CN102071217B - Plant polygene expression vector composition and application thereof - Google Patents

Abstract

The invention discloses a plant polygene expression vector composition and application thereof. The vector composition consists of a vector A and a vector B; the nucleotide sequence of the vector A comprises a T-DNA terminal sequence, a multiple cloning site sequence and a specific recombinant site sequence sequentially connected in series; and the nucleotide sequence of the vector B comprises a specific recombinant site sequence, a multiple cloning site sequence and a T-DNA terminal sequence sequentially connected in series. The plant polygene expression vector composition is reduced to minimum, and copy number, stability and conversion efficiency of plasmids are improved. Under the condition of ensuring that the conversion efficiency is not remarkably reduced, the smaller the plasmids are, the longer the fragment of the containable exogenous DNA is, so the vector composition is very favorable for experiment operation.

Description

A kind of plant polygene expression vector composition and application thereof

Technical field

The present invention relates to a kind of plant polygene expression vector composition and application thereof.

Background technology

In the past few decades, scientist has been developed be suitable in a large number the clone in vegetable cell, shifts and the carrier of expression alien gene.Have operability although these carriers are proved to be at phytology research and biological technical field, the Basic Design of these carriers all exists very large restricted, and seldom allows the clone and shift more than a goal gene (except screening-gene).Because to analyzing the gradually concern of complicated study metabolic pathways, and excavate polygenic character to the potentiality of plant biological engineering, causing needs some new Method and kit fors to transform simultaneously a plurality of genes.

As the main method of genetically engineered plant, agrobacterium vector in the past few decades in along with the difference of research purpose has been carried out continuous improvement and modification.Make a general survey of various Agrobacterium binary vectors, all have basic structure a: T-DNA and carrier framework.T-DNA is limited by border sequence (LB, RB), comprises multiple clone site (MCS), plant selectable marker gene, reporter gene and interested goal gene.Length is when the Agrobacterium infected cell generally at 12～24kb, cuts down the section of DNA sequence of transferring to the Plant Genome from Ti-plasmids.Carrier framework comprises plasmid replication functional unit (ColE1), the selectable marker gene of bacterium (Kan and Amp), plasmid is moved (the Toshiyuki Komori et al of subsidiary unit (OriT and bom) with function with some, 2007, PlantPhysiology, December 2007, Vol.145, pp.1155-1160).Be the interact process of developmental biology effect of bacterium and vegetable cell by the transfer of agriculture bacillus mediated T-DNA and integration, have bacterium in the prokaryotic organism concurrently in conjunction with the characteristics that shift and eukaryotic cell processing is modified.

The binary vector that makes up the earliest is pBin19 (Bevan M (1984) Binary Agrobacterium vectorsfor plant transformation.Nucleic Acids Res 12:8711-8721), has wherein produced pBI121 (Jefferson RA (1987) Assaying chimericgenes in plants:the GUS gene fusion system.Plant Mol Biol Rep 5:387-405) by a marker gene is incorporated into subsequently.As time goes on, also produced pPZP carrier (Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for planttransformation.Plant Mol Biol 25:989-994) and modified version pCAMBIA carrier series, the carrier of namely commonly using now, they generally have the food antiseptic scope of wide spectrum, in intestinal bacteria, keep high copy number, host strain there is higher compatibility, the very high features such as transformation efficiency.

Since the eighties in 20th century, the widespread use of the plant gene engineering technology take transgenic technology as representative has been opened up wide prospect as the genetic improvement of plant.Traditional transgenic plant overwhelming majority is that the genetic transformation by single goal gene comes the orderly improvement single traits to obtain.For a long time, cultivation has multiple fine quality, is the ultimate aim that scientist carries out genetic modification of plants such as high yield, high-quality, high resistance, many crop varieties anti-, balanced in nutrition, that possess characteristic useful in other productions always.And in the realization of this target, two levels are arranged again:

At first be the several genes that how to pass through to express on the pathways metabolism, strengthen the expression of a certain single high-quality proterties. because at occurring in nature, growing of organism is comprised of a rule pathways metabolism, and every pathways metabolism is by one by one genomic constitution, they are expressed according to the order of sequence, be connected successively, consist of the metabolism network of biology self. and specific to a certain proterties, can be directed to a certain section pathways metabolism, and then several key genes, obtain the proterties that we need by expressing or suppressing key gene.

Next is that the key gene of how to express simultaneously some specific trait obtains polymerization high yield is arranged, the plant of many good characters such as high-quality, i.e. so-called plant multiple goal molecule aggregation breeding.For example can pass through the plant salt tolerance pathways metabolism, the drought resisting pathways metabolism, the key gene clone in the disease-resistant pathways metabolism is integrated in the new plant, realizes whole raising of multiple goal of plant quality.

For this reason, multiple gene assembly and transformation technology just arise at the historic moment, and express in order to a plurality of goal gene are imported in the Plant Genome and in plant.The vector construction strategy that polygene transforms probably can be divided into the following aspects

1. based on the conversion of single-gene list carrier

Be exactly that each carrier carries a foreign gene (except the screening-gene), in order to reach the purpose that a plurality of gene integrations is entered organism, this utilizes diverse ways to operate with regard to many carriers of needs.For example: the sexual hybridization polymerization, repeat conversion method, cotransformation method, functional domain fusion method etc.

2. based on the conversion of polygene list carrier

A plurality of goal gene are structured on the carrier, only just several genes can be transformed in the genome that enters the host simultaneously by once transforming. also avoided simultaneously the sexual hybridization method, repeating the loaded down with trivial details time-consuming disadvantages such as conversion method, can also overcome cotransformation method integration mode complexity and the uncertain integration of offspring and the shortcoming of separating. the method that transforms based on polygene list carrier also can be divided into two aspects: protein level and gene level.

