CN102477443A - Method for effectively reducing gene drift of transgene plant through pollen mediation - Google Patents
Method for effectively reducing gene drift of transgene plant through pollen mediation Download PDFInfo
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- CN102477443A CN102477443A CN2010105635559A CN201010563555A CN102477443A CN 102477443 A CN102477443 A CN 102477443A CN 2010105635559 A CN2010105635559 A CN 2010105635559A CN 201010563555 A CN201010563555 A CN 201010563555A CN 102477443 A CN102477443 A CN 102477443A
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Abstract
The invention provides a method for effectively reducing environmental risk caused by pollen propagation of transgene plant. In the method, two tightly linked gene expression cassettes of 1) a pollen lethal gene ZM-AA1 driven by a pollen growth anaphase singular promoter PG47 and 2) a fluorescence screening mark gene FP driven by a callus / seed coat singular promoter END 2 are transferred to a Zhonghua 11 wild type material. During selfing fructification of a single plant carrying a single copy transgene, a pollen grain without carrying the transgene can be fertilized with a female gamete normally, but a pollen grain carrying the transgene is abortive in pollen growth anaphase and can not be fertilized with a female gamete, so as to reduce risk of transgene drift in the environment.
Description
Technical field
The invention provides and a kind ofly stop transgenic plant and foreign gene is passed to the method for other plant, thereby effectively reduce the environmental exposure of transgenic plant, belong to biological technical field through pollen.
Background technology
In recent years, along with the swift and violent increase of transgenic plant kind and cultivated area, the extensive environment of genetically modified organism discharges and the environmental organism safety-problems that possibly bring has become one of field with dispute of greatest concern, the present whole world day by day.Foreign gene in the transgenic plant is transferred in the non-transgenic kind or its wild relatives (comprising the weeds kind) in the environment through genetic drift (or natural hybrization); And through heredity gradually ooze in the colony of wild relatives, retain or further the diffusion, thereby bring the environmental organism safety-problems.The genetic drift of pollen-mediated extensively is present between the floristics with cross compatibility, comprises the genetic drift between same species different groups and the different plant species.Compare with the genetic drift of other form (like the genetic drift of seed mediation), the genetic drift of pollen-mediated be owing to can be undertaken by wind-force and insect, thereby avoids least easily.
Have been found that at present some reduce the method for foreign gene diffusion in pollen, common as chloroplast(id) transform, the removal etc. of foreign gene in the pollen.
The chloroplast(id) of most plants is matrocliny, if therefore exogenous origin gene integrator on the chloroplast gene group, just can not can avoid the diffusion of foreign gene through pollen to other plant diffusion.Yet chloroplast(id) only transforms and in the minority plant, utilizes particle bombardment to obtain success.In addition; Chloroplast(id) transforms carrier and includes the chloroplast(id) homologous fragment; Fixed point to guarantee foreign gene makes up, and the chloroplast gene group sequence of most plants is unclear, therefore; Can't confirm that the homologous fragment and the foreign gene that are used for vector construction insert the site, can only accomplish through conserved sequence unknown nucleotide sequence chloroplast gene group.The crucial problem of another one is; The most plants cell contains 10-100 chloroplast(id); Each chloroplast(id) contains again up to 100 plastom copies; And the plastom that has only minority can transform success, and foreign gene is entirely integrated in each DNA copy of chloroplast(id) needs effective tissue culture method and triage techniques, and for most of monocotyledonss, does not also set up effective conversion and screening system at present.
Specific site reorganization (like Cre/loxP system, FLP/FRTs system and Zinc finger nuclease (ZFNs) system) is the method that external source gene-splicing in the pollen is fallen.In this type system, pollen-specific promoters driven site differential recombination enzyme accurately excises the transgenic element of its recognition site side, and does not influence the normal generation and the function of pollen, but the efficient of this method is lower.The loxP/FRTs system of recent development can be compared Cre/loxP and FLP/FRTs by Cre or any recombinase identification of FLP, have higher efficient (Luo, K. et al., 2007), but this technology has only obtained checking at present in tobacco.
