI1210-GUS carrier.Deliver order-checking company after the detection again,, determine positive monoclonal, the corresponding bacterium colony of preserving is the purpose transformant.
Two, the functional verification of expression vector pCRI1210-GUS
(1) the transient expression analysis of expression vector
(1) the bacterium liquid of the positive monoclonal that above-mentioned steps one is obtained utilizes TIANprep Mini Plasmid Kit to extract the pCRI1210-GUS plasmid.
(2) the particle gun bombardment transforms
Utilize the particle gun conversion method that the pCRI1210-GUS plasmid is imported in cotton embryo callus and the onion epidermis.The cotton embryo callus oozes pre-cultivation 4h (interpolation 0.6molL for preceding 28 ℃ high in conversion
-1N.F,USP MANNITOL and 0.6molL
-1Sorbyl alcohol), adopt Bio-RadPDS 1000/He particle gun to bombard, can split film pressure is 1.1kPa.Cotton embryo callus after the particle gun bombardment is being carried out GUS dyeing behind 37 ℃ of dark cultivation 48h on the MS substratum; Onion epidermis fluorescence microscopy after cultivating 24h under 28.0 ℃ of low light levels on the MS substratum is observed.(Fig. 6: A is the onion epidermis fluorescent microscopic imaging picture that has bombarded carrier pCRI1210-GUS plasmid to the result as shown in Figure 6, B is the onion epidermis fluorescence microscopy picture that has bombarded carrier pBI121-GUS plasmid, a is the corresponding white light visual field picture in the A figure visual field, b is the corresponding white light visual field picture in the B figure visual field), as can be known from the results, reporter gene EGFP obtains stronger expression in carrier pCRI1210-GUS, prove that the EGFP reporter gene in the expression vector can have expression effect preferably.
Contrast as empty carrier with cotton embryo callus and the onion epidermis that transforms pCRI1210 simultaneously, with the cotton embryo callus of unconverted and onion epidermis as blank.The result: green fluorescent protein is not seen in the empty carrier contrast; Do not see green fluorescent protein in the blank.
(3) GUS of callus dyeing
The GUS dyeing procedure is: with callus be soaked in the GUS staining fluid (the 0.1mol sodium phosphate buffer, pH 7.0; 1gL
-1X-Gluc; 2%DMF; 0.2% polysorbas20) in, 37 ℃ of incubation 12h, rinsing in the GUS washing lotion again with 37 ℃ of decolourings of 95% ethanol, 8～10h, repeats after the rinsing once then, is stored in 75% ethanol room temperature storage.
Simultaneously with the cotton embryo callus that transforms pBI121 and onion epidermis as empty carrier contrast (being positive control), with the cotton embryo callus of unconverted and onion epidermis as blank (being negative control).
The result as shown in Figure 7, (Fig. 7: A is for transforming the embryo callus subculture of pCRI11210-GUS, B is for transforming the embryo callus subculture (positive control) of pBI121, and C is the embryo callus subculture (negative control) of unconverted any carrier), the result shows that the expression efficiency of GUS in the embryo callus subculture that transforms pCRI11210-GUS and the embryo callus subculture (positive control) that transforms pBI121 is suitable.
(2) contain the Agrobacterium of pCRI1210-GUS carrier to the conversion of tobacco
1, Agrobacterium-mediated Transformation
The preparation of competent cell: 1. use the single bacterium colony of inoculating needle streak culture Agrobacterium LBA4404 on YEB solid medium flat board; 2. picking list colony inoculation contains in the suitable antibiotic YEB liquid nutrient medium in 5ml, and 28 ℃, the 250rpm shaking culture is spent the night; 3. by 1: 25-50 is inoculated in 100ml and contains in the suitable antibiotic YEB liquid nutrient medium, continues to cultivate 4-6h; 4. with bacterium liquid ice bath 30min, during constantly shake; 5. change 2 50ml centrifuge tubes over to, resuspended precipitation, 4 ℃, 4000rpm, 10min; 6. remove supernatant, add the 10-15ml sterile purified water, resuspended precipitation, 4 ℃, 4000rpm, 10min; 7. remove supernatant, add 10ml 10% glycerine of sterilizing, resuspended precipitation, 4 ℃, 4000rpm, 10min; 8. remove supernatant, add 10ml 10% glycerine of sterilizing, resuspended precipitation, 4 ℃, 4000rpm, 10min; 9. remove supernatant, with the resuspended precipitation of glycerine that refluxes; 10. use the packing of 0.5ml centrifuge tube, every pipe 20 μ l ,-70 ℃ of preservations are standby.