Protein level: encoding sequence (coding sequences, CDSs) the assembling fusion of several albumen is become a special expression cassette, and then in cell, can only produce a transcription unit.In the process of translation, the polyprotein polymer of coding can by special proteolytic enzyme processing and cutting, finally produce independently albumen.

Genomic level: will need the two ends of the foreign gene assembled to put respectively the controlling elements such as promotor, terminator, enhanser, and form one by one independently open reading frame, then they being together in series to transform enters in the plant materials.Each gene is transcribed respectively in plant materials inside, translates into separately product and effect.This method can also can also utilize the polycistron strategy to carry out the assembling of gene, and the goal gene that needs are expressed is connected in turn under the same promotor, expresses in turn in plant materials.

At present, when utilizing existing plant expression vector to carry out vector construction, the method that just gene or dna fragmentation need to be cut connection according to the multiple clone site on the carrier by traditional enzyme is assembled into final expression vector.Because multiple clone site only has one on the final expression vector, so the restriction enzyme site that can select has quantitative restriction, especially in the time will connecting simultaneously a plurality of genes or dna fragmentation, along with increasing of fragment, the restriction enzyme site that can select is just few.

Summary of the invention

The purpose of this invention is to provide a kind of convenient, fast structure and can express the plant expression vector combination of a plurality of genes.

Plant polygene expression vector composition of the present invention is comprised of carrier A and carrier B; Described carrier A and carrier B must exist simultaneously, are integrated into as a whole.The nucleotide sequence of described carrier A comprises successively a T-DNA end sequence, multiple clone site sequence and the special recombination site sequence of series winding; The nucleotide sequence of described carrier B comprises successively special recombination site sequence, multiple clone site sequence and a T-DNA end sequence of series winding; Wherein, when the T-DNA end sequence in the carrier A was T-DNA LB end sequence, the T-DNA end sequence in the described carrier B was T-DNA RB end sequence; When the T-DNA end sequence in the carrier A was T-DNA RB end sequence, the T-DNA end sequence in the described carrier B was T-DNA LB end sequence.

Described special recombination site is preferably the Loxp site; Described Loxp site sequence is sequence 3 in the sequence table.

Described special recombination site can be but be not limited to modify Loxp, modifies FRT, modifies R, modifies attB, modifies attP.

Described T-DNA LB end sequence is sequence 1 in the sequence table; Described T-DNA RB end sequence is sequence 2 in the sequence table.

Multiple clone site sequence in the described carrier A is sequence 4 in the sequence table; Multiple clone site sequence in the described carrier B is sequence 5 in the sequence table.

On described carrier A and the carrier B also respectively with the bacterial resistance gene; Described carrier A is not identical with bacterial resistance gene on the carrier B.

Carrier A or carrier B can form suicide plasmid with the replicon that plays the screening effect, such as replicon R6K γ, in order to carry out the feminine gender screening.

Described carrier A is inserted among the pUNI20 for a T-DNA end sequence, multiple clone site sequence and the special recombination site sequence of will be described contacting successively, and the recombinant vectors that obtains with the bacterial resistance gene among the needed bacterial resistance Gene Replacement pUNI20.

Described carrier B is for to be inserted into described special recombination site sequence between the SacII and EcoRI restriction enzyme site of pCambia1302, and the recombinant vectors that the excision of GFP expression casette is obtained.Also can keep the GFP expression casette, namely utilize existing 35S promoter expression cassette, can before the GFP gene, insert easily other foreign genes by restriction enzyme NcoI and BglII, make things convenient for the later stage Vector construction.

On the described carrier A also with the foliage filter screening gene, such as kantlex, Totomycin, antiweeds etc. are so that conversion foliage filter screening gene as required.

Plant polygene expression vector composition of the present invention, gene or dna fragmentation can be connected on the less plant polygene expression vector composition carrier of two self scales, then utilize the locus specificity restructuring of recombinase-mediated that two dna fragmentations (intermediate carrier) are assembled into a complete final expression vector.Even a same like this cover multiple clone site also can be utilized at intermediate carrier respectively, also can utilize the conveniently plant selectable marker gene of the final expression vector of conversion of intermediate carrier simultaneously.The size of plant polygene expression vector composition of the present invention is reduced to minimum, along with the reduction of plasmid DNA size, and the copy number of plasmid, stability and transformation efficiency all increase to some extent.Guaranteeing that plasmid is less under the not significantly reduced condition of transformation efficiency, the section of open ended foreign DNA is just longer, and the operation that is very beneficial for testing.Also proved this point by transgenic experiments, plasmid vector of the present invention can change at least four foreign genes in the plant simultaneously and reach stable expression.

The present invention is owing to the dna sequence dna on two restructuring elements is LB-MCS-Loxp and Loxp-MCS-RB successively, effect by recombinase, the plasmid at restructuring element place is integrated, dna sequence dna carries out new arranging, can form following relative position: LB-MCS-Loxp-MCS-RB, i.e. complete T-DNA fragment, and then be fit to carry out the genetic transformation of plant.Structure for the polygene conversion carrier, we can be connected to interested goal gene on the carrier by traditional molecular cloning method according to the multiple clone site on carrier A and the carrier B first, because this moment, self scale of carrier was less, conventional molecular cloning method is easily quick.Then carrier A and carrier B are carried out recombining reaction at the external recombinase that utilizes, by the recombination site generation recombination and integration of self, transform competent escherichia coli cell, carry out the screening of recombinant plasmid according to the selectable marker gene on carrier A and the carrier B, and then obtain required final expression vector.Carrier A also can select to contain the rear negative cloning vectors that screen of can recombinating such as replicon R6K γ, can greatly improve screening efficiency like this.A series of foliage filter screening expression casette will be made up on the carrier A, kalamycin resistance gene for example, hygromycin gene, (including but not limited to above screening-genes) such as antiweed resistant genes, can change easily according to the breeding needs like this plant screening mark gene of final expression vector, and need not again do, greatly accelerated the transgenosis process.

