CN106434741A - Plant expression vector skeleton and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a plant expression vector skeleton. The plant expression vector skeleton is characterized in that: a deletion site 1, a terminator 1, a selection marker or reporter gene 1, a promoter 1, a promoter 2, a selection marker or reporter gene 2, a terminator 2, a deletion site 2, a promoter 3, a polyclone site and a terminator 3 are sequentially arranged between a right boundary and a left boundary of T-DNA. The vector skeleton can change the selection marker gene, the reporter gene, the promoter capable of controlling a target gene expression unit and a terminating sequence easily, is suitable for genetic transformation of various plants, and is relatively high in biosecurity.

Description

A kind of plant expression vector skeleton and its application

Technical field

The invention belongs to field of plant genetic is and in particular to a kind of plant expression vector skeleton.

Background technology

Plant genetic engineering can carry out genetic modification to plant cell, and then cultivates the new varieties with merit. Plant expression vector is the fundamental carrying out plant genetic engineering operation, the T-DNA section (transfer-DNA) being comprised Have and shift from Agrobacterium and be incorporated into the characteristic Plant Genome, this section generally comprises target gene expression unit and sieve Apply for accusing gene expression element, play very crucial effect during the genetic transformation of plant.

With the development of plant transgenic technology, the floristics that can carry out genetic transformation operation gets more and more, at present Conventional plant expression vector shows certain limitation.First, in genetic transformation operating process, different types of plant Thing is larger to selective agent sensitivity differences.For example, kalamycin is more common in the resistance screening of dicotyledon, and paddy rice, The monocot crops such as corn have certain resistance to kalamycin, are such as used kalamycin as selective agent, false positive easily Plant.Second, numerous studies show, the spatial and temporal expression characteristic of target gene, larger to genetically modified plants Phenotype.This requires The promoter of control targe gene expression can relatively easily be changed in plant expression vector.3rd, consumer is to transgenosis The security of commodity is increasingly paid attention to, and contains the DNA sequence dna unit of clone from virus, bacterium in conventional plant expression vector Part, easily causes the worry of consumer.4th, national correlation department has drafted the resistant gene register that can use with commercialization, Genetically modified plants with resistance screening gene beyond register will be difficult to commercialization and promote the use of.Therefore, build and be easy in molecule Level carries out operating the T-DNA modifying, and reduces the DNA beyond introducing target gene expression unit in Plant Genome simultaneously, There is important value.

Content of the invention

The purpose of the present invention provides a kind of new selection for building plant expression vector.

The technical scheme is that a kind of plant expression vector skeleton, the right margin of this T-DNA between left margin according to Secondary comprise following element：Delete site 1, terminator 1, selection markers or reporter gene 1, promoter 1, promoter 2, selection markers Or reporter gene 2, terminator 2, delete site 2, promoter 3, MCS, terminator 3.

Specifically, described deletion site 1 and deletion site 2 are loxp site.

Specifically, described promoter 1 and promoter 2 are pCaMV35S：：PGOS2 (promoter of corn GOS2 gene) Merge bidirectional promoter.

Specifically, described promoter 3 is promoter pOsHISTONE3 (pH3) of paddy rice H3 gene, cauliflower Mosaic virus CaMV35S promoter pCaMV35S, ubiquitin gene promoter pUBI, the promoter of fruit specific expression gene FBP7 PFBP7 or actin gene promotor pACTIN.

Specifically, described terminator is NOS, PolyA or HST.

Specifically, described selection markers or reporter gene 1 are hygromycin phosphotransferase gene HPT gene, neomycin Phosphoric acid transferase gene NPT II, double alanyl phosphorus resistant gene Bar, green fluorescence protein gene GFP, red fluorescent protein base Because of RFP or beta-glucuronidase gene GUSplus gene；Described selection markers or reporter gene 2 are HPT gene, NPT II, Bar, GFP, RFP or GUSplus gene；And selection markers or reporter gene 1 and selection markers or reporter gene 2 are not same Gene.

Specifically, the restriction enzyme site between right margin and terminator 3 is Pvu I, EcoR I.

Specifically, described MCS is Sma I, Sac I, Kpn I, Apa I, BamH I, Sal I, Pst I, Hpa I, Xba Ⅰ.

Specifically, the restriction enzyme site between promoter 3 and loxp site is Hind III.

Specifically, the restriction enzyme site between terminator 2 and selection markers or reporter gene 2 is Xho I.

Specifically, selection markers or reporter gene 2 and pCaMV35S：：PGOS2 merges the digestion position between bidirectional promoter Point is Xho I.

Specifically, pCaMV35S：：PGOS2 merges the digestion position between bidirectional promoter and selection markers or reporter gene 1 Point is Mlu I.