In Agrobacterium LB4404 competent cell, add the plasmid 10ul that from positive monoclonal, extracts in the step 2 (1), placed on ice 30 minutes; Immersed in the liquid nitrogen 5 minutes, and took out 37 ℃ of water-baths 5 minutes rapidly, ice bath is 2 minutes then; Add 500ul YEB liquid nutrient medium, 28 ℃, the 175rpm concussion was cultivated 3-5 hour; Utilize aseptic elbow glass stick bacterium liquid to be uniformly coated on the YEB solid medium flat board that contains Streptomycin sulphate, Rifampin and kantlex, 28 ℃ of cultivations are until growing single bacterium colony; Picking list bacterium colony carries out PCR and detects, and target stripe all appears in each swimming lane.The single bacterium colony of the positive is shaken bacterium spend the night, be cultured to OD600=0.3-0.6 and be used for infecting.
2, contain the Agrobacterium of pCRI1210-GUS carrier to the conversion of tobacco
The Agrobacterium that contains the pCRI1210-GUS carrier that obtains with above-mentioned steps 1 infects that (be cut into 0.5 * 0.5cm) 3 minute, (lucifuge was cultivated 48 hours for 28 ℃ altogether among MS minimum medium+6-BA (2mg/L)+IAA (0.2mg/L) to be moved into common substratum with the tobacco leaf after the mercuric chloride sterilization.The tobacco leaf that designs unconverted any carrier is blank, transform the positive contrast of tobacco leaf of pBI121 carrier and transform contrasting for empty carrier of pCRI1210.Blade is changed in the young shoot propagation solid medium (MS minimum medium+6-BA (2mg/L)+IAA (0.2mg/L)+Kan (100mg/L)+CB (500mg/L)), place 28 ℃ of growth cabinets to induce the young shoot differentiation.After ten days, young shoot appears.
Resistance screening: add kantlex (100mg/L) and screen positive young shoot in young shoot propagation solid medium, positive young shoot growth is normal, is peak green, and it is slow that unconverted successful young shoot then obviously breaks up, and is shallow white.
EGFP screening: tear and get transgene tobacco young shoot blade and make the water load, directly under fluorescent microscope, observe EGFP fluorescence, microscope model: Axioskop 40 fluorescent microscope (Zeiss, Germany), excitation wavelength (Excitation Wavelength:480 ± 20nm), emission wavelength (Emission Wavelength): 510 ± 20nm utilizes CCD to carry out the collection of image.What vein presented bright green fluorescence is the transgenic positive plant.
Add up positive transformation efficiency.The result: change 213 of the young shoots that the normal and vein of growth in the young shoot of pCRI1210-GUS carrier presents bright green fluorescence, 19 of undesired and non-blooming young shoots, positive young shoot accounts for 91.8% of total young shoot; The normal young shoot of growth is 241 in the young shoot of commentaries on classics positive control carrier pBI121, and 20 of undesired young shoots, positive transformation efficiency are 92.3%; Normal young shoot is 235 in the young shoot of commentaries on classics empty carrier pCRI1210, and 23 of undesired young shoots, positive transformation efficiency are 91.1%.The positive transformation efficiency of three kinds of carriers is suitable, all reaches more than 90%.
The Gus dye liquor is put in the young shoot cutting-out of the resistance screening positive dyeed, 37 ℃ of heat insulating culture are spent the night the back with the decolouring of 70% alcohol, organize to present the navy blue GUS of the being detection positive.The result shows, the tobacco young shoot (positive control) that the tobacco young shoot that the pCRI1210-GUS carrier transforms and pBI121 carrier transform all presents mazarine, and the tobacco young shoot (blank) that transforms and the tobacco young shoot that the transforms pCRI1210 translucent (Fig. 8: the tobacco young shoot (blank) that A Guan Weiwei transforms that becomes colorless, the tobacco young shoot (positive control) that B and C transform for the pBI121 carrier, the tobacco young shoot that D and E transform for the pCRI1210-GUS carrier), the CaMV 35S in the presentation of results pCRI1210-GUS carrier can normally start its downstream gus gene normal expression in the tobacco young shoot.