Description of drawings

Fig. 1: restructuring element pElementA and pElementB.By the left end (LB) of T-DNA, multiple clone site (MCS) and recombination site (Loxp) form the pElementA fragment successively.The pElementB fragment is successively by recombination site (Loxp), and the right-hand member (RB) of multiple clone site (MCS) and T-DNA forms.Wherein foreign gene is inserted on the multiple clone site.

Fig. 2: upper foliage filter screening gene kind on the carrier A.Kan: kalamycin resistance gene, HptII: hygromycin gene, Bar: antiweed resistant gene.

Fig. 3: the assembling flow path of final expression vector.Carrier A and carrier B transform reactant and enter intestinal bacteria after external effect through recombinase CRE, and screening obtains being integrated with the clone of the final expression vector of carrier A and carrier B.RES1 and RES2: different bacterial resistance genes.The two ends of LB and RB:T-DNA.R6K γ Ori: intestinal bacteria R6K γ replicon.Ori: intestinal bacteria replicon.Loxp: recombination site.MCS1 and MCS2: multiple clone site, both can be the same or different.

Fig. 4: carrier construction A schematic diagram.PMD18-T, pBluescript SK: plasmid vector.Amp: ampicillin resistance gene.Kan: kalamycin resistance gene.R6K γ Ori: intestinal bacteria R6K γ replicon.F1 ori:f1 replicon.MCS: multiple clone site.

Fig. 5: carrier construction B schematic diagram.PCambia1302: plant expression vector.PVS1: Agrobacterium replicon.PBR322 ori: intestinal bacteria replicon.Hyg: hygromycin gene.MCS: multiple clone site.GFP: green fluorescent protein.

Fig. 6: contain GFP, GUS, BADH, the final expression vector T-DNA of the plant area schematic of four foreign genes of BAR.

Fig. 7: the RT-PCR of transgene tobacco detects electrophorogram.Among Fig. 7, swimming lane 0 is molecular weight 1kb Marker, swimming lane 1 is the contrast of antiweed resistant gene fragment, swimming lane 2 is the contrast of betaine aldehyde dehydrogenase gene fragment, swimming lane 3 is the contrast of gus gene fragment, swimming lane 4 is the contrast of non-transgenic tobacco for GFP gene fragment contrast, swimming lane 5-8, and swimming lane 9-12 is for turning the pModularB-bar-badh-gfp-gus tobacco plant.

Fig. 8: utilize GUS histochemical staining method (A) and laser confocal microscope (B) that transfer-gen plant is carried out the expression analysis result.

Embodiment

Below in conjunction with concrete example content of the present invention is described, but the application that the invention is not restricted to provide below, following description is not construed as limiting claim of the present invention.

Method among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

" the Molecular Cloning:A Laboratory Manual (Third Edition) " that the genetically engineered basic fundamental that adopts in the present embodiment is all write from Joseph Sambrook and David W.Russell, some reagent product etc. carries out to specifications.

The structure of embodiment 1, plant polygene expression vector composition

The present invention considers time-consuming with it, effort with gene or dna fragmentation assembly unit to self scale on the final carrier of very large (approximately 10kb), not as first gene or dna fragmentation being connected on the less intermediate carrier of two self scales, then utilize the locus specificity restructuring of recombinase-mediated that two dna fragmentations (intermediate carrier) are assembled into a complete final expression vector.Even a same like this cover multiple clone site also can be utilized at intermediate carrier respectively, also can utilize the conveniently plant selectable marker gene of the final expression vector of conversion of intermediate carrier simultaneously.Therefore, plant polygene expression vector composition of the present invention is comprised of two expression vectors, carrier A and carrier B, and specific features has:

Both all have a special recombination site (such as Loxp)

Both have respectively a plant expression vector T-DNA terminal (LB or RB)

Both all have a multiple clone site (MCS)

Respectively with the bacterial resistance gene, but both are different on carrier A and the carrier B.

Carrier A or carrier B can be carried out the feminine gender screening with the replicon that plays the screening effect such as R6K γ etc.

Can be with different foliage filter screening genes (kantlex, Totomycin, antiweed etc., but be not limited to) on the carrier A, so that conversion foliage filter screening gene as required.

Building process is as described below:

One, the structure of carrier A

1, the acquisition of Amp resistant gene fragment

According to upper ampicillin resistance gene (Amp) the primers P1 of pMD18-T (Takara company) and P2 (invitrogen company), primer sequence is as follows:

P1:CG ACGCGTTTACCAATGCTTAATCAG, the mark underscore partly is restriction enzyme MluI site.

P2:CTA GCTAGCGACGAAAGGGCCTCGTGATAC, the mark underscore partly is restriction enzyme NheI site.

Take pMD18-T as template, take P1 and P2 as primer, carry out PCR reaction amplification, amplified fragments carries out sequence verification, showing that amplification obtains the Amp resistant gene fragment of 1078bp, and two ends are respectively MluI and NheI restriction enzyme site.

2, the acquisition of multiple clone site (MCS)

According to multiple clone site (MCS) primers P3 and P4 on pBluescript SK (Clotech company) carrier, primer sequence is as follows:

P3: GGTACCTggcaggata tattgtggtg taaaca GGGCCCCCCCTCGAGGTCG, the mark underscore partly be restriction enzyme Acc65I site, lowercase is that LB holds (in the sequence table shown in the sequence 1).