Specifically, the restriction enzyme site between selection markers or reporter gene 1 and terminator 1 is Mlu I.

Specifically, the restriction enzyme site between loxp site and left margin is Pvu I.

Wherein, target gene expression unit area is made up of promoter 3, MCS, terminator 3, selection markers or report Accuse gene expression units area by terminator 2, selection markers or reporter gene 2, pCaMV35S：：PGOS2 fusion bidirectional promoter, Selection markers or reporter gene 1, terminator 1 are constituted.The terminator 3 in target gene expression unit area can be by EcoR I and polyclonal Any Restriction Enzyme excision in site, and it is replaced by other terminators, including but not limited to NOS, PolyA, HST etc. have and turn The DNA fragmentation of record expiry feature；Promoter 3 by any Restriction Enzyme excision in Hind III and MCS, and can be changed For other promoters, the including but not limited to composing type such as pCaMV35S, pUBI, pFBP7, pACTIN or tissue specific expression opens Mover；MCS is used for inserting target DNA fragments, including but not limited to possess the gene of complete ORF frame, RNAi fragment, Antisense sequences etc..Reserved Hind III and EcoR I site can be used for excising target gene expression unit, and introduce other DNA pieces Section.

GUSplus the and HPT gene two ends of screening or reporter gene expression cellular zone have reserved Xho I, Mlu I digestion respectively Site, can be deleted by corresponding restriction enzyme single endonuclease digestion, and is replaced by other screenings or reporter gene, including but not limited to NPTII, Bar, GFP, RFP etc.；pCaMV35S：：PGOS2 merges bidirectional promoter by the high expression of composing type extensive in dicotyledon PCaMV35S promoter and pGOS2 (promoter of corn GOS2 gene, expression water that in monocotyledon, composing type height is expressed Flat higher) promoter merges and forms, and has two-way high expression characterization.Two loxp sites can be identified by Cre recombinase, is used for deleting Except screening and reporter gene expression unit, in order to reduce the introducing of non-targeted sequence in genetically modified plants genome as far as possible, design , the loxp site of the other end and Pvu I site adjacent with HindIII site near the loxp site in target gene expression unit area Adjacent.Two reserved Pvu I restriction enzyme sites can be used for cutting whole T-DNA region (not comprising left margin and right margin), and root Arrive other plasmid vector skeletons according to needing displacement.

Beneficial effects of the present invention：The readily replaceable riddled basins of carrier framework of the present invention, reporter gene, control targe The promoter of gene expression units and terminator sequence, are suitable to carry out genetic transformation to various plants, and have higher biology Security.

Brief description

Fig. 1, pD plant expression vector difference digestion with restriction enzyme electrophoretograms；M is Beijing ancient cooking vessel state prosperous biology skill Art Co., Ltd product Middle DNA Marker I.

The T-DNA plot structure of Fig. 2, pD plant expression vector；Restriction enzyme site 1 and 2,3 and 4,5 and 6,7 and 8 can be respectively used to Excision or replacing selection markers or reporter gene 1 or 2, and promoter 3, terminator sequence 3.Restriction enzyme site 1 and 2,3 and 4,5 and 6th, 7 and 8 every groups of enzymes be not limited to identical restriction enzyme site.Specific to pD carrier, restriction enzyme site 1,2,3,4,5,6,7,8 is respectively Mlu I, Mlu I, Xho I, Xho I, Hind III, Xba I, Sma I, EcoR I, reporter gene 1 and 2 is respectively HPT and GUSplus, opens Mover 3 is pH3, and terminator sequence 3 is HST.

Fig. 3, pD plant expression vector is using HPT as the genetic transformation to plants such as tobacco, corns during riddled basins Situation.

When Fig. 4, pD plant expression vector is using GUSplus as reporter gene, the histochemical stain situation of transformant.Left Figure is that pD carrier is used for tobacco genetic transformation, positive plant with compare blade GUS staining conditions, right figure is pD carrier for corn Genetic transformation, positive plant with compare callus GUS staining conditions.

Specific embodiment

Below in conjunction with accompanying drawing, the present invention is described in further detail.Unless otherwise stated, present example In reagent chemicals be commercially available, the equal reference of method《Molecular Cloning:A Laboratory guide》(Sambrook and Russell, 2001).

