CN1375010A - Targeted gene removal - Google Patents
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- CN1375010A CN1375010A CN00813040.XA CN00813040A CN1375010A CN 1375010 A CN1375010 A CN 1375010A CN 00813040 A CN00813040 A CN 00813040A CN 1375010 A CN1375010 A CN 1375010A
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Abstract
A method of removing a part of a transgene after its integration into a genome comprising flanking the part of the transgene on each side with an attachment P region (attP) of bacteriophage lambda and inducing intrachromasomal homologous recombination between the flanking attP regions so that the part of the transgene sandwiched between the attP regions is removed.
Description
The present invention relates to from plant especially transgenic plant, remove the method for selectable marker gene or the auxiliary nucleic acid of external source, device for this reason and product thereof.
Background of invention
In Plant Transformation, be extensive use of the recombination of tolerance antibiotic or weedicide as selected marker.In case selected transgenic line by its resistance, marker gene is just unnecessary.The existence of antibiotic or Herbicid resistant mark makes the people worry the patience kind and may be diffused into the wild-type plant kind in transgenic plant
1Or other kind
2
Owing to many reasons are expected to remove the resistance marker gene from transgenic plant very much.Relevant interracial cross-pollination may cause the resistance features convey to give seed
3Thereby, jeopardize the life-time service of genetically modified crops and cause the potential ecological problem.For example, the sign of coding antibiotic or Herbicid resistant may become transgenic plant weeds type pest, thereby destroys the eubiosis.Human consumers worry widely-used resistance sign in food, might laterally send the danger of passing intestinal bacteria with transgenosis in theory.If theoretic danger becomes a reality, then marker gene may be transformed into microorganism and increase the people and/or animal intestine in the quantity of resistance pathogenic micro-organism.
The resistance marker gene is removed in expectation from transgenic plant Another reason is, owing to can be used for the selection limited amount of the marker gene of Plant Transformation, often make plant produce the many copies that contain the identical selection marker that is connected in the different effect gene by the multiple transgenosis combination of features of the hybridization of different transgenic strains.Because the use of this sign in combination subsequently got rid of in the existence of special sign gene in the transgenosis, this just makes each combination all need to use different selection marker systems.Therefore in the long term, may need to introduce than relating to the more transgenosis of obtainable suitable detected sign quantity.Another problem is the inhibition that a plurality of homologous sequences of existing in the plant will increase homology-dependent gene
4, this will seriously limit the reliable life-time service of genetically modified crops.
By the reason that need remove the resistance sign from transgenic plant that as above provides, developed many systems and guaranteed to remove selectable marker gene.For example, two kinds of different cotransformations that constitute thing can obtain being integrated with this two kinds of genetically modified transgenic strains
5, but the application of this method is limited, because it depends on the efficient (this is that they are separately needed in genetic cross) in these two kinds of transgenosiss insertion different genes group zones.In addition, so another defective of this method is owing to need genetic cross to waste time and energy.
As substituting of cotransformation, remove sign with some transposable element systems and locus specificity recombination system
6These systems need mediate the transposase of included zone disappearance between reorganization or swivel base target sequence or the expression of recombinase, remove this auxiliary gene by gene isolation subsequently, and this makes these systems relatively consuming time.In addition, the fragment of leaving out also can be inserted other genome position again, and recombinase or transposase protein might cause bad side effect.
The method that another kind is removed selectable gene marker is based on intrachromosomal homologous recombination (ICR) inducing DNA disappearance between two homologous sequences.Though by stimulating repair system can increase ICR, the inherent defective of ICR is that the too low deficiency of its frequency is so that this system is effectively applied to produce the disappearance in transgenosis zone.For example in tobacco, on average being less than 10 ICR incidents in all cells of the tobacco plant in 6 ages in week is can be detected.
Except the limitation of above-mentioned ICR method, we have also developed the ICR strategy based on the untapped so far zone reorganization of lambda particles phage, and we can produce surprising and enough high efficiency ICR, thereby can simplify the evaluation of disappearance transgenosis marker gene tissue.
Summary of the invention
One aspect of the present invention provides a kind of method of removing the transgenosis part after transgenosis is integrated into genome, comprise step: with the both sides that P zone (attP) places described transgenosis part of adhering to of lambda particles phage, and the homologous recombination between each flank attP zone of generation in the induced chromosome, thereby remove therebetween described transgenosis part.