P4: TCGCGA

AATTAACCCTCACTAAAGG, the mark underscore partly is restriction enzyme NruI site, that mark mark of emphasis is recombination site Loxp.

Take pBluescript SK as template, take P3 and P4 as primer, carry out PCR reaction amplification, amplified fragments carries out sequence verification, showing that amplification obtains the MCS fragment of 205bp, and two ends are respectively Acc65I and NruI restriction enzyme site.This MCS fragment is by the terminal LB of T-DNA, pBluescript SK multiple clone site and the recombination site Loxp of series winding form (concrete structure pElementA as shown in Figure 1) successively.This MCS fragment has the fragment shown in the sequence 4 in the sequence table, is the terminal LB of T-DNA from 5 of sequence 4 ' end 7-32 position Nucleotide, is the sequence of pBluescript SK multiple clone site from 5 of sequence 4 ' end 33-134 position Nucleotide; Be recombination site Loxp from 5 of sequence 4 ' end 166-199 position Nucleotide.

3, the structure of carrier A

Carrier A comprises the element of structure shown in the pElementA among Fig. 1, the terminal LB of the T-DNA that namely contacts successively, pBluescript SK multiple clone site and recombination site Loxp.The final expression vector of the rear formation of carrier B restructuring that can need with insertion foreign gene behind carrier A insertion expressing gene and bacterial resistance gene, the foliage filter screening mark.Selection markers can be selected different genes as required with the bacterial resistance gene, we take the Amp resistant gene, select the antiweed marker gene this technology to be described as example.We are with the basic framework of pUNI20 as carrier, because this carrier has R6K γ replicon, in the restructuring screening in later stage, can play the effect of negative screening.Concrete grammar is as follows:

The Amp resistant gene fragment that step 1 is obtained is behind MluI and NheI double digestion, be connected Arabidopsis Biological Resource Center (ABRC) with same with NheI double digestion pUNI20 with MluI) dna fragmentation that obtains is connected, transform intestinal bacteria competence DH5 α (Tiangen company), obtain recombinant vectors, this recombinant vectors is carried out sequence verification, order-checking is shown correct recombinant vectors called after pUNI20-Amp.

The MCS fragment that step 2 is obtained is behind Acc65I and NruI double digestion, reclaim purifying, be inserted between the Acc65I and NruI restriction enzyme site of carrier pUNI20-Amp, transform intestinal bacteria competence DH5 α (Tiagen company), obtain recombinant vectors, this recombinant vectors is carried out sequence verification, obtain correct recombinant vectors called after pModularA.PModularA contains the successively terminal LB of T-DNA, pBluescript SK multiple clone site and the recombination site Loxp (namely containing pElementA component structure as shown in Figure 1) of series winding.The pModularA concrete structure as shown in Figure 3 and Figure 4.

Clone's antiweed marker gene (BAR) expression cassette, enzyme is cut and is connected on the carrier pModularA, produces pModularA-bar.Concrete grammar is:

Design primer P5 and P6, sequence is P5: GGTACCGCTGAAAGCGACGTTGGATG, the mark underscore partly is restriction enzyme Acc65I site; P6: GGTACCTCCTGCTGAGCCTCGACATG, the mark underscore partly is restriction enzyme Acc65I site.Take pSATlA-ocsAocsP-bar-ocsT (available from ABRC) as template, carry out pcr amplification take P5 and P6 as primer, amplification obtains the fragment of 1683bp, show that through order-checking it is the 412nd to 2094 nucleotide sequences of 5 of DQ005457 ' end that this fragment has from GENBANK number, i.e. amplification obtains antiweed marker gene (BAR) expression cassette, after cutting, Acc65I restriction enzyme (NEB company) enzyme is connected to the Acc65I restriction enzyme point of carrier pModularA, through sequence verification, with the correct insertion of checking the recombinant vectors called after pModularA-bar of antiweed marker gene (BAR) expression cassette.

Two, the structure of carrier B

Carrier B comprises the element of structure shown in the pElementB among Fig. 1, namely comprises the successively terminal RB of recombination site Loxp, multiple clone site and T-DNA of series winding.In order to verify this carrier effect and to make things convenient for vector construction, we have selected pCambia1302 (available from www.cambia.org mechanism) as final carrier framework, because it has had a multiple clone site and has contained the GFP expression casette of 35S promoter, wherein can insert easily other foreign gene by NcoI and BglII or SpeI again before the GFP gene, this carries out follow-up Vector construction with regard to being beneficial to very much us.The concrete construction process of carrier B is as described below:

According to the sequence of plant expression vector pCambia1302 (www.cambia.org), because it has contained a complete multiple clone site (GAATTCGAGC TCGGTACCCG GGGATCCTCT AGAGTCGACC TGCAGGCATGCAAGCTT; Sequence 5 in the sequence table) and T-DNA right-hand member (RB) (gtaaacctaa gagaaaagag cgttta; Sequence 2 in the sequence table), just need the dna fragmentation that one of subclone contains recombination site Loxp to enter carrier replacement T-DNA left end (LB).Artificial-synthetic DNA's linker fragment (invitrogen company is synthetic), sequence is as described below: CCGCGG

GAATTC, the mark underscore partly is respectively restriction enzyme SacII site and EcoRI site, and that mark mark of emphasis is recombination site Loxp.These sequence two ends are respectively SacII and EcoRI restriction enzyme site, contain recombination site Loxp in the fragment, this fragment is connected between the SacII and EcoRI restriction enzyme site of plant expression vector pCambia1302, transform intestinal bacteria competence DH5 α, obtain recombinant vectors, recombinant vectors is carried out sequence verification, the carrier called after pModularB-gfp that checking is correct (owing to self contain a GFP expression cassette on the pCambia1302 carrier, so contained a foreign gene GFP on the carrier B).The GFP gene also can be replaced by easily other gene of interest or with this GFP expression cassette excision, obtain not contain the carrier B of foreign gene according to restriction enzyme site as required.PModularB-gfp concrete structure and building process are as shown in Figure 5.

