CN1793376A - Process for removing selecting mark in transferring gene plant and special carrier - Google Patents
Process for removing selecting mark in transferring gene plant and special carrier Download PDFInfo
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- CN1793376A CN1793376A CN 200510115092 CN200510115092A CN1793376A CN 1793376 A CN1793376 A CN 1793376A CN 200510115092 CN200510115092 CN 200510115092 CN 200510115092 A CN200510115092 A CN 200510115092A CN 1793376 A CN1793376 A CN 1793376A
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Abstract
The invention discloses a transgenic plant selecting sign removing method and its special vector. The vector contains selecting sign gene. And the tow sides of the gene are respectively connected with identical cocurrent repetitive sequence. The invention uses homologous recombination principle to remove transgenic plant selecting sign. Its period is short. And it can be applied to the plant without generativ propagation such as trees, and so on.
Description
Technical field
The present invention relates to a kind of method and dedicated carrier thereof of removing selective marker in the transgenic plant.
Background technology
At present, transgenic plant have brought huge economic and social benefit in the outstanding behaviours aspect the pest-resistant antiweed to human society, meanwhile, also brought risk, the selective marker of using in transgeneic procedure mostly is antibiotic resistance gene or anti-herbicide gene, if these genes are propagated by biologic chain, to cause great destruction to ecotope, jeopardize human society at last.Therefore how to remove selectable marker gene and become the problem that must solve.At present, comparatively the method for Chang Yong removal plant selectable marker gene has the double T-DNA method, promptly on a plant expression vector, selectable marker gene and functional gene are structured in respectively in two T-DNA structures, cotransformation, utilize the isolating principle of offspring in transgenic progeny, to select not plant (T-DNA integration intothe barley genome from single and double cassette vectors Rainer Stahl with selective marker, Henriette Horvath, Jennifer Van Fleet, Michael Voetz, Diter von Wettstein, and Norbert Wolf.Proc Natl Acad Sci U S A.2002February 19; 99 (4): 2146-2151); Utilize the site-specific recombinase of bacterium origin to remove selective marker in addition, as the Cre-loxP method, connect the loxP sequence at the selective marker two ends, by the proteic effect of Cre, obtain offspring (Chemical-regulated, the site-specific DNA excision in transgenic plants.NatBiotechnol.2001 Feb of marker-free; 19 (2): 157-61.Zuo J, Niu QW, Moller SG, Chua NH.) or the like.Hybridization of these methods or needs or offspring's separating process, or can in plant, stay microbe-derived fragment.
The mode of action different according to selectable marker gene, be divided into positive selection markers and negative selection markers to it so long, make converting material under the effect of particular screen agent or under specific environment, survive, this selectable marker gene is positive selection markers, as neomycin phosphotransferase gene, hygromycin phosphotransferase gene, acetyl-CoA transferase gene etc.; Make converting material dead under the effect of particular screen agent or under the specific environment, this selectable marker gene is for negative selection markers, as cytosine deaminase gene, amidohydrolase gene, alcohol dehydrogenase gene etc.
Summary of the invention
The purpose of this invention is to provide a kind of method and dedicated carrier thereof of removing selective marker in the transgenic plant.
The carrier of selective marker contains positive selection markers gene in the removal transgenic plant provided by the present invention, wherein, is connected with one section identical direct repeat respectively in the both sides of described selectable marker gene.
Described tumor-necrosis factor glycoproteins can derive from plant, animal, microorganism or synthetic.
The length of described tumor-necrosis factor glycoproteins is at least 300bp, and long more recombination frequency is high more.
Described positive selection markers gene can be resistant maker gene or metabolic marker gene.
Described resistant maker gene comprises antibiotics resistance gene (as neomycin phosphotransferase gene, hygromycin phosphotransferase gene, streptomycin phosphotransferase gene or the like) and anti-herbicide gene (as acetyl-CoA transferase gene gene, careless glycosides phosphorus oxidation reductase gene, the fine hydratase gene of ammonia or the like).
Described metabolic marker gene can be isopentyl transferring enzyme, histidine kinase or the like.
Whether remove for the ease of the selective marker in the checking transgenic plant, between described two sections identical direct repeats, be connected with negative selection markers gene.
Described negative selection markers gene can be cytosine deaminase gene or amidohydrolase gene, alcohol dehydrogenase gene etc.
The method of selective marker in the removal transgenic plant provided by the present invention, it is carrier conversion purpose plant with selective marker in the above-mentioned removal transgenic plant, screening obtains transgenic plant, the selection markers screening of answering with described positive selection markers gene pairs again can not growing plants be the transgenic plant of removing selective marker.
In the described method, also comprise following verification step: the transgenic plant of the removal selective marker that will obtain are screened in the selective marker of negative selectable marker gene correspondence again, can growing plants be the transgenic plant of removing selective marker.