Embodiment 1, the structure of pD carrier

(1) amplification of element and assembling

From Maize genome, amplification obtains pGOS2 promoter, and introduces Hpa I and Xba I recognition site at two ends.Amplification Obtain NOS terminator 3, and introduce Spe I, Mlu I recognition site at 5 ' ends, introduce loxp, Pvu I site at 3 ' ends.Extension increasing sequence TA is cloned into pMD19-T carrier, after sequencing identification, pMD19-pGOS2 Xba I and Hind III double digestion, pGOS2 fragment even Enter the pMD19-NOS3 carrier with Spe I and Hind III double digestion, obtain pMD19-pGOS2::NOS3 plasmid.Design primer exists HPT gene two ends introduce Mlu I site, after the identification of TA cloning and sequencing, obtain the HPT gene piece with corresponding cohesive end with Mlu I digestion Section.PMD19-pGOS2 simultaneously::NOS3 dephosphorylation after Mlu I digestion, is converted by connecting, and obtains pMD19-pGOS2:: HPT::NOS3 recombinant plasmid.

Artificial synthesized pCaMV35S promoter, GUSplus gene and NOS terminator 2.Wherein in pCaMV35S promoter two End introduces Hind III, Sma I and Xba I site respectively, introduces Xho I site at GUSplus gene two ends, in NOS terminator 2 5 ' ends introduce SpeI and XhoI, and 3 ' ends introduce loxp, Hind III, EcoR I, Pvu I and Kpn I site.PCaMV35S promoter is used Hind III and Xba I double digestion, NOS terminator 2 Spe I and Kpn I double digestion, pMD19 carrier Hind III and Kpn I double digestion Reclaim carrier, three fragments connect conversion Escherichia coli, obtain recombinant plasmid pMD19-pCaMV35S::NOS2.pMD19- pCaMV35S::NOS2 XhoI single endonuclease digestion dephosphorylation, is connected with the GUSplus gene of XhoI single endonuclease digestion, converts large intestine bar Bacterium, screening obtains recombinant plasmid pMD19-pCaMV35S::GUSplus::NOS2.

pMD19-pGOS2::HPT::NOS3 reclaims fragment, pMD19-pCaMV35S with Hpa I and Kpn I digestion:: GUSplus::NOS2 is reclaimed with Sma I and Kpn I digestion, connects the recombinant plasmid pMD19- obtaining comprising two expression units pGOS2::HPT::NOS3-pCaMV35S::GUSplus::NOS2.This plasmid is partially digested with Pvu I, reclaims pGOS2:: HPT::NOS3-pCaMV35S::GUSplus::NOS2 fragment, pCAMBIA1305 plasmid is reclaimed with Pvu I digestion dephosphorylation and carries Body, obtains pCAMBIA1305-pGOS2 by connecting conversion::HPT::NOS3-pCaMV35S::GUSplus::NOS2 plasmid.

With oryza sativa genomic dna and pBI121 plasmid as template, design specific primer expands acquisition pH3 promoter respectively With NOS1 terminator.Wherein pH3 promoter two ends introduce Hind III and Xba I site respectively, and NOS1 terminator two ends introduce respectively MCS MCS and EcoR I site.PH3 promoter Hind III and Xba I digestion, NOS1 terminator Xba I and EcoR I Digestion, is connected with the pMD19 carrier of Hind III and EcoR I digestion, and connection product converts Escherichia coli, and screening obtains pMD19- pH3::NOS1 recombinant plasmid.

pCAMBIA1305-pGOS2::HPT::NOS3-pCaMV35S::GUSplus::NOS2 plasmid is with Hind III He EcoR I digestion, pMD19-pH3::NOS1 reclaims pH3 with Hind III and EcoR I digestion::NOS1 Expression element.Both turn in connection Change Escherichia coli, Screening and Identification recombinant plasmid is pD.

(2) pD carrier restriction enzyme digestion and electrophoresis

PD carrier uses Xho I, Hind III+Xba I, Hind III+EcoR more than I group enzyme to be incubated 15min at 37 DEG C respectively, and digestion is produced With 1% agarose gel electrophoresis 20min under 100V voltage, ethidium bromide staining is shown in Fig. 1 to thing.Digestion result shows permissible with Xho I Excision GUS screening-gene, can excise the promoter of target gene expression unit, with Hind III+EcoR I with Hind III+Xba I Promoter and the terminator sequence of target gene expression unit can be excised, this is with SEQ ID NO.1 nucleotide sequence information and Fig. 2 The T-DNA plot structure shown is consistent.

The replacing of the replacing of reporter gene or expression unit promoter in embodiment 2, pD carrier

Digestion has all been reserved at reporter gene in pD carrier, screening-gene, the promoter of expression unit and terminator two ends Site, can require to change other genes or element according to specifically used.So that GUSplus reporter gene to be replaced by RFP base Because example, pD carrier Xho I single endonuclease digestion, delete GUSplus reporter gene, reclaim carrier dephosphorylation standby.Set by primer Meter amplification RFP gene, two ends introduce Xho I restriction enzyme site.With Xho I digestion RFP fragment, and the pD carrier being reclaimed with Xho I digestion It is attached reacting.Connection product converts Escherichia coli, and picking single bacterium colony, with digestion and amplification checking recombinant plasmid.