This paper adheres to P zone (attP) and refers to comprise the lambda bacteriophage dna zone relevant with high recombination efficiency.
Preferably, described genetically modified described part comprises the auxiliary nucleic acid of marker gene and/or carrier sequence and/or other external source.
Preferably, this genome is a Plant Genome.
Preferably, the marker gene pointer is to the resistance of antibiotic and/or the resistance of weedicide.
Be appreciated that the term marker gene refers to comprise gene that participates in the specific biological route of synthesis and/or the gene that participates in environmental tolerence.
Preferably, marker gene is selected from nptII, Ble, dhfr, cat, aphIV, SPT, aacC3, aacC4, bar, EPSP, bxn, psbA, tfdA, DHPS, AK, sul, crsl-1 and tdc.
Preferably, this method can be deleted the zone of two interregional 10kb at the most of attP, more preferably is the zone of 7kb.
Preferably, when removing an above marker gene and/or carrier sequence and/or other auxiliary nucleic acid, wait that the flank of deleting each undesirable part of transgenosis all has the attP zone.Therefore be appreciated that method of the present invention can remove genome undesirable part more than simultaneously in the identical time.
Preferably, the 3052bp that is positioned at 27492 of lambda particles phages and 27844 interdigits is contained in the attP zone.
Preferably, attP contains in the zone the suitable fragment of the nucleotide sequence shown in the SEQ ID NO:1 or its function or can and have nucleic acid as the attP regional function with SEQ ID NO:1 hybridization under stringent condition, or because of the genetic code degeneracy variant and play the nucleic acid that the attP zone acts on the DNA of SEQ ID NO:1.
Method of the present invention provide a kind of after transgenosis is integrated into Plant Genome the novel strategy of the undesirable and/or other parts of this transgenosis of deletion.Method of the present invention has been developed lambda particles phage and has been adhered to the high recombination efficiency of the Unidentified so far potential in P zone (attP), deletes after the reorganization in the karyomit(e) in two attP zones.Proved that the attP system deleted a 5.9kb zone from the recombinant vectors that inserts two different genes group zones.With other based on the bacterium recombination system or use transposable element
6System's difference, need not the to recombinate expression of auxiliary protein of attP system causes disappearance.Therefore, this attP delet method of the present invention is that a kind of simply and effectively instrument improves the potentiality of Plant Transformation and solves many problems (human consumer who comprises the use of worry resistance sign).
Preferably, the attP zone is in the sequence box.
Preferably, this box also comprises conversion enhancement sequences (TBS) or its fragment that improves homology and unusual reorganization.
Usually TBS derives from green winter eggplant (Petunia hybrida).
Preferably, this very also comprises effector such as oryzacystastin-I or its function equivalent.
Another aspect of the present invention provides a kind of usefulness plant that method as herein described produces, and removed and be transformed into its genomic part, and this plant randomly also has preferred feature as herein described.
Preferably, this plant grows, and from its results plant product.
Another aspect of the present invention provides a kind of usefulness vegetable cell that method as herein described produces, thereby has removed a part that is transformed into this cell, and optional preferred characteristics with this paper the following stated.
Preferably, this plant grows, and from its results plant product.
Another aspect of the present invention provides a kind of attP reorganization box, and it contains marker gene and/or carrier sequence and/or other flank auxiliary nucleic acid of external source in each end of attP zone.
Preferably, this box also has arbitrary preferred characteristics as herein described.
Another aspect of the present invention provides plant or vegetable cell or the plant tissue that contains reorganization attP zone.The present invention also comprises seed in addition.
Another aspect of the present invention provides attP reorganization box, is used to remove the part that is integrated into Plant Genome and randomly has arbitrary preferred characteristics as herein described.
Another aspect of the present invention provides a kind of test kit, is used for removing this genetically modified part after transgenosis is integrated into the Plant Genome that comprises the above-mentioned attP reorganization box as this paper, and randomly has preferred characteristics as herein described.
Another aspect of the present invention provides a kind of plant or vegetable cell or plant tissue, and it contains and is integrated into its genomic reorganization transgenosis, and this transgenosis links to each other with lambda particles phage attP zone.