Described tumor-necrosis factor glycoproteins has the nucleotide sequence of sequence 1 in the sequence table.
The nucleotides sequence of sequence 1 is classified 3 ' terminal fragment of paddy rice rubisco gene as in the sequence table, and long is 732bp.
3 ' terminal fragment of described paddy rice rubisco gene can oryza sativa genomic dna be a template, carries out pcr amplification with forward primer and reverse primer and obtains; The 1st to the 26th deoxynucleotide from 5 ' end of described forward primer is 5 ' AGCATGCTGGCAACTAAGCCGTCATC 3 '; The 1st to the 21st deoxynucleotide from 5 ' end of described reverse primer is 5 ' TATCATCTGCCAACCTTTCTG 3 '.
Described tumor-necrosis factor glycoproteins has the nucleotide sequence of sequence 2 in the sequence table.
The nucleotides sequence of sequence 2 is classified 3 ' terminal fragment of paddy rice ubiquitin gene as in the sequence table, and long is 1535bp.
3 ' terminal fragment of described paddy rice ubiquitin gene can oryza sativa genomic dna be a template, with primer to 1 or primer carry out pcr amplification to 2 and obtain; Described primer to 1 is: forward primer 5 ' TGGTGGTCAGTAATCAGCCAGTT 3 ', reverse primer 5 ' ATGCATCTAGTATTTATAGTCCTGAGCTTACTTA 3 '; Described primer to 2 is: forward primer 5 ' GCATGCTGGTGGTCAGTAATCAGCC3 ', reverse primer 5 ' ATGCATCTAGTATTTATAGTCCTGAGCTTACTTA 3 '
Method of the present invention is suitable for dicotyledons, as tobacco, potato, cotton, lettuce, tomato, muskmelon, cucumber, pea, oil grain, beet or Sunflower Receptacle etc., also be suitable for monocotyledons, as tall grass, other turfgrass, wheat, barley, oat, paddy rice or corn etc.
The clone, the host bacterium that import the carrier of selective marker in the removal transgenic plant of the present invention all belong to protection scope of the present invention.
The present invention utilizes principle of homologous recombination to remove the transgenic plant selective marker, can remove selective marker the present age in transgenosis, not only the cycle short, and can in trees etc. can't carry out the plant of sexual propagation, use.
Description of drawings
Fig. 1 is the physical map of selective marker carrier pNE103
Fig. 2 is for changeing the PCR product electrophorogram of pNE103 plasmid tobacco
Fig. 3 is the physical map of selective marker carrier pNE101::Pubi-gusA
Fig. 4 is the active detected result of the GUS that changes pNE101::Pubi-gusA Festuca Arundinacea callus
Embodiment
Method among the following embodiment is ordinary method if no special instructions.
The selective marker of going of embodiment 1, commentaries on classics pNE103 tobacco is tested
One, removes the structure of selective marker carrier pNE103
Remove selective marker carrier pNE103 (Fig. 1) with the ordinary method structure.Negative selection markers gene cytosine deaminase gene (codA) is obtained through PCR by intestinal bacteria in this carrier.The PCR primer is codA-S:5 '-GTCGACATGTCGAATAACGCTTTACAAA-3 ', codA-A:5 '-CTCGAGTCAACGTTTGTAATCGATGG-3 '.The PCR reaction system is: 25ul contains e. coli k12 thalline, 1.5mM MgCl altogether
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M codA-S and 1 μ M codA-A, the Taq polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.The 1296bp dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, insert between the XhoI and KpnI site of pUC19 plasmid with XhoI and this fragment of KpnI double digestion, order-checking (three rich polygala root biotech companies), sequencing result shows the plasmid that obtains containing cytosine deaminase gene (codA), called after pUC19::codA.
3 ' end sequence with PCR method amplification 35S.With pCAMBIA1300 (CAMBIA company) is template, and the PCR primer is: 35SpolyA-S:5 '-GATCTGTCGATCGACAAGCTC-3 ', 35SpolyA-A1:5 '-GGTACCGCATGCTAATTCGGGGGATCTGGAT-3 ' carries out pcr amplification.The PCR reaction system: 25ul contains 100ng pCAMBIA1300 (CAMBIA company) plasmid DNA, 1.5mM MgCl altogether
2, 20mMTris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M 35SpolyA-S and 1 μ M35SpolyA-Al, the Taq polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.The dna fragmentation of the 227bp that amplification obtains, the scheme that reclaims test kit (sky, Beijing latitude Time Technology company limited) and provide by test kit with gel reclaims this fragment, to reclaim fragment and be connected to pGEM-Teasy, order-checking (three rich polygala root biotech companies), and with behind SalI and the KpnI double digestion, insert between the XhoI and KpnI site of pUC19::codA, show that through order-checking the plasmid that obtains contains the 3 ' end sequence of the 35S that amplification obtains, this plasmid called after pUC19::codA-35SpolyA.