15 days one-period of the tobacco young shoot of above-mentioned cultivation are changed substratum, grow into seedling about 4cm until young shoot, seedling is gone to root media, get blade simultaneously and carry out Gus dyeing.The result shows, the tobacco leaf (positive control) that the tobacco leaf that the pCRI1210-GUS carrier transforms and pBI121 carrier transform all presents mazarine, (Fig. 9: A is the tobacco leaf that the pCRI1210-GUS carrier transforms and the tobacco leaf of the tobacco leaf that transforms (blank) and conversion pCRI1210 does not present mazarine, the tobacco leaf (positive control) that B transforms for the pBI121 carrier, the tobacco leaf (blank) of C for transforming; For improving Color, blade all cuts off the edge and punches with glass capillary), the CaMV 35S in the presentation of results pCRI1210-GUS carrier can normally start its downstream gus gene normal expression in tobacco leaf.
Above-mentioned tobacco seedling adventive root occurred after seven days, occurred a large amount of adventive root after 20 days, opened the bottle cap hardening 24 hours, and seedling is taken out back water flushing root, removed substratum, was transplanted in the basin.Simultaneously, getting the part root puts into the Gus dye liquor and dyes.The result shows, the tobacco root (positive control) that the tobacco root that the pCRI1210-GUS carrier transforms and pBI121 carrier transform all presents mazarine, (Figure 10: A is the tobacco root that the pCRI1210-GUS carrier transforms and the tobacco root of the tobacco root (blank) that transforms and conversion pCRI1210 does not present mazarine, the tobacco root (positive control) that B transforms for the pBI121 carrier, the tobacco root (blank) of C for transforming; For improving observing effect, adopt different background colours), the CaMV 35S in the presentation of results pCRI1210-GUS carrier can normally start its downstream gus gene normal expression in the tobacco root.
5, the molecular Biological Detection of pCRI1210-GUS carrier transformation of tobacco
Get the young leaflet tablet for the empty carrier contrast of the tobacco plant that the pCRI1210-GUS carrier transforms in the step 4, the tobacco plant (positive control) that the pBI121 carrier transforms, the tobacco plant (blank) that transforms and conversion pCRI 1210 respectively, extract DNA, specific detection primer GUSF2 and the GUSR2 with gus gene carries out the PCR detection respectively.The GUS primer sequence is:
GUSF2:5 '-GAAAGCCGGGCAATTGCT-3 ' and
GUSR2：5’-CACATCACCACGCTTGGGT-3’。
Detected result shows that (Figure 12: the A swimming lane is MakerIII, the tobacco plant (blank) of B swimming lane for transforming, the C-E swimming lane is the different individual plants of the tobacco of pCRI1210-GUS carrier conversion, the F-G swimming lane is the different individual plants (positive control) of the tobacco of pBI121 carrier conversion), the tobacco (positive control) that the tobacco that the pCRI1210-GUS carrier transforms and pBI121 carrier transform amplified band all occurred at the 1100bp place, and the tobacco plant that transforms (blank) and transform pCRI1210 amplified band does not appear herein for empty carrier contrasts, this result shows that the carrier pCRI1210 at goal gene GUS place successfully is incorporated among the tobacco gene group DNA, the tobacco plant well-grown of the pCRI1210-GUS carrier conversion simultaneously (tobacco plant (blank) of Figure 13: A for transforming, the tobacco plant that B transforms for the pCRI1210-GUS carrier, the tobacco plant that C transforms for the pBI121 carrier).
(3) contain the Agrobacterium of RI1210-GUS carrier to the conversion of cotton aseptic seedling hypocotyl segment
Infected cotton aseptic seedling hypocotyl segment 3 minutes with the Agrobacterium that contains the RI1210-GUS carrier that step 3 in above-mentioned (two) obtains, be moved in the common substratum (MS minimum medium+6-BA (2mg/L)+IAA (0.2mg/L)) 28 ℃ and cultivate 48 hours (designing blank simultaneously) altogether.Hypocotyl is gone to dedifferentiation substratum (MSB substratum, IAA, KT, 2, each 0.1mg/L of 4-D, 25g/L glucose, 2.2g/L Gel pH=6.5) goes up dark the cultivation about 20 days, on Bechtop, extract a spot of callus with tweezers, make the water load, directly observe EGFP fluorescence under fluorescent microscope, excitation wavelength (Excitation Wavelength) is 480 ± 20nm, emission wavelength (Emission Wavelength) is 510 ± 20nm, utilizes CCD to carry out the collection of image.The result shows, compare with unconverted cotton healing tissue (blank), reporter gene EGFP obtains than strongly expressed (Figure 11: A is unconverted cotton healing tissue (blank), and B is for transforming the cotton healing tissue of pCRI1210-GUS carrier) in the cotton healing tissue of conversion pCRI1210-GUS carrier.