Taking change the pH3 promoter of expression unit as a example, with Hind III and Xba I digestion pD carrier, remove pH3 promoter, Reclaim carrier DNA standby.CaMV35S promoter is expanded by design of primers, two ends introduce Hind III and Xba I digestion position respectively Point, digestion obtains the CaMV35S promoter fragment with Hind III and Xba I cohesive end.With T4DNA ligase by pD carrier segments and CaMV35S promoter fragment connects, and obtains the generic plant table for CaMV35S promoter for the pH3 promoter changing expression unit Reach carrier.

Embodiment 3, pD carrier make genetic transformation to tobacco, corn etc.

According to Bio-RAD MicroPulser instruction manual book, the pD plant expression vector of structure is divided using electric shocking method Zhuan Hua not agrobacterium tumefaciens lba4404.With the Agrobacterium LBA4404 of above-mentioned conversion, infect the acceptor materials such as tobacco, corn, ginseng Genetic transforming method according to report is operated, and screens hygromycin resistance transfer-gen plant.Fig. 3 shows pD carrier transformation of tobacco And corn, it is obtained in that resistant transformant with hygromycin as selective agent.

Embodiment 4, the GUS histochemical stain of transformant

The blade taking transgene tobacco makees GUS histochemical stain, have detected about 120 plants of hygromycin resistance plant, and GUS contaminates The color positive is more than 80%.GUS is positive to be also detected that to corn and rice conversion rataria and callus dyeing.These result tables Bright pD carrier being capable of successful conversion unifacial leaf and dicotyledon.Fig. 4 shows transformant GUS histochemical stain result, wherein Tobacco dyeing top is negative control, and the corn dyeing left side is negative control.

Claims (10)

1. a kind of plant expression vector skeleton it is characterised in that：The right margin of this T-DNA comprises as follows between left margin successively Element：Delete site 1, terminator 1, selection markers or reporter gene 1, promoter 1, promoter 2, selection markers or reporter gene 2, terminator 2, delete site 2, promoter 3, MCS, terminator 3.

2. plant expression vector skeleton as claimed in claim 1 it is characterised in that：Described deletion site 1 and deletion site 2 For loxp site.

3. plant expression vector skeleton as claimed in claim 1 or 2 it is characterised in that：Described promoter 1 and promoter 2 For pCaMV35S：：PGOS2 merges bidirectional promoter；Described promoter 3 be pH3, pCaMV35S, pUBI, pFBP7 or pACTIN.

4. the plant expression vector skeleton as described in any one of claims 1 to 3 it is characterised in that：Described terminator is NOS, PolyA or HST.

5. the plant expression vector skeleton as described in any one of Claims 1 to 4 it is characterised in that：Described selection markers or Reporter gene 1 is HPT gene, NPT II, Bar, GFP, RFP or GUSplus gene；Described selection markers or reporter gene 2 For HPT gene, NPT II, Bar, GFP, RFP or GUSplus gene, and selection markers or reporter gene 1 and selection markers or report Accusing gene 2 is not same gene.

6. the plant expression vector skeleton as described in any one of Claims 1 to 5 it is characterised in that：Right margin and terminator 3 it Between restriction enzyme site be Pvu I, EcoR I.

7. the plant expression vector skeleton as described in any one of claim 1～6 it is characterised in that：Described MCS For Sma I, Sac I, Kpn I, Apa I, BamH I, Sal I, Pst I, Hpa I, Xba I.

8. the plant expression vector skeleton as described in any one of claim 1～7 it is characterised in that：Promoter 3 and loxp site Between restriction enzyme site be Hind III.

9. the plant expression vector skeleton as described in any one of claim 1～8 it is characterised in that：Terminator 2 and selection markers Or the restriction enzyme site between reporter gene 2 is Xho I.

10. the plant expression vector skeleton as described in any one of claim 1～9 it is characterised in that：Selection markers or report base Because of 2 and pCaMV35S：：The restriction enzyme site that pGOS2 merges between bidirectional promoter is Xho I.

Specifically, pCaMV35S：：The restriction enzyme site that pGOS2 merges between bidirectional promoter and selection markers or reporter gene 1 is Mlu Ⅰ.

Specifically, the restriction enzyme site between selection markers or reporter gene 1 and terminator 1 is Mlu I.

Specifically, the restriction enzyme site between loxp site and left margin is Pvu I.