Promoter sequence with PCR method amplifying rice actin gene.With CTAB method (Steiner JJ, PoklembaCJ, Fjellstrom RG, Elliott LF., A rapid one-tube genomic DNA extractionprocess for PCR and RAPD analyses.Nucleic Acids Res.1995 Jul11; 23 (13): 2569-70.) extract oryza sativa genomic dna, concrete grammar is as follows: water intaking rice blade, with liquid nitrogen grinding, add 5 milliliters in the vegetable material of per 1 gram through the CTAB of 68 ℃ of preheatings solution (with before adding the 10mM mercaptoethanol), and 60 ℃ were heated 30 minutes.Add the equal-volume chloroform then: primary isoamyl alcohol (24: 1), centrifugal 15 minutes of 15000g gets supernatant.The Virahol that in supernatant liquor, adds 2/3 times of volume, mixing stirs out the DNA silk with the entry needle needle point and to place new pipe, adds 75% ethanol again, and washing precipitation and centrifugal tube wall blot ethanol then, and air is dried remaining ethanol, adding TE dissolving DNA.And in-20 ℃ of preservations.With this oryza sativa genomic dna is template, and primer is: Actin-S:5 '-CTCGAGTCTTCTACCTACAAAAAAGCTCC-3 ', act in-A:5 '-CTCGAGGTCATTCATATGCTTGAG-3 ' carries out pcr amplification.The PCR reaction system is: be total to 25ul, contain 100ng genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M Actin-S and 1 μ M actin-A, the Taq polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.The dna fragmentation of the 1410bp that amplification obtains, the scheme that reclaims test kit (sky, Beijing latitude Time Technology company limited) and provide by test kit with gel reclaims this fragment, and be connected to pGEM-Teasy vector, order-checking (three rich polygala root biotech companies), sequencing result shows that the fragment that this amplification obtains is the fragment with promoter sequence of actin gene, it is cut the XhoI site that pUC19::codA-35SpolyA is inserted in the back with the XhoI enzyme, obtain plasmid pUC19::Pactin-codA-35SpolyA.
With two sections 3 ' end sequence fragments of PCR method amplification with the paddy rice ubiquitin gene of different restriction enzyme sites.Oryza sativa genomic dna with said extracted is a template, and primer is: Tubi-S1:5 '-TGGTGGTCAGTAATCAGCCAGTT-3, Tbui-A:5 '-ATGCATCTAGTATTTATAGTCCTGAGCTTACTTA-3 ' carries out pcr amplification.The PCR reaction system: 25ul contains 100ng genomic dna, 1.5mM MgCl altogether
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M Tubi-S1 and 1 μ M Tubi-A, the Taq polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.Amplification obtains the dna fragmentation of 1541bp, the dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, insert the HincII site of pBluescriptII KS (-) (STRATAGENE company), order-checking (three rich polygala root biotech companies), sequencing result shows that amplified production is to have in the sequence table 3 ' end sequence fragment of the paddy rice ubiquitin gene of the 1st to 1535 nucleotide sequence in the sequence 2, will contain this segmental pBluescriptIIKS (-) plasmid called after pBS::Tubi-A.
With the oryza sativa genomic dna is template, and with Tubi-S2:5 '-GCATGCTGGTGGTCAGTAATCAGCC-3 ', Tbui-A:5 '-ATGCATCTAGTATTTATAGTCCTGAGCTTACTTA-3 ' is a primer, carries out pcr amplification.PCR reaction system 25ul altogether contains 100ng genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M Tubi-S2 and 1 μ M Tubi-A, the Pfu enzyme of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.Amplification obtains the dna fragmentation of 1547bp, the dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, be inserted into the EcoRV site of pBluescriptII KS (-) (STRATAGENE company), order-checking (three rich polygala root biotech companies), sequencing result shows that amplified production is to have in the sequence table 3 ' end sequence fragment of the paddy rice ubiquitin gene of the 1st to 1535 nucleotide sequence in the sequence 2, will contain this segmental pBluescript II KS (-) plasmid called after pBS::Tubi-B.
3 ' end sequence with PCR method amplification 35S.With pCAMBIA1300 (CAMBIA company) is template, and primer is: 35SpolyA-S:5 '-GTCGACGATCTGTCGATCGACAAGCTC, 35SpolyA-A2:5 ' GTCGACTAATTCGGGGGATCTGGAT.PCR reaction system: contain 100ngpCAMBIA1300 (CAMBIA company) plasmid DNA, 1.5mM MgCl among the 25ul altogether
2, 20mM Tris-HCl (pH8.4), 50mMKCl, 0.8mM dNTP mixture, 1 μ M 35SpolyA-S and 1 μ M 35SpolyA-A2, the Pfu polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.The 221bp dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, and after cutting with the SalI enzyme, insert the same loci of pBluescript II KS (-) (STRATAGENE company), order-checking (three rich polygala root biotech companies), sequencing result shows the segmental plasmid of 3 ' end sequence that has obtained containing amplified production 35S, with its called after pBS::35SpolyA2.