Preferably, plant or vegetable cell or plant tissue comprise at least one λ attP zone and effect transgenosis (being integrated into its genome).Described as can be seen plant or vegetable cell or plant tissue comprise at least two attP zones that influence ICR ideally, but described plant or vegetable cell or plant tissue can comprise an attP zone, thereby and side joint can be introduced subsequently in this transgenosis in the 2nd attP zone.
Preferably, be characterized as λ attp zone and a transgenosis of plant or vegetable cell or plant tissue do not assist nucleic acid to be connected with marker gene and/or carrier sequence and/or external source.
Preferably, this transgenosis also can with can improve conversion enhancement sequences or its fragment of homology and be connected with unusual reorganization.
Detailed Description Of The Invention
Now also further specify the present invention with reference to the accompanying drawings with embodiment.
Fig. 1 has shown the T-DNA zone of pattP-ICR.
Fig. 2 has shown the selection of no marks rotaring gene tobacco plant.
Fig. 3 has shown the pcr analysis to km-resistance and km-sensitive plant, and has comprised the nucleotide sequence (SEQ ID NO:1) in lambda particles phage attP zone; With
Fig. 4 has shown the Southern engram analysis to km-resistance and km-sensitive plant.
Fig. 1 has shown the T-DNA zone of pattP-ICR.At carrier pPCV002
8In the two ends of NPTII insert the 352bp zone (being positioned at genomic 27492 and 27844 of lambda particles phage) in two attp zones.In the attP box, we have inserted coding region and the polyadenylic acid zone of tms-2
9Coding region with GFP
10(being connected in no polyadenylic acid zone).By these two genes of two 1 '-2 ' promoter transcriptions.The attP box is embedded in 0.6kb DS element, in the presence of active A c transposase 5 ', enables to remove this complete box, and in the attP box, insert TBS fragment (thereby improving unusual and homologous recombination) and be example action effect gene with the oryzacystatin-I gene.Arrow indicates the zone by the primer specificity deletion amplification of effector (PE1 and PE2), NPTII gene (PN1 and PN2) and attP box (P5 ' and P3 ').To produce the disappearance of 5.9kb at two interregional ICR of attP, and only keep a zone and remove a zone between attP.The black arrow coding is used for the probe (Fig. 4) of Southern blot hybridization.
Fig. 2 has shown the selection (A) of no marks rotaring gene tobacco plant.Fig. 1 and 2 has cultivated green and white bud containing on the substratum of km.The activity (B) of the tms-2 gene of the white tissue of the potential disappearance of further analysis NPTII sign.Have the active bud of tms2 on the substratum of NAM and produce a large amount of callus rather than root (left side) containing, and the blastogenesis that lacks the white tissue regeneration of tms2 gene certainly becomes normal root (right side).
Fig. 3 has shown to Fig. 1 (swimming lane 2) and Fig. 2 (swimming lane 4) deutero-km resistance tissue and from the pcr analysis of the km-sensitive plant of Fig. 1 (swimming lane 3) and the white tissue derived of Fig. 2 (swimming lane 5).Swimming lane 1 contains the λ DNA of HindIII digestion as the size sign.Represent the segmental size of PCR with bp.
A. the PCR with primer PE1 and PE2 shows that all four swimming lanes all contain effector.
B. the PCR with primer PN1 and PN2 shows that the km-sensitive plant has lacked the NPTII gene.
C. use the PCR of primer P5 ' and P3 ' to show that two km resistance swimming lanes contain complete attP box, the km-sensitive plant of two about 6kb has then lacked the attP box.
D.PCR product order-checking shows that the swimming lane 3 among the C shows that reorganization has accurately interregionally kept an attP zone 5 ' and 3 '.
Fig. 4 has shown the Southern engram analysis from the DNA of km-susceptibility (swimming lane 3) separate tissue of km-resistance (swimming lane 2) and Fig. 3.
A. make zone " a " (see figure 1) probe with the genomic dna of ScaI digestion, the left junction fragment of the T-DNA that integrates with mark.
B. the genome SDNA with ScaI digestion makes zone " b " (see figure 1) probe, the right junction fragment of the T-DNA that integrates with mark.
In km susceptibility tissue, the left-end point fragment remains unchanged (A), and right terminal fragment is then owing to the disappearance in zone between two attP zones (B) has been lacked about 6kb.