Male sterile is a kind of in addition method that is widely used in avoiding in the commercialization transgenic plant drift of pollen-mediated transgenic.Mariani etc. utilize the Barnases gene to create the male sterile line of tobacco and rape, and female parent is done by male sterile plant system, and male parent is done by wild-type strain system, and the cross-fertilize seed of acquisition recovers fertility by the Barstar gene.Yet its negative effect shows, compares with contrast, is that the insect of food such as quantity that pollen beetle grows up to adult obviously reduce with pollen.And, evidence suggests that barnase has toxicity to animal and human's class cell, therefore needing the position of plant specifically expressing barnase is not the part that is eaten.In addition; Barnase also exists a lot of problems in application facet; For example: male-sterile character has temperature sensitivity; Even affected by environment and unstable under the pollen-specific promotor easily, the toxicity of barnase also might be revealed and be expressed in other position of plant materials, thereby causes that there are unstable etc. in the variation of various Main Agronomic Characters, barnase in the offspring separates.Therefore, this method neither the ideal control transgenic method of drift.
Summary of the invention
The invention provides the method for a kind of effective prevention transgenic plant through pollen transmission to other rice varieties, wild relatives, weeds and other plant.The core technology of this method is to utilize pollen to form later stage specific promoter PG47 to drive pollen lethal gene ZM-AA1, will carry genetically modified pollen and kill, and not be with genetically modified pollen still can work orderly.
The invention has the advantages that: 1) have genetically modified pollen granule because carry glucose metabolism genes and the complete deactivation of a plant origin, efficient is up to 100%, reduced the environmental exposure that the genetic drift by pollen-mediated causes; 2) the pollen lethal gene is driven by pollen development later stage specific promoter, and half formation and function of not carrying genetically modified pollen granule is not in addition had influence fully; 3) foreign gene can pass through the megagamete transmission.
The present invention realizes through following scheme: at first make up conversion carrier, with the pollen lethal gene
ZM-AA1Place pollen development later stage specificity promoter PG47 downstream, red fluorescent protein
FPGene places callus and plants skin specificity promoter END2 downstream, two expression casette close linkages; Utilize then and spend 11 ratarias or mature embryo inductive callus in the wild-type rice varieties, utilize agriculture bacillus mediated method for transformation that foreign gene is imported as transforming explant; Recipient cell utilizes fluorescent microscope to carry out three screenings in after cultivating altogether 30 days, and the callus of stably express red fluorescence is divided into seedling; Further carry out the evaluation of molecular biology identification and pollen activity to transforming seedling.
The present invention realizes through following steps successively:
1) vector construction: the pollen lethal gene is connected in pollen development later stage specificity promoter downstream, makes the pollen abortion of expressing this fusion gene.With the selection markers gene fusion in callus with plant skin specificity promoter downstream, thereby can be to transforming individual callus and planting skin and screen.The two close linkage is implemented in the T-DNA district of expression vector.
2) utilize agriculture bacillus mediated conversion method rice transformation, screen through selection markers gene pairs transformant, and then be divided into regeneration plant.
3) molecular Biological Detection: utilize PCR, Southern blot method is screened transfer-gen plant and is identified.
4) mensuration of the deadly ability of pollen: adopt I
2-KI staining is observed the pollen types on the transfer-gen plant and is judged its fertility.
5) utilize χ
2Whether the ratio that seed kind fluorescent seeds (transgenic seed) and non-fluorescent seeds (non-transgenic seed) are tied in the selfing of-inspection statistics transfer-gen plant conforms to 1:1.
Description of drawings
Fig. 1 has shown plant expression vector pSPT05 structural representation among the embodiment 1.
Fig. 2 has shown the rice callus tissue of after Agrobacterium-mediated Transformation, expressing red fluorescent protein among the embodiment 2.
Fig. 3 has shown the reuse water rice plants that after Agrobacterium-mediated Transformation, obtains among the embodiment 2.
Fig. 4 has shown that the PCR of part plant among the embodiment 3 identifies (M:DNA molecular weight standard;-: negative/negative control; +: just/positive control).
Fig. 5 has shown that the Southern blot of part transfer-gen plant among the embodiment 4 identifies (N: negative control, M: molecular weight standard; Plasmid: to transform plasmid) as positive control.
Fig. 6 has shown pollen I among the embodiment 5
2Pollen granule after the-KI dyeing.