With pCAMBIA1300 (CAMBIA company) is template, and primer is: TBL-S:5 '-CTGCAGATTCGGCGTTAATTCAGTACA-3 ', TBL-A:5 '-ATACCTTAGCAGGAGACATTCC-3 '.25ul PCR reaction system contains 100ng pCAMBIA1300 (CAMBIA company) plasmid DNA, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M TBL-S and 1 μ M TBL-A, the Pfu polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.Amplification obtains the fragment of 616bp, the dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, insert the EcoRV site of pBluescriptII KS (-) (STRATAGENE company), order-checking (three rich polygala root biotech companies), sequencing result shows the plasmid of the dna fragmentation that has obtained comprising the T left margin, with its called after pBS::TBL.
Plasmid pBS::35SpolyA2 with the XhoI site that the SalI enzyme downcuts the fragment insertion pBS::Tubi-A of the 3 ' end sequence that contains 35S, is obtained plasmid pBS::35SpolyA2-TubiA.
Plasmid pBS::TBL with the NsiI/SacII site that the PstI/SacII enzyme downcuts the sequence fragment insertion pBS::35SpolyA2-TubiA that contains the T left margin, is obtained plasmid pBS::35SpolyA2-TubiA-TBL.
The 35SpolyA2-TubiA-TBL fragment that contains among the plasmid pBS::35SpolyA2-TubiA-TBL is obtained plasmid pNE3 with the same loci that SacII/XhoI downcuts insertion pCAMBIA1300 (CAMBIA company).
Hygromycin gene on the pCAMBIA1300 is downcut the same loci place that inserts pNE3 with XhoI, obtain plasmid pC1300::35SpolyA-Tubi-TBL.
Plasmid pBS::Tubi-B with the SbfI site that the NsiI enzyme downcuts 3 ' the end sequence fragment insertion plasmid pUC19::Pactin-codA-35SpolyA that contains paddy rice ubiquitin gene, is obtained plasmid pUC19::Tubi-Pactin-codA-35SpolyA.
Go up the BstxI site that contains the fragment of Tubi-Pactin-codA-35SpolyA sequence and insert pCAMBIA1300 with the SphI cutting-out from plasmid pUC19::Tubi-Pactin-codA-35SpolyA, obtain plasmid pC1300::Tubi-codA.
Upward downcut the same loci that contains the fragment of 35SpolyA-Tubi-TBL sequence and insert pC1300::Tubi-codA from plasmid pC1300::35SpolyA-Tubi-TBL, obtain plasmid pNE103 with AatII/SacII.
Negative selection markers gene codA gene is subjected to the regulation and control of paddy rice actin promotor and links to each other with positive selection markers gene hygromycin gene in the pNE103 carrier, at the outer survey two ends of these two expression structures are two sections 3 ' terminal fragments of multiple paddy rice ubiquitin gene in the same way, and long is 1535bp.
Two, the selective marker of going of changeing the pNE103 tobacco is tested
PNE103 is transformed Agrobacterium LBA4404, obtain agrobacterium strains TpNE103.Be cut into the leaf dish of the about 1cm of four length of sides with the sterile razor blade tobacco aseptic seedling blade that the children is tender, soaked 10 minutes in TpNE103 bacterium liquid, culture medium is altogether gone in switching, in 25 ℃ of dark cultivations 3 days, change in the subculture medium illumination cultivation again over to three days, and changed screening culture medium over to.Continue to cultivate 7 days, change regeneration culture medium again over to.When treating that blade edge grows the regeneration bud of growing thickly to 2-3cm, with scalper regeneration bud is downcut, and change in the root media, get 2 transfer-gen plants and continue subsequent experimental.
Every liter the culture medium composition is as follows altogether: the MS minimum medium adds 6-benzyl aminopurine (Sigma, the U.S.) 1.0mg, indolylacetic acid (Gibco, the U.S.) 0.1mg, sucrose (Beijing Yili Fine Chemicals Co., Ltd.) 30g, pH5.2.
Every liter of succeeding transfer culture based component is as follows: the MS minimum medium adds 6-benzyl aminopurine (Sigma, the U.S.) 1.0mg, indolylacetic acid (Gibco, the U.S.) 0.1mg, Pyocianil (Beijing is glad through Bioisystech Co., Ltd of section) 250mg, sucrose (Beijing Yili Fine Chemicals Co., Ltd.) 30g, pH5.8.