Claims (10)
1. a plant binary expression vector is to obtain as follows: make up fluorescent protein report gene expression cassette and exogenous gene expression box in the carrier pBI121 that sets out; Described exogenous gene expression box is connected in sequence by promotor, intron and terminator, has several restriction enzyme sites between described promotor and the described intron, has several restriction enzyme sites between described intron and the described terminator; The restriction enzyme site of the restriction enzyme site of other position and described intron both sides is all inequality in the plant binary expression vector that structure obtains.
2. plant binary expression vector according to claim 1, it is characterized in that: several restriction enzyme sites between described promotor and described intron are: Xba I, Age I, Kpn I, AvrII and BamH I, several restriction enzyme sites between described intron and the described terminator are: EcoR I, Sac I, Sal I and SnaB I.
3. plant binary expression vector according to claim 2, it is characterized in that: the nucleotide sequence of described exogenous gene expression box is shown in sequence 4.
4. plant binary expression vector according to claim 1 and 2, it is characterized in that: the method for the expression cassette of described structure fluorescent protein report gene obtains the expression cassette of described fluorescent protein report gene for replace the gus gene in the gus gene expression cassette among the described carrier pBI121 that sets out with fluorescent protein report gene.
5. plant binary expression vector according to claim 4 is characterized in that: described fluorescent protein report gene be the EGFP reporter gene.
6. plant binary expression vector according to claim 1 and 2, it is characterized in that: the expression direction of described exogenous gene expression box is opposite with the expression direction of the expression cassette of described fluorescent protein report gene.
7. the application of arbitrary described plant binary expression vector in plant that the goal gene importing is set out among the claim 1-6.
8. the application in the goal gene afunction of arbitrary described plant binary expression vector in making the plant that sets out among the claim 1-6.
9. application according to claim 8 is characterized in that: described goal gene afunction in the plant that sets out disturb to be realized by RNA.
10. according to claim 7,8 or 9 described application, it is characterized in that: the described plant that sets out is dicotyledons or monocotyledons; Described dicotyledons is specially tobacco or cotton; Described monocotyledons is specially onion.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104263750A (en) * | 2014-09-28 | 2015-01-07 | 江苏农林职业技术学院 | RNAi (ribonucleic acid interference) intermediate vector for inhibiting expression of target genes and preparation method of RNAi intermediate vector |
| CN104805119A (en) * | 2014-01-23 | 2015-07-29 | 杭州纽贝生物科技有限公司 | Novel endogenous gene expression substituting lentivirus vector and construction method thereof |
| CN105274101A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of leptinotarsa decemlineata, method for constructing RNAi expression vector and application thereof |
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| CN1844403A (en) * | 2006-04-13 | 2006-10-11 | 中国农业科学院植物保护研究所 | Plant RNAi binary expression vector with forward and reverse promoters |
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| CN1844403A (en) * | 2006-04-13 | 2006-10-11 | 中国农业科学院植物保护研究所 | Plant RNAi binary expression vector with forward and reverse promoters |
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| AN等: "AtNHX8, a member of the monovalent cation:proton antiporter-1 family in arabidopsis tha;iana, encodes a putative Li/H antiporter", 《THE PLANT JOURNAL》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805119A (en) * | 2014-01-23 | 2015-07-29 | 杭州纽贝生物科技有限公司 | Novel endogenous gene expression substituting lentivirus vector and construction method thereof |
| CN104263750A (en) * | 2014-09-28 | 2015-01-07 | 江苏农林职业技术学院 | RNAi (ribonucleic acid interference) intermediate vector for inhibiting expression of target genes and preparation method of RNAi intermediate vector |
| CN105274101A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of leptinotarsa decemlineata, method for constructing RNAi expression vector and application thereof |
| CN105274101B (en) * | 2015-11-04 | 2018-06-05 | 重庆大学 | The RNAi expression vector and its construction method of colorado potato bug TOR genes and application |
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| CN103205456B (en) | 2015-11-18 |
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