The compliance test result of embodiment 2, plant polygene expression vector composition of the present invention

Plant polygene expression vector composition carrier A and the carrier B of embodiment 1 preparation can add respectively the exogenous gene expression box that need to change plant over to, and recombination and integration becomes the plant expression vector that can express a plurality of genes.Wherein, pModularB-gfp has carried a foreign gene GFP.

Take following method as example, the effect of plant polygene expression vector composition of the present invention is described.

1, in carrier A, inserts the exogenous gene expression box

1) contains the acquisition of the recombinant vectors of betaine-aldehyde dehydrogenase (BADH) expression casette

According to GENEBANK DQ497233, betaine-aldehyde dehydrogenase (BADH) gene in synthetic (invitrogen company) the plant prunella asiatica, these sequence two ends are respectively with XhoI and BamHI restriction enzyme digestion sites, and betaine-aldehyde dehydrogenase (BADH) gene order in the plant prunella asiatica of synthetic is shown in sequence 6.

Synthetic P7 and P8:P7:G TTAATTAATGAGATTTTTCAAATCAG, the mark underscore partly is restriction enzyme PacI site; P8: GGCGCGCC TCGATTTGGTGTATCGAG, the mark underscore partly is restriction enzyme AscI site, the part of mark mark of emphasis is restriction enzyme XhoI site.

Take pEarleyGate100 (Arabidopsis Biological Resource Center (ABRC)) as template, take P7 and P8 as primer, carry out pcr amplification, obtain the mas promoter fragment of 410bp, this fragment is connected among the carrier pMD18-T, obtains carrier pMD18-T-mas-pro.

Synthetic primer P9 and P10:P9: CTCGAGC

TCTTGGACTCCCATGTTGG, the mark underscore partly is restriction enzyme XhoI site, the part of mark mark of emphasis is restriction enzyme BamHI site.

P10: GGCGCGCCGATAATTTATTTGAAAATTC, the mark underscore partly is restriction enzyme AscI site.Take pEarleyGate100 (Arabidopsis Biological Resource Center (ABRC)) as template, take P9 and P10 as primer, carry out pcr amplification, obtain the mas terminator fragment of 275bp, this fragment is connected among the carrier pMD18-T, obtains pMD18-T-mas-ter.

Utilize XhoI and AscI double digestion the mas terminator fragment on the pMD18-T-mas-ter to be downcut between the XhoI and AscI restriction enzyme digestion sites that is connected on the pMD18-T-mas-pro, obtain containing the expression cassette of mas promotor and terminator, called after pMD18-T-mas ORF after order-checking shows correctly.Betaine-aldehyde dehydrogenase (BADH) gene in the above-mentioned synthetic plant prunella asiatica is utilized XhoI and BamHI double digestion, obtain containing the recombinant vectors of BADH expression cassette in being connected between the XhoI of pMD18-T-mas and the BamHI restriction enzyme site, this carrier contains successively mas promotor, BADH gene and the mas terminator fragment of series winding, will show correct carrier called after pMD18-T-mas pro-BADH-mas ter through order-checking.

2) in pModularA-bar, insert betaine-aldehyde dehydrogenase (BADH) expression casette

Synthetic primer: P11: GCGGCCGCTGAGATTTTTCAAATCAG, the mark underscore partly is restriction enzyme NotI site; P12:GATAATTTATTTGAAAATTC, the mark underscore partly is restriction enzyme SacI site.

Take pMD18-T-mas pro-BADH-mas ter as template, take P11 and P12 as primer, carry out pcr amplification, obtain the BADH expression cassette sequence of 2185bp, it is correct rear by NotI and SacI double digestion that enzyme is cut sequence verification, endonuclease bamhi is connected between the NotI and SacI restriction enzyme site of carrier pModularA-BAR, has obtained inserting the recombinant vectors of BADH expression cassette, will show correct recombinant vectors called after pModularA-BAR-BADH through order-checking.

2, in carrier B, insert the exogenous gene expression box

Because had a foreign gene GFP on the pModularB-GFP, this gene is expressed under common plant composition type expression promoter 35S effect.Simultaneously can insert easily other foreign gene by existing restriction enzyme NcoI and BglII or SpeI before the GFP gene, so adopt the pCambia1302 carrier to have certain superiority as pModularB Vector construction skeleton.Insert beta-Glucuronidase gene (GUS) at pModularB-GFP more herein.Concrete grammar is as described below:

1) contains the acquisition of the expression vector of beta-Glucuronidase gene (GUS) expression cassette

Synthetic primer P13 and P14:P13: GAATTCGATCATGAGCGGAGAATTAAGG, the mark underscore partly is restriction enzyme EcoRI site; P14: AAGCTT

AGATCCGGTGCAGATTATTTG, the mark underscore partly is restriction enzyme HindIII site, the part of mark mark of emphasis is restriction enzyme XhoI site.Take pBI121 (Arabidopsis BiologicalResource Center (ABRC)) as template, carry out pcr amplification take P13 and P14 as primer, obtain the no promoter fragment of 325bp, this fragment is connected among the carrier pMD18-T, contains the carrier pMD18-T-nos pro that obtains the no promoter fragment.