Every liter of screening and culturing based component is as follows: the MS minimum medium adds 6-benzyl aminopurine (Sigma, the U.S.) 1.0mg, indolylacetic acid (Gibco, the U.S.) 0.1mg, Pyocianil (Beijing is glad through Bioisystech Co., Ltd of section) 250mg, Totomycin (Beijing is glad through Bioisystech Co., Ltd of section) 30mg, sucrose (Beijing Yili Fine Chemicals Co., Ltd.) 30g, pH5.8.
Every liter of regeneration culture medium composition is as follows: the MS minimum medium adds 6-benzyl aminopurine (Sigma, the U.S.) 1.0mg, Pyocianil (Beijing is glad through Bioisystech Co., Ltd of section) 250mg, Totomycin (Beijing is glad through Bioisystech Co., Ltd of section) 30mg, sucrose (Beijing Yili Fine Chemicals Co., Ltd.) 30g, pH5.8.
Every liter of root culture based component is as follows: the MS minimum medium adds Totomycin (Beijing is glad through Bioisystech Co., Ltd of section) 30mg, sucrose (Beijing Yili Fine Chemicals Co., Ltd.) 20g, pH5.8.
With CTAB method (Steiner JJ, Poklemba CJ, Fjellstrom RG, Elliott LF., Arapidone-tube genomic DNA extraction process for PCR and RAPD analyses.NucleicAcids Res.1995 Jul 11; 23 (13): 2569-70.) extract tobacco gene group DNA.With the 100ng genomic dna is that template is carried out the PCR reaction, and the part of amplification 35s promotor and hygromycin gene is to confirm transgenic plant.Reaction solution contains 100ng genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M, 5 ' primer and 1 μ M, 3 ' primer, and the Taq polysaccharase of 1 unit (Shanghai Shen You biotech company).Primer sequence is as follows:
5 ' primer: 5 '-cgtaagggatgacgcacaa-3 '
3 ' primer: 5 '-ttggcgacctcgtattgg-3 '
In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation a part of dna fragmentation according to following scheme with amplification 35s promotor and hygromycin gene: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.Amplified production is carried out electrophoresis, and the result is shown in Figure 2, and changeing the pNE103 tobacco has band clearly at the 846bp place that estimates, shows that pNE103 is transferred in the tobacco and can expresses in tobacco.Among Fig. 2, swimming lane 1 is the PCR product of not transgene tobacco, and swimming lane 2 is the PCR product of pNE103 transgene tobacco 1, and swimming lane 3 is the PCR of pNE103 transgene tobacco 2, and swimming lane M is 1kb DNA marker
Get the tobacco leaf evoked callus on substratum that changes the pNE103 plasmid, behind the one and a half months, with the callus lines morsel, and be added with at no Totomycin on the substratum of negative selective agent 5 flucytosines of 1mg/ml and screen callus, the survival frequency of callus lines is 0.4%, the callus of survival is got a part to be put back on the substratum of 30mg/l Totomycin again, the death of discovery callus, reorganization has taken place in explanation in these callus, lost codA and hygromycin gene.
The selective marker of going of embodiment 2, commentaries on classics pNE101::Pubi-gusA Festuca Arundinacea callus is tested
One, removes the structure of selective marker carrier pNE101::Pubi-gusA
Remove selective marker carrier pNE101::Pubi-gusA with the ordinary method structure.
3 ' end sequence with PCR method amplifying rice rubisco small ylidene gene.With the oryza sativa genomic dna is template, and primer is: TrbcS-S:5 '-AGCATGCTGGCAACTAAGCCGTCATC-3 ', TrbcS-A:5 '-TATCATCTGCCAACCTTTCTG-3 ' carries out pcr amplification.The PCR reaction system is 25ul altogether, contains 100ng genomic dna, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M TrbcS-S and 1 μ M TrbcS-A, the Pfu polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.Amplification obtains the dna fragmentation of 732bp, the dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, with (Promega company of pGEM-Teasy vector system, the U.S.) scheme that provides by this system is cloned this fragment, order-checking (three rich polygala root biotech companies), show through order-checking, amplified fragments has the sequence of sequence 1 in the sequence table, is the 3 ' end sequence (shown in sequence table 1) of paddy rice rubisco small ylidene gene.With the EcoRV site that this fragment is inserted pBluescriptIIKS (-) (STRATAGENE company), obtaining plasmid is pBS::TrbcS.
The plasmid pUC19::Pactin-codA-35SpolyA of embodiment 1 is downcut the KpnI/NsiI site that the fragment that contains the Pactin-codA-35SpolyA expression structure is inserted pBS::TrbcS with the KpnI/SbfI enzyme, obtain plasmid pBS::TrbcS-Pactin-codA-35SpolyA.
The pBS::35SpolyA2 of embodiment 1 is downcut the XhoI site that contains the terminal sequence fragment insertion pBS::TrbcS of 35S3 ' with the SalI enzyme, obtain plasmid pBS::35SpolyA-TrbcS.