Material and method
Plant Transformation
It is described to press people such as Koncz
12Joint, the pattP-ICR multipolymer is introduced among Agrobacterium tumefaciens (A.tumefacien) the bacterial strain GV3101 (pMP90PK).It is described to press people such as Horsch
13, carry out leaf sequin transformation of tobacco (Nicotiana tabacum) and Petit Havana SR1.
The preparation of DNA and analysis
Behind small-sized preparation DNA
14, contain 10mM TRIS-HCl PH 8.3 at 50 μ l, 50mMKCl, 2mM MgCl2, the 0.1%w/v gelatin, each Nucleotide of 0.2mM carries out PCR in the reaction volume of each primer of 25pmol and 1U Taq polysaccharase (Promega).Detecting the used PCR circulation of effector is: 94 ℃ 4 minutes; 94 ℃, 30 took turns 1 minute; 55 ℃ 30 seconds; 72 ℃ 30 seconds; With 72 ℃ 10 minutes; The PCR circulation of NPT II gene of being used to increase is: 94 ℃ 4 minutes; Took turns for 94 ℃ 30 1 minute; 60 ℃ 1 minute; 72 ℃ 1 minute; 72 ℃ 10 minutes.For the attP box that increases, grow template PCR with ExpandTM LongTemplate PCR System (Boehringer Mannheim) by manufacturers's explanation.The primer sequence that is used for PCR is:
PE1：?TCA?TCA?GAC?GGA?GGA?CCA?GTT?TTG?G (SEQ?ID?NO：2)
PE2：?ATC?CAT?GGT?TTT?TCC?CAA?ACT?TTA?G (SEQ?ID?NO：3)
PN1：?CCA?TGA?TCA?TGT?CGA?TTG?AAC?AAG?ATG (SEQ?ID?NO：4)
PN2：?CCA?TTT?TCC?ACC?ATG?ATA?TTC?GGC?AAG (SEQ?ID?NO：5)
P5’：GAA?TTC?TAA?TTC?GGG?ATG?ACT?GCA?ATA?TGG(SEQ?ID?NO：6)
P3’：GGA?TCC?AAC?GGG?ATA?TAC?CGG?TAA?CGA?AAA?GG(SEQ?ID?NO：7)
The Southern engram analysis
As people such as van Bloklad
15Described isolation of genomic DNA.Digest 15 μ g genomic dnas with ScaI, and on 0.7% sepharose, carry out the electrophoresis classification.Behind the electrophoresis, the DNA point sample on Nybond N filter, is used the UV radiation crosslinking, and press people such as Kose
16Described, 65 ℃ with
32The 0.3kb Eco RI/Sac I dna fragmentation hybridization of the effector of P mark.
The result
By the reorganization between phage connecting zone (attP) and bacterium connecting zone (attB), lambda particles phage is integrated in intestinal bacteria (E.coli) genome.Integration (Int) gene that in attP integrates, needs bacterium integration host factor (IHF) and encoding viral, and need add viral excisionase (Xis) protein when cutting.Three kinds of recombinant proteins are incorporated into 250bp attP fragment
17Defined in DNA.IHF has subsidiary function, makes the attP zone
18Crooked and may assist Int among the organism attP to enter the structure (this is required with the effective joint conference of attB) of nucleosome class
19By exchange with the protein mediated actual chain that between attP and attB nucleus, is taken place in the homology of 7bp of the active Int of topoisomerase I.The tyrosine residues of Int is incorporated into the 3 ' end of DNA, and is similar with Mammals topoisomerase I type and opposite with intestinal bacteria topoisomerase I (being incorporated into the 5 ' end of DNA)
20
In the present invention, we design and have made up plant conversion carrier pattP-ICR, and wherein we insert two 352bp flanks in the attP zone of NPTII resistance sign, GFP gene and tms2 gene (Fig. 1).We have settled conversion enhancement sequences (TBS) (to strengthen homology and unusual reorganization on an attP side
21) and oryzacystastin-I gene 22 (as the example of ' effect ' gene, finally being transformed into genome by the attP system).Two interregional ICR of attP can remove the interregional 5.9kb zone of attP, and produce the transgenosis that keeps effector and TBS sequence.Transform with the leaf sequin, this construction is introduced tobacco, and on kantlex (km) substratum, select resistant calli.Transform back 2 months, 11 km resistant callis (0.5cm diameter) are transferred in the substratum of no km.When callus length arrives the 5-6cm diameter, they are cut into less part transfer to again in the shoot regeneration substratum that contains km.After 3-5 month, all grown a plurality of sproutings at all on these 11 callus, we find to have two clones (clone) to mix and produce green and white sprouting.Further these two clones of research (swimming lane 1 and 2 among Fig. 2) assess the tissue whether white sprouting contains disappearance NPTII resistance sign and tms2 gene.