Embodiment
Specifically describe technical scheme of the present invention below in conjunction with embodiment and accompanying drawing, but be not limited thereto.
Be specifically related to two genes in the embodiment of the invention,
ZM-AA1With
RP ZM-AA1Coding glycase; Under the driving of pollen development later stage specificity promoter, it can give expression to glycase in the mature pollen later stage, thus the starch in the degraded pollen granule; Finally make pollen abortion, its nucleotide sequence and aminoacid sequence are seen SEQ ID NO.1 and SEQ ID NO.2 respectively;
RPGene is as the selection markers gene; Derive from reef coral (Discosoma sp.); Its codified red fluorescent protein; Can be inspired red fluorescence, be convenient to the screening of transgenic callus and seed, its nucleotide sequence and aminoacid sequence are seen SEQ ID NO.3 and SEQ ID NO.4 respectively.
Embodiment 1: construction of expression vector
Transform the expression vector name of using among the present invention and be called pSPT05 (accompanying drawing 1).This carrier is artificial constructed on the pPZP basis.2 expression casettes are arranged on the expression vector:
ZM-AA1Expression casette; This expression cassette drives (its nucleotide sequence is seen SEQ ID NO.5) by the pollen development later stage specificity promoter PG47 from corn; And by stopping transcribing (its nucleotide sequence is seen SEQ ID NO.6) from the terminator IN2-1 of corn; Transit peptides ZM-BT1 is blended between ZM-AA1 gene and the PG47 promotor in addition, and the nucleotide sequence of ZM-BT1 is seen SEQ ID NO.7, report/selection markers gene
RPExpression casette; This expression cassette drives (its nucleotide sequence is seen SEQ ID NO.8) by callus and the seed specific promoters END2 from corn; And by stopping transcribing from the terminator PIN II of yam, the nucleotide sequence of PIN II is seen SEQ ID NO.9.Do not contain the antibiotics resistance marker gene in the T-DNA section, do not contain the herbicide screening marker gene yet.
Embodiment 2: obtain transgenic rice plant
Conversion carrier among the embodiment 1 is imported among the agrobacterium strains AGLO with electric shocking method, be used for transforming wild-type and spend 11 inductive callus.Utilize red fluorescent protein that transformed calli is screened, accompanying drawing 2 is the callus of expression alien gene, further with its differentiation regenerate transformed plant (accompanying drawing 3) of expression alien gene.
Embodiment 3: the PCR of transfer-gen plant detects
Get the transgenic rice plant blade among the embodiment 2, extract total DNA.With
DsRED2Gene order is a template design primer, to T
0Carry out pcr amplification for the transgenic paddy rice genomic dna.The amplified production fragment is 789bp.Amplification program is: 94 ℃ of 10min; 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 1min; 37 circulations; 72 ℃ of 10min.The forward primer sequence is 5 '-GGACTTGAACTCCACCAGG-3 '; The reverse primer sequence is: 5 '-ATAATGCCAATACGACACC-3 '.Accompanying drawing 4 is part transfer-gen plant pcr amplification result, explains that foreign gene has been integrated in the paddy rice acceptor gene group.
Embodiment 4: the Southern Blot of transfer-gen plant detects
To T
0Carry out Southern Blot for the transgenic paddy rice genomic dna and detect, to identify the integration situation of foreign gene in the rice conversion acceptor.Get T among the embodiment 2
0For the transgenic rice plant blade, extract total DNA.With
FP526bp nucleotide fragments in the gene order is probe, selects NEB company for use
XbaThe I restriction enzyme carries out digestion reaction.The endonuclease reaction system is 200ul: genomic dna 15ug, NE Buffer 20ul, BSA 2ul,
XbaI 4ul (20U/ul) also complements to 200 ul with vaal water with system.37 ℃ of incubations 6 hours.Accompanying drawing 5 is a part transfer-gen plant Southern Blot detected result.The result shows that foreign gene has been integrated in the acceptor rice genome.