The pBS::TBL of embodiment 1 is cut the NsiI/SacII site that contains T left margin sequence insertion pBS::35SpolyA-TrbcS with the PstI/SacII enzyme, obtain plasmid pBS::35SpolyA-TrbcS-TBL.
The 35SpolyA-TrbcS-TBL fragment that contains among the plasmid pBS::35SpolyA-TrbcS-TBL is obtained plasmid pNE with the same loci that SacII/XhoI downcuts insertion pCAMBIA1300.Hygromycin gene on the pCAMBIA1300 is downcut the same loci place that inserts pNE with XhoI, obtain plasmid pNE10.
Plasmid pBS::TrbcS-Pactin-codA-35SpolyA with the BstXI site that the SphI enzyme downcuts the fragment insertion plasmid pNE10 that contains TrbcS-Pactin-codA-35SpolyA, is obtained plasmid pNE101.
Go up the SmaI site of downcutting the gus gene and inserting pNE101 from plasmid pBI121 (AF485783), obtain plasmid pNE101::gusA with the flat back of Klenow enzyme benefit with SmaI/EcoRI.
Extract corn gene group DNA, with PCR method amplification ubiquitin promotor.PCR reaction system 25ul altogether contains 100ng corn gene group DNA, 1.5mM MgCl
2, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.8mM dNTP mixture, 1 μ M Ubi-S and 1 μ M Ubi-A, the Taq polysaccharase of 1U (Shanghai Shen You biotech company).In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation according to following scheme: earlier 94 ℃ 5 minutes; Again 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ 10 minutes.Ubi-S：5’-CTGCAGAAGTAACACCAAACAACAGG-3’，Ubi-A：5’-CTGCAGTGCAGCGTGACCCGGTCG-3’。The 1998bp dna fragmentation that amplification is obtained reclaims this fragment with the scheme that gel reclaims test kit (sky, Beijing latitude Time Technology company limited) and provides by test kit, with (Promega company of pGEM-Teasyvector system, the U.S.) scheme that provides by this system is cloned this fragment, order-checking (three rich polygala root biotech companies), and, obtain plasmid pNE101::Pubi-gusA with the SbfI site that Pst I enzyme is cut insertion pNE101::gusA.(Fig. 3).
In pNE101::Pubi-gusA, negative selection markers gene cytosine deaminase gene is subjected to the regulation and control of actin promotor and links to each other with positive selection markers gene hygromycin gene, at the outer survey two ends of these two expression structures are two sections 3 ' terminal fragments of multiple paddy rice rubisco gene in the same way, and long is 732bp.Outer survey corn ubiquitin promoter regulation gus expression of gene at two sections tumor-necrosis factor glycoproteinss.
Two, the selective marker of going of changeing pNE101::Pubi-gusA Festuca Arundinacea callus is tested
PNE101::Pubi-gusA is transformed Agrobacterium LBA4404, obtain agrobacterium strains TpNE101::Pubi-gusA.Choose tall grass (the Festuca arundinacea Schreb.) seed of mature and plump, sterilization.Under aseptic condition, with embryo complete peeling from the seed that disinfects, crosscut inserts callus inducing medium in the back into two again.Every liter of callus inducing medium composed as follows: on the basis of MS minimum medium, add the auxin substance 2,4 dichlorophenoxyacetic acid (2,4-D) 3-8mg, caseinhydrolysate concentration is at 0.5-2g, pH5.8.Per two to three weeks are changeed ware once with callus.Infecting preceding callus is transferred on the callus state adjustment substratum cultivated 30 days.Every liter of callus state is adjusted the composed as follows of substratum: add auxin substance 2 on the basis of MS minimum medium, 4-dichlorphenoxyacetic acid (2,4-D) 0.5-2mg, cytokinin-like substance 6-benzyl aminopurine 0.1-0.5mg, KT concentration 0-0.5mg, caseinhydrolysate concentration is at 0.5-2g, pH5.8.State adjusted cultured calli places agrobacterium tumefaciens TpNE101::Pubi-gusA bacterium liquid to infect 10 minutes on the substratum, remove bacterium liquid, on callus inducing medium, 25 ℃ of dark cultivations after 3 days induce screening culture medium that callus is screened with resistant calli.2-3 week, as seen the resistant calli that is fresh ivory buff grows out, glucuronidase activity qualitative detection test-results as shown in Figure 4, the callus glucuronidase activity qualitative detection result who changes pNE101::Pubi-gusA is blue, and the callus that does not change pNE101::Pubi-gusA still presents ivory buff, show the callus that the gus gene has inserted changes pNE101::Pubi-gusA, and at the genomic dna and the stably express of resistant calli.