Synthetic primer P15 and P16:P15: CTCGAG

GATCGTTCAAACATTTGGC, the mark underscore partly is restriction enzyme XhoI site, the part of mark mark of emphasis is restriction enzyme XbaI site.P16: AAGCTTCCCGATCTAGTAACATAG, the mark underscore partly is restriction enzyme HindIII site.Take pBI121 (Arabidopsis Biological Resource Center (ABRC)) as template, take P15 and P16 as primer, carry out pcr amplification, obtain the no terminator fragment of 274bp, this fragment is connected among the carrier pMD18-T, obtains containing the carrier pMD18-T-nos ter of no terminator fragment.

Utilize XhoI and HindIII double digestion the no terminator fragment on the pMD18-T-nos ter to be downcut between the XhoI and HindIII restriction enzyme digestion sites that is connected on the pMD18-T-nos pro, obtain containing the expression cassette of no promotor and terminator, called after pMD18-T-nos-ORF after order-checking shows correctly.

Synthetic primer: P17: CTCGAGATGGTCCGTCCTGTAGAAAC, the mark underscore partly is restriction enzyme XhoI site; P18: TCTAGATCATTGTTTGCCTCCCTG, the mark underscore partly is restriction enzyme XbaI site.Take pENTR-gus (invitrogen company) as template, take P17 and P18 as primer, carry out pcr amplification, obtain the gus gene of 1824bp, it is correct rear by XhoI and XbaI double digestion that enzyme is cut sequence verification, endonuclease bamhi is connected between the XhoI and XbaI enzyme recognition site of carrier pMD18-T-nos-ORF, obtains containing the recombinant vectors of GUS expression cassette, the recombinant vectors called after pMD18-T-nos pro-GUS-nos ter that sequence verification is correct.

2) insert beta-Glucuronidase gene (GUS) expression cassette at pModularB-gfp

With pMD18-T-nos pro-GUS-nos ter by EcoRI and HindIII double digestion, obtain GUS expression cassette fragment, endonuclease bamhi is inserted between the EcoRI and HindIII enzyme recognition site of carrier pModularB-GFP, obtain containing the recombinant vectors that obtains beta-Glucuronidase gene (GUS) expression cassette and GFP expression cassette, order-checking is shown correct carrier called after pModularB-GFP-GUS.

3, utilize the CRE recombinase that two carriers are assembled into final expression vector

The schematic diagram of two carrier assembly units as shown in Figure 3.Concrete grammar is as described below:

Utilize the CRE recombinase that pModularA-BAR-BADH and pModularB-GFP-GUS are carried out recombining reaction, reaction system is 20 μ L, by 2 μ L, 10 * CRE recombinase damping fluid (NEB company), 0.5 μ L (0.3ug) pModularA-bar-badh, 0.5 μ L (0.3ug) pModularB-gfp-gus, 1 μ L CRE recombinase (20units, NEB company), 16 μ L water.

Place 37 ℃ to react half an hour reaction system, 70 ℃ of temperature are bathed and were made CRE recombinase inactivation in 10 minutes, then transform competent escherichia coli cell DH5 α, at the enterprising row filter of the substratum that contains penbritin, the positive colony that screening is obtained extracts plasmid and carries out that enzyme is cut and sequence verification, will the correct recombinant vectors called after pModularB-bar-badh-gfp-gus (structural representation as shown in Figure 6) of checking.This carrier contains two reporter genes (GFP and GUS), a foliage filter screening gene (BAR), a plant salt tolerance functional gene (BADH).

4, multigene carrier transformation of tobacco checking

Final expression vector pModularB-bar-badh-gfp-gus is transformed the Agrobacterium that the Agrobacterium competent cell obtains containing pModularB-bar-badh-gfp-gus, utilize this Agrobacterium infestation method transformation of tobacco.Concrete grammar is as described below:

1) preparation of Agrobacterium bacterium liquid: the Agrobacterium that will contain plant expression vector pModularB-bar-badh-gfp-gus is inoculated in YEB liquid medium (the 10g/L yeast extract that contains 100 μ g/mL Kan (kantlex) and 100 μ g/mL Rifampins, the 10g/L peptone, 5g/L sodium-chlor) in, 28 ℃ of shaking culture are diluted 10 times with MS liquid nutrient medium (pH 7.0) with bacterium liquid to OD600＞1.0;

2) aseptic tobacco (Nicotiana tabacum L.cv Xanth is available from Tobacco Institute, Chinese Academy of Agricultural Science) tissue cultured seedling blade is cut edge and main vein, be cut into 0.5 * 0.5cm ²Size;

3) explant that cuts soaks 5min in Agrobacterium bacterium liquid;

4) blot the bacterium liquid on vegetable material surface with aseptic filter paper, the MS minimum medium that changes upper berth one deck filter paper over to (contains macroelement: saltpetre 1900mg/L, ammonium nitrate 1650mg/L, potassium primary phosphate 170mg/L, sal epsom 370mg/L, CaCl ₂2H2O 44mg/L; Trace element: KI0.83mg/L, H ₃BO ₃6.2mg/L, MnSO ₄4H ₂O 22.3mg/L, ZnSO ₄7H ₂O 8.6mg/L, Na ₂MoO ₄2H ₂O 0.25mg/L, copper sulfate CuSO45H ₂O 0.025mg/L, cobalt chloride CoCl ₂0.025mg/L, molysite: disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate FeSO ₂7H ₂O 27.8mg/L; Organic composition: inositol 100mg/L, glycine 2mg/L, vitamin (VB ₁) 0.1mg/L, pyridoxine hydrochloride VB6 0.5mg/L, nicotinic acid 0.5mg/L, sucrose 30g/L, agar 7g/L), 25 ℃ of dark cultivations.