The white blade of these two series is placed on the regeneration culture medium of no km, the sprouting that produces is placed on the substratum that is supplemented with naphthalene niacinamide (NAM).Because the activity of tms2 changes into growth hormone NAA with NAM
9What the plant of expressing the tms2 gene produced that thereby high-caliber growth hormone prevented that the growth of root from replacing then is the generation of having induced callus.Organizing of the intersegmental disappearance of two attP sheets zone also lacks the tms2 gene, thereby can be identified containing the ability that forms root on the NAM substratum by it.12 of 32 sproutings of 11 and series 2 produce roots in series 20 sproutings of 1, and this 23 strain seedling is further analyzed as the material standed for of disappearance NPTII gene and tms2 gene.Pcr analysis shows that all these 23 strain seedlings have all lost NPTII gene and tms2 gene really.Having in series 1 the 11 strain seedlings in the 12 strain seedlings of 2 strains and series 2 has 1 strain also to keep effector, and all other series have all lacked effector.
With represent series 1 and series 2 keep effectors as further identifying the levels of precision of analyzing its disappearance incident.We with series 1 and series 2 km-resistance sprouting in contrast.The pcr analysis that carries out in the primer of attP dipolymer box regiospecificity with pairing effect gene, NPTII gene and flank shows and kept effector, and lacked the NPTII gene and be reduced about 6kb (2 attP zone reorganization as expected) (Fig. 3) in 2 intersegmental zones of attP sheet.Two kinds of clones produce identical PCR pattern, show that they are all produced by identical disappearance product.To a PCR sequencing fragment, show in 5 ' and 3 ' interregional accurate the kept attP zone of flank in the attP box, this with estimate consistent promptly in 5.9kb zone that the intersegmental ICR of two attP sheets has deleted the two.The green of Southern engram analysis series 1 and white tissue have shown PCR data (Fig. 4).
The seedling in fact most of disappearance NPTII/tms2 zone also lacks the transgenosis zone outside the attP box, shows ICR not with homologous recombination is relevant accurately between two attP, but may produce a large amount of the disappearance because of unusual reorganization.But ICR can clearly identify in the interregional accurate karyomit(e) of generation where of 2 attP and recombinate.As if owing to can find identical disappearance in two different transformant, the interregional effective reorganization of attP not only occurs in the transgenosis that is integrated into specific gene group integration zone.Our data show with the less callus enlightenment of relative populations, can select the accurately same source material of disappearance, and owing to the feasibility of round pcr easily distinguishes them with bad disappearance (also having other disappearance the disappearance except that between two attP sites) mutually.At least 35% sprouting is the tissue regeneration that shows disappearance from white, comes preliminary election to lack to it seems it is unnecessary with the tms2 gene, can substitute it with PCR.
The reason of the high relatively ICR efficient of attP fragment it be unclear that, but estimates that this effect may be because the accessibility that improve in the attP zone.Reorganization in the tobacco somatic chromosome is the part by the impaired stimulation repair mechanism of DNA
7The efficient of ICR is usually than the low several magnitude of extrachromosomal reorganization in the plant, and this shows DNA is packaged into has significantly protected it to avoid reparation/recombination mechanism in the chromatin
23The accessibility in the DNA zone of improving owing to the change of chromatin Structure has a significant effect to the efficient of ICR.The support of this hypothesis derives from the experiment of human fibroblasts's knurl, and its nucleosome that shows that DNA reparation synthetic improves than the acetylize reduction in the super acetylizad nucleosome has more open chromatin Structure
24
The attP fragment has high relatively AT content (67%), and this may just improve its accessibility potentially.The zone that to be rich in AT is considered as unusual transgenosis of recombinating mediation and is integrated into the preferred target position of Plant Genome, and estimates that the curvature of space that high AT content can increase by the chromosomal DNA structure carries out the possibility that DNA integrates.As for explaining the working hypothesis of the high recombination efficiency in attP site, we think because the sequence composition in attP zone or experienced the change of conformation with the nucleoprotein that the attP zone links to each other, and attP has improved in the zone its accessibility to reparation/recombinase.In bacterium, by the bending in IHF mediation attP zone, little basic protein constitutes two dissimilar subunits.These two kinds of subunits all show the bacterioprotein of class histone
26Characteristic, and can suppose that attP zone and plant histone or class histone proteinic react to each other can cause warp architecture equally.