Every liter of resistant calli is induced the composed as follows of screening culture medium: add 2 on the basis of MS minimum medium, 4-dichlorphenoxyacetic acid (2,4-D) 0.5-8mg, cytokinin-like substance 6-benzyl aminopurine 0-0.5mg, KT concentration 0-0.5mg, caseinhydrolysate concentration is at 0.5-2g, Totomycin 50-100mg, Pyocianil 250mg, pH5.8.
Above-mentioned resistant calli is placed let alone growth on the callus of induce substratum of no any selective agent, after 2-3 month, callus placed on the substratum that contains negative selective agent 5 flucytosines of 2mg/ml screen, the survival frequency of callus lines is 0.1%, the callus of survival is got a part carry out glucuronidase activity qualitative detection result for blue, other gets a part and puts back on the substratum that contains the 50-100mg/l Totomycin, callus death.Illustrating in the genome of these callus lines has gus gene, homologous recombination takes place lost codA and hygromycin gene.
Sequence table
<160>2
<210>1
<211>732
<212>DNA
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>1
tggcaactaa?gccgtcatcg?tcatatatag?ccttgtttaa?ttgttcatct?ctgattcgat 60
gatgtctccc?accttgtttc?gtgtgttccc?agtttgttca?tcgtcttttg?attttaccgg 120
ccgtgctctg?cttttgtttt?tgtttcacct?gatctctctc?tgacttgatg?taagagtggt 180
atctgctacg?actatatgtt?gttgggtgag?gcatatgtga?atgaaatata?tggaagctcc 240
ggctatatat?atttatacaa?agggtacgag?atggatgtga?atcctagagc?atatgtgtcc 300
aacaatcaat?tcgtcgacaa?tgaaaatttg?atcatggaat?taaaaaatca?tgcttctgtt 360
gttcatcgga?aatgcctttc?tatactgatt?agtggatttc?caaactgaat?tcacaagaaa 420
acatatataa?ttgttgtgtt?catcaatcta?cttcttttga?tcaaataaaa?gataatttga 480
cttggcaaaa?aacaaaaaat?acagtttttc?aggtatggag?ggagtacttt?ttttttttca 540
agattgtcac?atgcaaatag?gcaataatac?actcttttta?taacttgtaa?gtgttttttt 600
accttttttt?tgctacatat?taaataccaa?aacagtgtat?cgaaaagcaa?atctgtacga 660
cgaccaaatg?gctaagtggt?gtaattgtgt?gtcagtccag?tccaagtcca?acagaaaggt 720
tggcagatga?ta 732
<210>2
<211>1535
<212>DNA
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>2
tggtggtcag?taatcagcca?gtttggtgga?gctgccgatg?tgcctggtcg?tcccgagcct 60
ctgttcgtca?agtatttgtg?gtgctgatgt?ctacttgtgt?ctggtttaat?ggaccatcga 120
gtccgtatga?tatgttagtt?ttatgaaaca?gtttcctgtg?ggacagcagt?atgctttatg 180
aataagttgg?atttgaacct?aaatatgtgc?tcaatttgct?catttgcatc?tcattcctgt 240
tgatgtttta?tctgagttgc?aagtttgaaa?atgctgcata?ttcttattaa?atcgtcattt 300
acttttatct?taatgagctt?tgcaatggcc?tatgggatat?aaaagattat?tctggaggga 360
agtgatgctg?gaaggactat?gctgtcctga?tttatatttg?gagccactat?gagcatttgg 420
gctttctttt?cagaacgctg?taggcgtgtg?ttgaaatctt?tgcgacattc?aatttgatat 480
atgattcgag?gtaattgggc?tttaatttgt?catctcatgt?aacatctttt?tgtttcttcg 540
ctgcttgatt?tctctatttc?gtagcattgg?aagataatag?tagaatgatg?atatactcca 600
atacttgcaa?tttcaaaacc?gttagaaaga?aaggaaaatc?acggctaggg?gaaaaattct 660
ctctgatgcg?tgcatcacca?aaatctgatt?gattgacata?agcattggaa?cacaaataga 720
agatggagag?gtggacatgt?ttccgagaca?tagcatttga?gctcacattg?tttttgatcc 780
gtggctgtgc?catacgcgat?aatttactaa?actttccctt?gccccttggt?aatataaagc 840
ttataatgta?tacaccgaaa?ttgaaaaacg?tattagttca?ctcttcttcc?tttttctcag 900
caaagcgtgt?tcatcaattt?tattccacat?gaatgtgatt?gcgaattgtg?accaggaatg 960
atttaacaat?cccgatcaac?cctgttgtcg?gtggtggcac?taaaaattac?caaacagcaa 1020
cctatcaagc?aatgacaaat?actttccaat?ccaaaaaatg?atccaagcca?aaagtctgac 1080
aaaagggagg?aggaagcttc?agagctttgg?aaccttgcct?aagcaaggca?atggcaggaa 1140
agaggaggaa?aggatcaagt?tcttttcttg?ctctttcttc?cacccacgat?gtaccaatcg 1200
atcacccatc?aattcatcat?caagattgca?aaagttaaac?atgattcgaa?aaagactaaa 1260
agaagctaga?tataaagcag?agaaattaaa?gcgaaagaaa?tagatagagg?aatcaagtga 1320
cactgtcaca?cgcttctccc?ggttccattt?tgcgtgcgtg?catgtggagg?catatttttc 1380
actgttcctt?tcaaaatcat?tggcgaatcc?aagttaaatt?accctgcttt?ttatactgca 1440
tttttctctc?aaaaaataga?tttcgaacta?agtttgaagt?aatctcactc?tctgaagaaa 1500
tgtctgttaa?gtaagctcag?gactataaat?actag 1535
Claims (10)
1, a kind of carrier of removing selective marker in the transgenic plant contains positive selection markers gene, it is characterized in that: the both sides of described selectable marker gene are connected with one section identical direct repeat respectively.