5) after three days, material forwarded in the division culture medium (MS minimum medium+2mg/L6-BA (6-benzyl aminopurine)+0.5mg/L NAA (naphthylacetic acid)+300mg/L Cb (Pyocianil)) that suitable screening presses carry out illumination cultivation, 25 ± 2 ℃ of temperature, photoperiod 14hr illumination/8hr is dark;

6) treat that resistant buds grows to 2-3cm when high, downcut budlet and change root induction in the root media (MS minimum medium+200mg/L Cb (Pyocianil)) that suitable screening presses over to;

7) root grows to 10cm above (approximately 4 weeks), and have some amount and can remove sealed membrane, in group training chamber, took exercise 2-3 days, then transplant in flowerpot and at room temperature grow 2-3 week, transplant at last to field planting, obtain turning pModularB-bar-badh-gfp-gus tobacco T ₀For plant.

10 strains of extracting above-mentioned acquisition turn pModularB-bar-badh-gfp-gus tobacco T ₀For the genomic dna of plant, the transgenosis of utilizing the polymerase chain reaction to carry out genomic level detects.Concrete grammar is the pModularB-bar-badh-gfp-gusT that turns that sprouts after 20 days ₀The transgene tobacco spire extracts genomic dna, and with primer barz and the bary shown in the table 1, gusz and gusy carry out respectively the polymerase chain reaction, and amplification obtains the fragment of size as shown in table 1, is the transgenic positive plant.The result shows, obtains the 6 strain positives and turns the pModularB-bar-badh-gfp-gus tobacco plant.

Table 1. transgenosis detects primer and amplified fragments size thereof

The positive of utilizing trizol reagent (invitrogen company) to extract to specifications above-mentioned acquisition turns the pModularB-bar-badh-gfp-gus tobacco or tobacco (Nicotiana tabacum L.cv Xanth) wild-type is sprouted 20 days plant leaf RNA, and the transgenosis RT-PCR that utilizes the polymerase chain reaction to carry out the mRNA level detects.Primer and annealing temperature adopt is four pairs of primers in the table 1, PCR product electrophoresis detection, and amplification obtains the fragment of size as shown in table 1, is the transgenic positive plant.The result shows that four foreign genes that all positives turn the pModularB-bar-badh-gfp-gus tobacco plant are all expressed.Wherein the result of number one sample plant as shown in Figure 7.Swimming lane 1,5,9 is the BAR gene test; Swimming lane 2,6,10 is the BADH gene test; Swimming lane 3,7,11 are the gus gene detection; Swimming lane 4,9,12 is the GFP gene test.Wherein swimming lane 1,2, and 3,4 are the detection of plasmid positive control; Swimming lane 5,6,7,8 is that wild-type non-transgenic tobacco (Nicotianatabacum L.cv Xanth) detects; Swimming lane 9,10,11,12 for turning the detection of pModularB-bar-badh-gfp-gus tobacco.

With above-mentioned RT-PCR detected result positive turn pModularB-bar-badh-gfp-gus tobacco T ₀Utilizing GUS histochemical stain and laser confocal microscope respectively transfer-gen plant to be carried out Phenotypic Observation for the plant spire detects.With reference to the method for molecular biology of plants experiment guide methods in plant molec μ lar biology:Alaboratory course manual (Ma Li adds D.F. claisen W. Crewson nurse A.R. Cashmore A.R. Wa Erna), slightly improve.Concrete grammar is: with N, dinethylformamide joins among the X-Gluc, stir, until dissolving (approximately needing 2min), again 0.1M phosphate buffered saline buffer, the 50mM Tripotassium iron hexacyanide and 50mM potassium ferrocyanide solution are joined in the X-Gluc solution, stir, add at last TritonX-100 (now joining with front).Get the GUS positive plant of different treatment, immerse in the X-gluc solution, occur to blue in 37 ℃ of dark reaction 2-24hr, the phosphoric acid buffer flushing once, with the fixing 50min of 2% (v/v) formaldehyde, 0.5% (v/v) glutaraldehyde, 100mM phosphoric acid buffer room temperature, then use (v/v) each 5min of rinsing of series concentration ethanol (50%, 70%, 100%), be immersed at last in 75% (v/v) ethanol.Converting material is observed under Zeiss LSM510META type confocal laser scanning microscope, CLSM.With 488nm argon-ion laser excitation, the 545nm spectroscope filters, and 505-530nm detects GFP green fluorescence signal.

X-Gluc (5-bromo-4-chloro-3-indyl-β-glucuronide) solution (1ml): 50 μ l N, dinethylformamide, 1mg X-Gluc, 749 μ l 0.1M phosphate buffered saline buffers (pH7.0), the 100 μ l 50mM Tripotassium iron hexacyanides (molecular weight 329.3), 100 μ l 50mM yellow prussiate of potash (molecular weight 422.4), 1 μ l TritonX-100.

The result shows, the positive blade that turns the pModularB-bar-badh-gfp-gus tobacco plant of all RT-PCR detected results all can have been dyed blueness by GUS, and confocal laser scanning microscope is to green fluorescence, the T-DNA zone that shows final expression vector pModularB-bar-badh-gfp-gus has transformed and has entered in the Plant Genome, and stably express (Fig. 8).

The present invention is reduced to minimum with the size of plant expression plasmid carrier, brings many advantages, along with the reduction of plasmid DNA size, and the copy number of plasmid, stability and transformation efficiency all increase to some extent.Guaranteeing that plasmid is less under the not significantly reduced condition of transformation efficiency, the section of open ended foreign DNA is just longer.So, the operation that less plasmid is very beneficial for testing.Above-mentioned transgenic experiments has also proved this point, and plasmid of the present invention can change over to four foreign genes in the plant simultaneously and reach stable expression.