Though causing the molecular mechanism of the high ICR efficient in attP zone still fails to determine, but the discovery to this accidental action provides a kind of useful instrument, comes to remove from transgenic plant rapidly and effectively resistance marker gene or any other bad transgenosis zone.
Reference
1.Dale, the distribution of P.J. cydorge gene and wild relations, Plant Physiol.100,13-15 (1992).
2.Hoffmann, T., Golz, C.﹠amp; Schieder, O. and transgenosis higher plant are cultivated back aspergillus niger wild type strain altogether and have obtained exogenous DNA array, Curr Genet 27,70-6 (1994).
3.Gressel J. is used to sow wild-type avenaceous selectable marker does not have difference? TIBTECH10,282 (1992).
4.Matzke, M.A.﹠amp; Matzke, the transgenosis of A.J.M. plant is by the difference deactivation of the homologous sequence that contains two kinds of suppressor gene locus and methylate Plant Mol.Biol.16,821-830 (1991).
5.De Block, M.﹠amp; Debrouwer, D. mainly is integrated into identical locus, Theor.Appl.Genet.82,257-263 (1991) by two kinds of T-DNA that two edaphic bacilluss infection corotation dissolve turnip (brassicanapus).
6.Yoder, J.I.﹠amp; Goldsbrough, A.P. produces the conversion system of the transgenic plant of no marks gene, and BIO/TECHNOLOGY 12,263-267 (1994).
7.Puchta, H., Swoboda, P., Gal, S., Blot, M.﹠amp; Hohn, the homologous recombination in the equal kind of B. plant in the somatic chromosome, Plant Mol Biol 28,281-92 (1995).
8.Konz, C.﹠amp; Schell, the promotor control of J.TL-DNA gene carries the expression of the mosaic gene of the two carriers of new soil bacillus, Mol.Gen Genet.204,383-396 (1986).
9.Dipicker, A.G., Jacobs, A.M.﹠amp; Van Montagu, M.C. is to expressing the negative selection flow process of the cell of T-DNA gene, Plant Cell Reports 7,63-66 (1988) from the tobacco protoplast deutero-.
10.Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W.﹠amp; Prasher, D.C. egfp are as the mark of genetic expression, and Science 263,802-5 (1994).
11.Velten, J.﹠amp; Schell, J. separates two plant promoter fragments from the Ti-plasmids of Agrobacterium tumefaciens, EMBO J.3,2723-2730 (1984).
12.Koncz people such as C. are used for the idiosyncratic carrier of gene target and expression study, the biological handbook B2 of plant molecular, 1-22 (1994).
13.Horsch people such as R.B. go into the simple and regular method of plant, Science227,1229-1231 (1985) with gene transformation.
14.Dellaporta, S.J., Wood, J.﹠amp; Hicks, the small-sized preparation of J.B. DNA of plants, PlantMol.Biol.Rep.1,19-21 (1983).
15.Van Blokland, R., Ross, S., Corrado, G., Scollan, C.﹠amp; Meyer, P. and the transgene tobacco Desoxyadenosine relevant heteroplasia that methylates, The Plant Journal 15,543-551 (1998).
16.Koes, R.E., Spelt, C.E.Mol.J.N.M.﹠amp; Gerats, the chalcone synthase multigene family of G.M.Petunia hybrida (V30): sequence homology, chromosomal localization and its evolution aspect, Plant MolecularBiology 10,159-169 (1987).
17.Gardner, J.F.﹠amp; Nash, the effect of H.A. intestinal bacteria IHF protein in the reorganization of λ locus specificity, J.Mol.Biol.191,181-189 (1986).
18.Yang, C.﹠amp; H., N. intestinal bacteria IHF protein engages the interaction between the site with its specificity, and Cell 57,869-880 (1989).
19.Craig, N.L.﹠amp; Nash, the mechanism of H.A. lambda particles phage locus specificity reorganization: the Int topoisomerase is to the locus specificity fracture of DNA, and Cell 39,707-716 (1984).