Embodiment 5: the mensuration of the deadly ability of pollen
Adopt I
2-KI staining is observed the form of transgenic paddy rice pollen and is judged its fertility (accompanying drawing 6).Investigated 1995 of pollen altogether, normal fertile flower powder is 1015 (50.8%), and pollen sterile is 980 (49.2%).Whether the two is met the 1:1 law of segregation carried out χ
2-check (degree of freedom is 1).χ
2Value is 0.614, and the threshold value 3.814 during far below level of signification 0.05 proves that the two ratio meets 1:1 in P≤0.05 level.Therefore think
ZM-AA1It is thorough that gene kills the pollen function.
Sequence table
< 110>Beijing Weiming Kaituo Crops Design Center Ltd
The Beijing Kaituo Dna Biotech Research Center Co., Ltd
< 120>a kind of effective reduction transgenic plant are through the method for pollen-mediated genetic drift
<160> 9
<170> PatentIn version 3.3
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<213>Corn (
Zea mays)
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atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60
ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120
gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180
gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcggcctg cggcctggtc 240
caggcacaag tcctcttcca ggggtttaac tgggagtcgt gcaagcagca gggaggctgg 300
tacaacaggc tcaaggccca ggtcgacgac atcgccaagg ccggcgtcac gcacgtctgg 360
ctgcctccac cctcgcactc cgtctcgcca caaggctaca tgccaggccg cctatacgac 420
ctggacgcgt ccaagtacgg cacggcggcg gagctcaagt ccctgatagc ggcgttccac 480
ggcaggggcg tgcagtgcgt ggcggacatc gtcatcaacc accggtgcgc ggaaaagaag 540
gacgcgcgcg gcgtgtactg catcttcgag ggcgggactc ccgacgaccg cctggactgg 600
ggccccggga tgatctgcag cgacgacacg cagtactcgg acgggacggg gcaccgcgac 660
acgggcgagg ggttcgcggc ggcgcccgac atcgaccacc tcaacccgcg cgtgcagcgg 720
gagctctccg cctggctcaa ctggctcagg tccgacgccg tggggttcga cggctggcgc 780
ctcgacttcg ccaagggcta ctcgccggcc gtcgccagaa tgtacgtgga gagcacgggg 840
ccgccgagct tcgtcgtcgc ggagatatgg aactcgctga gctacagcgg ggacggcaag 900
ccggcgccca accaggacca gtgccggcag gagctgctgg actggacgcg ggccgtcggc 960
gggcccgcca tggcgttcga cttccccacc aagggcctgc tgcaggcggg cgtgcagggg 1020
gagctgtggc ggctgcgcga cagctccggc aacgcggccg gcctgatcgg gtgggcgccc 1080
gagaaggccg tcaccttcgt cgacaaccat gacaccgggt cgacgcagaa gctctggccg 1140
ttcccatccg acaaggtcat gcagggctac gcctacatcc tcacccatcc aggagtcccc 1200
tgcattttct acgaccacat gttcgactgg aacctgaagc aggagatatc cacgctgtct 1260
gccatcaggg cgcggaacgg catccgcgcc gggagcaagc tgcggatcct cgtggcggac 1320
gcggacgcgt acgtggccgt cgtcgacgag aaggtcatgg tgaagatcgg gacaaggtac 1380
ggcgtgagca gcgtggtccc gtcggatttc cacccggcgg cgcacggcaa ggactactgc 1440
gtctgggaga aagcgagcct ccgcgtcccg gcggggcgcc acctctagca gctcagattg 1500
ctcagtcttg tgctgcattg caaacacagc agcacgacac tgcataacgt cttttccttg 1560
a 1561
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<213>Corn (
Zea mays)
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cacaacaccg tgaagctgaa ggtgacgaag ggcggccccc tgcccttcgc ctgggacatc 180
ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240
gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300
gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360
aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420
atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480
acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540
tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600
atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660
caccacctgt tcctgtag 678
<210> 4
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<212> DNA
<213>Corn (
Zea mays)
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ccagacagtc cggtgtgcca gatcagggca cccttcggtt cctttgctcc tttgcttttg 120
aaccctaact ttgatcgttt attggtttgt gttgaacctt tatgcacctg tggaatatat 180
aatctagaac aaactagtta gtccaatcat ttgtgttggg cattcaacca ccaaaattat 240
ttataggaaa aggttaaacc ttatttccct ttcaatctcc ccctttttgg tgattgatgc 300
caacacaaac caaagaaaat atataagtgc agaattgaac tagtttgcat aaggtaagtg 360
cataggttac ttagaattaa atcaatttat acttttactt gatatgcatg gttgctttct 420
tttattttaa cattttggac cacatttgca ccacttgttt tgttttttgc aaatcttttt 480
ggaaattctt tttcaaagtc ttttgcaaat agtcaaaggt atatgaataa gattgtaaga 540
agcattttca agatttgaaa tttctccccc tgtttcaaat gcttttcctt tgactaaaca 600
aaactccccc tgaataaaat tctcctctta gctttcaaga gggttttaaa tagatatcaa 660
ttggaaatat atttagatgc taattttgaa aatataccaa ttgaaaatca acataccaat 720
ttgaaattaa acataccaat ttaaaaaatt tcaaaaagtg gtggtgcggt ccttttgctt 780
tgggcttaat atttctcccc ctttggcatt aatcgccaaa aacggagact ttgtgagcca 840
tttatacttt ctccccattg gtaaatgaaa tatgagtgaa agattatacc aaatttggac 900
agtgatgcgg agtgacggcg aaggataaac gataccgtta gagtggagtg gaagccttgt 960
cttcgccgaa gactccattt ccctttcaat ctacgactta gcatagaaat acacttgaaa 1020
acacattagt cgtagccacg aaagagatat gatcaaaggt atacaaatga gctatgtgtg 1080
taatgtttca atcaaagttt cgagaatcaa gaatatttag ctcattccta agtttgctaa 1140
aggttttatc atctaatggt ttggtaaaga tatcgactaa ttgttctttg gtgctaacat 1200
aagcaatctc gatatcaccc ctttgttggt gatccctcaa aaagtgatac cgaatgtcta 1260
tgtgcttagt gcggctgtgt tcaacgggat tatccgccat gcagatagca ctctcattgt 1320
cacataggag agggactttg ctcaatttgt agccatagtc cctaaggttt tgcctcatcc 1380
aaagtaattg cacacaacaa tgtcctgcgg caatatactt ggcttcggcg gtagaaagag 1440
ctattgagtt ttgtttcttt gaagtccaag acaccaggga tctccctaga aactgacaag 1500
tccctgatgt gctcttccta tcaattttac accctgccca atcggcatct gaatatccta 1560
ttaaatcaaa ggtggatccc ttggggtacc aaagaccaaa tttaggagtg taaactaaat 1620
atctcatgat tcttttcacg gccctaaggt gaacttcctt aggatcggct tggaatcttg 1680
cacacatgca tatagaaagc atactatctg gtcgagatgc acataaatag agtaaagatc 1740
ctatcatcga ccggtatacc ttttggtcta cggatttacc tcccgtgtcg aggtcgagat 1800
gcccattagt tcccatgggt gtcctgatgg gcttggcatc cttcattcca aacttgttga 1860
gtatgtcttg aatgtacttt gtttggctga tgaaggtgcc atcttggagt tgcttgactt 1920
gaaatcctag aaaatatttc aacttcccca tcatagacat ctcgaatttc ggaatcatga 1980
tcctactaaa ctcttcacaa gtagatttgt tagtagaccc aaatataata tcatcaacat 2040
aaatttggca tacaaacaaa acttttgaaa tggttttagt aaagagagta ggatcggctt 2100
tactgactct gaagccatta gtgataagaa aatctcttag gcattcatac catgctgttg 2160
gggcttgctt gagcccataa agcgcctttg agagtttata aacatggtta gggtactcac 2220
tatcttcaaa gccgagaggt tgctcaacat agacctattc accccatttg atcacttttt 2280
tggtccttca ggatctaata gttatgtata atttagagtc tcttgtttaa tggccagata 2340
tttctaatta atctaagaat ttatgatatt ttttaatttt ttatcatgtc tgatgagaat 2400
taacataaag gctcaattgg gtcctgaatt aataatagag tgaaaattaa tccagaggct 2460
ctattagaac cttcaattag taataccaag atatatataa gatagtagag tatagtttaa 2520