Sequence table

<160>6

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

tggcaggata tattgtggtg taaaca 26

<210>2

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

gtaaacctaa gagaaaagag cgttta 26

<210>3

<211>34

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

ataacttcgt ataatgtatg ctatacgaag ttat 34

<210>4

<211>205

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

ggtacctggc aggatatatt gtggtgtaaa cagggccccc cctcgaggtc gacggtatcg 60

ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcca 120

ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattataac ttcgtatagc 180

atacattata cgaagttatt cgcga 205

<210>5

<211>57

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagctt 57

<210>6

<211>1515

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

ctcgagatgg cgttcccaat tcctgctcgt caactcttca tcgatggaga gtggagagaa 60

ccccttttaa aaaatcgcat acccatcatc aacccttcta ctgaagaaat catcggtgat 120

attcctgcag caactgcaga ggatgtggag gttgcagtgg tggcagctag aaaagccttt 180

aagaggaaca aaggcagaga ttgggctgca acttctggtg ctcatcgtgc taaatatttg 240

cgtgctattg ctgctaagat aacagagaaa aaagatcatt ttgttaaact ggaaaccctt 300

gattctggaa aaccacggga tgaagcagtg ttagatattg atgatgttgc tacatgcttt 360

gaatactttg ccggtcaagc agaagctctg gatgctaaac aaaaggctcc agtcaccctg 420

cctatggaaa gatttaaaag tcatgttctc aggcagccca ttggtgttgt tggattaata 480

tccccatgga attacccact tctaatggct acatggaaaa ttgctcccgc acttgctgct 540

ggatgcacag ctgtacttaa accatcagaa ttggcatctg tgacttgtct agaattcggt 600

gaagtgtgta atgaagtggg acttcctcca ggtgtgttaa atattttgac aggattaggt 660

cctgatgctg gtgccccaat agtatctcat cctgatattg acaaggtagc atttactggg 720

agtagtgcca ctggaagcaa gattatggct tctgctgccc aactagttaa gcctgttact 780

ttggagcttg gaggtaaaag tcctgttatc atgttcgaag atattgatat tgaaacagct 840

gttgaatgga ctctttttgg cgttttctgg acaaatggtc aaatctgtag tgcaacatct 900

agactgcttg tgcatgaaag cattgcagct gaatttgttg ataggatggt gaagtggaca 960

aaaaacataa aaatttctga tccatttgaa gaaggatgcc ggcttggccc cgttattagt 1020

aaaggacagt acgacaagat tatgaagttc atatcaacag cgaagagtga aggggcaaca 1080

attttgtgtg gaggctcccg tcctgagcat ttgaagaaag ggtattatat tgaacccacc 1140

attataactg atattaccac atccatgcaa atatggaaag aggaagtgtt tggccctgtc 1200

atatgtgtta aaacatttaa aactgaagat gaagccattg aattggcaaa tgatacagag 1260

tatggtttag ctggtgccgt gttttctaaa gatcttgaaa gatgtgagag ggtaactaag 1320

gctctagaag ttggagctgt ttgggttaat tgctcacaac catgctttgt tcatgctcca 1380

tggggaggag tcaagcgtag tggatttgga cgtgaacttg gggaatgggg tatcgagaat 1440

tacttgaata tcaagcaggt gacgagtgat atctctgatg aaccatgggg atggtacaag 1500

tctccttgag gatcc 1515

Claims (10)

1. plant polygene expression vector composition is comprised of carrier A and carrier B; The nucleotide sequence of described carrier A comprises successively a T-DNA end sequence, multiple clone site sequence and the special recombination site sequence of series winding; The nucleotide sequence of described carrier B comprises successively special recombination site sequence, multiple clone site sequence and a T-DNA end sequence of series winding; Wherein, when the T-DNA end sequence in the carrier A was T-DNA LB end sequence, the T-DNA end sequence in the described carrier B was T-DNA RB end sequence; When the T-DNA end sequence in the carrier A was T-DNA RB end sequence, the T-DNA end sequence in the described carrier B was T-DNA LB end sequence;

Described carrier A is inserted among the pUNI20 for a T-DNA end sequence, multiple clone site sequence and the special recombination site sequence of will be described contacting successively, and the recombinant vectors that the bacterial resistance gene among the needed bacterial resistance Gene Replacement pUNI20 is obtained.

2. plant polygene expression vector composition according to claim 1, it is characterized in that: described special recombination site is the Loxp site; Described Loxp site sequence is sequence 3 in the sequence table.

3. plant polygene expression vector composition according to claim 1 and 2, it is characterized in that: described T-DNA LB end sequence is sequence 1 in the sequence table; Described T-DNA RB end sequence is sequence 2 in the sequence table.

4. plant polygene expression vector composition according to claim 1 and 2 is characterized in that: the multiple clone site sequence in the described carrier A is for from 5 of sequence 4 ' end 33-134 position nucleotide sequence; Multiple clone site sequence in the described carrier B is sequence 5 in the sequence table.

5. plant polygene expression vector composition according to claim 1 and 2 is characterized in that: on described carrier A and the carrier B also respectively with the bacterial resistance expression casette; Described carrier A is not identical with bacterial resistance gene on the carrier B.

6. plant polygene expression vector composition according to claim 1 and 2 is characterized in that: the recombinant vectors of described carrier B for obtaining between the SacII that described special recombination site sequence is inserted into pCambia1302 and the EcoRI restriction enzyme site.

7. plant polygene expression vector composition according to claim 1 and 2, it is characterized in that: described carrier A or carrier B are also with the replicon that plays the screening effect.

8. plant polygene expression vector composition according to claim 1 and 2 is characterized in that: on the described carrier A also with the foliage filter screening gene.

9. the application of the described plant polygene expression vector composition of any one in cultivating transgenic plant among the claim 1-8.

10. the application of the described plant polygene expression vector composition of any one in making up the plant expression vector that to express a plurality of foreign genes among the claim 1-8.

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