2, carrier according to claim 1 is characterized in that: described direct repeat derives from plant, animal, and microorganism or synthetic, its length is at least 300bp.
3, carrier according to claim 1 is characterized in that: described positive selection markers gene is resistant maker gene or metabolic marker gene.
4, carrier according to claim 1 is characterized in that: be connected with negative selection markers gene between described two sections identical direct repeats; Described negative selection markers gene is cytosine deaminase gene or amidohydrolase gene or alcohol dehydrogenase gene.
5, carrier according to claim 4 is characterized in that: described tumor-necrosis factor glycoproteins has the nucleotide sequence of sequence 1 in the sequence table or sequence 2.
6, carrier according to claim 5 is characterized in that: the carrier of selective marker is as shown in Figure 1 pNE103 or pNE101 ∷ Pubi-gusA as shown in Figure 3 in the described removal transgenic plant.
7, a kind of method of removing selective marker in the transgenic plant, it is carrier conversion purpose plant with selective marker in arbitrary described removal transgenic plant in the claim 1 to 5, screening obtains transgenic plant, the selection markers screening of answering with described positive selection markers gene pairs again can not growing plants be the transgenic plant of removing selective marker.
8, method according to claim 7, it is characterized in that: in the described method, also comprise following verification step: the transgenic plant of the removal selective marker that will obtain are screened in the selective marker that negative selection markers gene pairs is answered again, can growing plants be the transgenic plant of removing selective marker.
9, according to claim 7 or 8 described methods, it is characterized in that: described purpose plant is monocotyledons or dicotyledons; Described monocotyledons comprises tall grass, turfgrass, wheat, barley, oat, paddy rice or corn; Described dicotyledons comprises tobacco, potato, cotton, lettuce, tomato, muskmelon, cucumber, pea, oil grain, beet or Sunflower Receptacle.
10, import clone, the host bacterium that the described carrier of arbitrary claim obtains among the claim 1-6.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015158115A1 (en) * | 2014-04-14 | 2015-10-22 | 中国科学院天津工业生物技术研究所 | Simple and efficient gene editing method |
| CN106554957A (en) * | 2015-09-30 | 2017-04-05 | 中国农业科学院深圳农业基因组研究所 | Sequencing library and its preparation and application |
| CN108759650A (en) * | 2018-04-23 | 2018-11-06 | 江苏大学镇江流体工程装备技术研究院 | A kind of magnetic drive pump bearing gap wear on-Line Monitor Device and its method |
| CN112592954A (en) * | 2020-12-22 | 2021-04-02 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of gene GliT as screening marker gene in resistance screening |
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2005
- 2005-11-28 CN CN 200510115092 patent/CN1793376A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015158115A1 (en) * | 2014-04-14 | 2015-10-22 | 中国科学院天津工业生物技术研究所 | Simple and efficient gene editing method |
| CN106554957A (en) * | 2015-09-30 | 2017-04-05 | 中国农业科学院深圳农业基因组研究所 | Sequencing library and its preparation and application |
| CN106554957B (en) * | 2015-09-30 | 2020-04-21 | 中国农业科学院深圳农业基因组研究所 | Sequencing library, preparation and application thereof |
| US11702690B2 (en) | 2015-09-30 | 2023-07-18 | Agricultural Genomics Institute At Shenzhen China Academy Of Agricultural Sciences | Sequencing library, and preparation and use thereof |
| CN108759650A (en) * | 2018-04-23 | 2018-11-06 | 江苏大学镇江流体工程装备技术研究院 | A kind of magnetic drive pump bearing gap wear on-Line Monitor Device and its method |
| CN108759650B (en) * | 2018-04-23 | 2020-11-03 | 江苏大学镇江流体工程装备技术研究院 | Magnetic pump bearing gap wear online monitoring device and method thereof |
| CN112592954A (en) * | 2020-12-22 | 2021-04-02 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of gene GliT as screening marker gene in resistance screening |
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