20.Weisberg, R.A.﹠amp; Landy, A. locus specificity reorganization (Hendrix.R.W.Roberts, J.W.Stahl, the F.W.﹠amp on the λ of lambda particles phage II; Weisberg, R.A. edits) 211-250 (Cold SpringHarbor, New York, 1983).
21.Galliano, H., Muller, A.E., Lucht, J.M.﹠amp; It is the derivative that is incorporated into the retrotransposon of nuclear framework that Meyer, P. transform enhancement sequences, Mol Gen Genet 247,614-622 (1995).
22.Urwin, P.E.Atkinson, H.J., Waller, D.A.﹠amp; McPherson, the engineering oryzacystatin-I that M.J. expresses in the transgenosis root of hair give Globodera pallida resistance, Plant J8,121-31 (1995).
23.Puchta, H.﹠amp; The substrate specificity of the plant recombinase that Meyer, P. determine in the recombination system outside karyomit(e), homologous recombination of plant and gene silencing (Paszkowski, J. edits), 123-155 (KluwerAcademic Publishers, Dortrecht, 1994).
24.Ramanathan, B.﹠amp; Smerdon, enhanced DNA repairs and synthesizes in the super acetylize nucleosome of M.J., and J.Biol.Chem 264,11026-34 (1989).
25.Takano, M.Egawa, H., Ikeda, J.E.﹠amp; Wakasa, the structure of integration site in the K. transgenosis rice, Plant J 11,353-61 (1997).
26.Drlica, K.﹠amp; Rouviere-Yaniv, the class histone protein in the J. bacterium, MicrobiolReview 51,301-319 (1987).
Claims (22)
1. after being integrated into genome, transgenosis removes transgenosis method partly for one kind, it is characterized in that, comprise step: with the both sides that P zone (attP) places described transgenosis part of adhering to of lambda particles phage, and the homologous recombination between each flank attP zone of generation in the induced chromosome, thereby remove therebetween described transgenosis part.
2. the method for claim 1 is characterized in that, described transgenosis contains the auxiliary nucleic acid of marker gene and/or carrier sequence and/or other external source.
3. method as claimed in claim 1 or 2 is characterized in that described marker gene is given the resistance at antibiotic and/or weedicide.
4. as claim 1,2 or 3 arbitrary described methods, it is characterized in that described marker gene participates in the specific biological route of synthesis and/or participates in environmental tolerence.
5. as the arbitrary described method of claim 1-4, it is characterized in that described marker gene is selected from: nptII, Ble, dhfr, cat, aphIV, SPT, aacC3, aacC4, bar, EPSP, bxn, psbA, tfdA, DHPS, AK, sul, crsl-1 and tdc.
6. as the arbitrary described method of claim 1-5, it is characterized in that described method is removed more than one marker gene and/or carrier sequence and/or exogenous nucleic acid part from transgenosis, and the flank of each deleted part all has the attP zone.
7. as the arbitrary described method of claim 1-6, it is characterized in that, described attP contain in the zone lambda particles phage 27492 and 27844 between 352 base pairs or the suitable fragment of its function.
8. as the arbitrary described method of claim 1-7, it is characterized in that, described attP contains in the zone the suitable fragment of the nucleotide sequence shown in the SEQID NO:1 or its function or can and have nucleic acid as the attP regional function with SEQ IDNO:1 hybridization under stringent condition, or because of the genetic code degeneracy variant and play the nucleic acid that the attP zone acts on the DNA of SEQ IDNO:1.
9. as the arbitrary described method of claim 1-8, it is characterized in that described attP zone is in box.
10. method as claimed in claim 9 is characterized in that, described box also contains conversion enhancement sequences or its fragment, to strengthen homology and unusual reorganization.
11. as claim 9 or 10 described methods, it is characterized in that, described box also contain effector such as oryazcyctastin-I or with its function equivalent.
12., it is characterized in that described genome is a Plant Genome as the arbitrary described method of claim 1-11.
13. plant, vegetable cell or plant tissue that produces with the arbitrary described method of claim 1-12.
14. one kind comprises the method for carrying out the described method generation plant of claim 12 or the described plant of claim 13 or vegetable cell or plant tissue being provided, it is characterized in that described method comprises growing plant and/or gathers in the crops its product.
15. contain plant or the vegetable cell or the plant tissue in reorganization attP zone.
16.attP the reorganization box is characterized in that, comprising flank is marker gene and/or the carrier sequence and/or the auxiliary nucleic acid of external source in attP zone.
17. the purposes of the described attP reorganization of claim 16 box is characterized in that, is used to remove the part that is integrated into Plant Genome.
18. treat that transgenosis is integrated into the test kit that is removed after the Plant Genome for one kind, it is characterized in that, contain attP reorganization box as claimed in claim 16.
Be integrated into the genetically modified plant of genomic reorganization or vegetable cell or plant tissue 19. contain, it is characterized in that, described transgenosis links to each other with lambda particles phage attP zone at its each end.
20. plant as claimed in claim 19 or vegetable cell or plant tissue is characterized in that, also comprise the lambda particles phage attP zone shown in and one and are integrated into its genomic effect transgenosis.
21. plant as claimed in claim 20 or vegetable cell or plant tissue is characterized in that, a described lambda particles phage attP zone and a transgenosis do not assist nucleic acid to link to each other with marker gene and/or carrier sequence and/or other external source.
22. as the arbitrary described plant of claim 19-21 or vegetable cell or plant tissue, it is characterized in that, described transgenosis also with can strengthen conversion enhancement sequences or its fragment of homology and link to each other with unusual reorganization.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9921937.0A GB9921937D0 (en) | 1999-09-17 | 1999-09-17 | Targeted gens removal |
| GB9921937.0 | 1999-09-17 |
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| EP (1) | EP1212443A2 (en) |
| JP (1) | JP2003510043A (en) |
| CN (1) | CN1375010A (en) |
| AU (1) | AU7532500A (en) |
| BR (1) | BR0014521A (en) |
| CA (1) | CA2384932A1 (en) |
| GB (1) | GB9921937D0 (en) |
| HU (1) | HUP0202805A2 (en) |
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| US6750379B2 (en) | 2000-03-09 | 2004-06-15 | Dekalb Genetics Corporation | Homologous recombination-mediated transgene alterations in plants |
| US6580019B1 (en) | 2000-03-09 | 2003-06-17 | Dekalb Genetics Corporation | Non-reciprocal recombination-mediated transgene deletion in transgenic plants |
| WO2001081600A2 (en) * | 2000-04-20 | 2001-11-01 | Btg International Limited | Transgenic plants |
| AU2006246975B2 (en) * | 2005-05-17 | 2011-08-11 | Ozgene Pty Ltd | Sequential cloning system |
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| US4673640A (en) * | 1984-04-30 | 1987-06-16 | Biotechnica International, Inc. | Regulated protein production using site-specific recombination |
| NL8701450A (en) * | 1987-06-22 | 1989-01-16 | Solvay | METHOD FOR TRANSFORMING CELLS. |
| JPH05507853A (en) * | 1990-06-12 | 1993-11-11 | ベイラー・カレッジ・オブ・メディシン | Homologous recombination methods in animal and plant cells |
| US5736367A (en) * | 1992-03-31 | 1998-04-07 | Medimmune, Inc. | Vectors and prokaryotes which autocatalytically delete antibiotic resistance |
| US5527695A (en) * | 1993-01-29 | 1996-06-18 | Purdue Research Foundation | Controlled modification of eukaryotic genomes |
| AU728915B2 (en) * | 1996-05-09 | 2001-01-18 | Nippon Paper Industries Co. Ltd. | Vector for introducing a gene into a plant from which a selectable marker gene can be optionally removed |
| CN100342008C (en) * | 1997-10-24 | 2007-10-10 | 茵维特罗根公司 | Recombinational cloning using nucleic acids having recombinatin sites |
| WO2001007572A2 (en) * | 1999-07-23 | 2001-02-01 | The Regents Of The University Of California | Dna recombination in eukaryotic cells by the bacteriophage phic31 recombination system |
-
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- 2000-09-15 CN CN00813040.XA patent/CN1375010A/en active Pending
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| EP1212443A2 (en) | 2002-06-12 |
| BR0014521A (en) | 2002-06-11 |
| CA2384932A1 (en) | 2001-03-29 |
| AU7532500A (en) | 2001-04-24 |
| GB9921937D0 (en) | 1999-11-17 |
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| WO2001021780A3 (en) | 2001-10-18 |
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