atgttggcat tgttcattct ttcttttgtt atttaattta tgctttccac ggtggttagt 2580
ggttacttct gaagggtcca aataatgcat gaagagtttg aggacaagaa gtctgcccta 2640
aaaatagcga tgcaaaggca tggtgtccaa gccatacata tagcgcacta attttatcag 2700
cagaacaatg gtatttatag gtcctagtgc ccaggcaaca agagacacga ataaagcatc 2760
gatcacgaca c 2771
<210> 6
<211> 348
<212> DNA
<213>Corn (
Zea mays)
<400> 6
gatctgacaa agcagcatta gtccgttgat cggtggaaga ccactcgtca gtgttgagtt 60
gaatgtttga tcaataaaat acggcaatgc tgtaagggtt gttttttatg ccattgataa 120
tacactgtac tgttcagttg ttgaactcta tttcttagcc atgccaagtg cttttcttat 180
tttgaataac attacagcaa aaagttgaaa gacaaaaaaa aaaacccccg aacagagtgc 240
tttgggtccc aagctacttt agactgtgtt cggcgttccc cctaaatttc tccccctata 300
tctcactcac ttgtcacatc agcgttctct ttcccctata tctccacg 348
<210> 7
<211> 225
<212> DNA
<213>Corn (
Zea mays)
<400> 7
atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60
ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120
gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180
gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcg 225
<210> 8
<211> 944
<212> DNA
<213>Corn (
Zea mays)
<400> 8
gatccagctt cgcttagttt ttagtttttg gcagaaaaaa tgatcaatgt ttcacaaacc 60
aaatattttt ataacttttg atgaaagaag atcaccacgg tcatatctag gggtggtaac 120
aaattgcgat ctaaatgttt cttcataaaa aataaggctt cttaataaat tttagttcaa 180
aataaatacg aataaagtct gattctaatc tgattcgatc cttaaatttt ataatgcaaa 240
atttagagct cattaccacc tctagtcata tgtctagtct gaggtatatc caaaaagccc 300
tttctctaaa ttccacaccc aactcagatg tttgcaaata aatactccga ctccaaaatg 360
taggtgaagt gcaactttct ccattttata tcaacatttg ttattttttg tttaacattt 420
cacactcaaa actaattaat aaaatacgtg gttgttgaac gtgcgcacat gtctccctta 480
cattatgttt ttttatttat gtattattgt tgttttcctc cgaacaactt gtcaacatat 540
catcattggt ctttaatatt tatgaatatg gaagcctagt tatttacact tggctacaca 600
ctagttgtag ttttgccact tgtctaacat gcaactctag tagttttgcc acttgcctgg 660
catgcaactc tagtattgac acttgtatag catataatgc caatacgaca cctgccttac 720
atgaaacatt atttttgaca cttgtatacc atgcaacatt accattgaca tttgtccata 780
cacattatat caaatatatt gagcgcatgt cacaaactcg atacaaagct ggatgaccct 840
ccctcaccac atctataaaa acccgagcgc tactgtaaat cactcacaac acaacacata 900
tcttttagta acctttcaat aggcgtcccc caagaactag taac 944
<210> 9
<211> 318
<212> DNA
<213>Yam (
Solanum tuberosum)
<400> 9
agacttgtcc atcttctgga ttggccaact taattaatgt atgaaataaa aggatgcaca 60
catagtgaca tgctaatcac tataatgtgg gcatcaaagt tgtgtgttat gtgtaattac 120
tagttatctg aataaaagag aaagagatca tccatatttc ttatcctaaa tgaatgtcac 180
gtgtctttat aattctttga tgaaccagat gcatttcatt aaccaaatcc atatacatat 240
aaatattaat catatataat taatatcaat tgggttagca aaacaaatct agtctaggtg 300
tgttttgcga atgcggcc 318
Sequence table
< 110>Beijing Weiming Kaituo Crops Design Center Ltd
The Beijing Kaituo Dna Biotech Research Center Co., Ltd
< 120>a kind of effective reduction transgenic plant are through the method for pollen-mediated genetic drift
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 1561
<212> DNA
<213>Corn (
Zea mays)
<400> 1
atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60
ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120
gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180
gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcggcctg cggcctggtc 240
caggcacaag tcctcttcca ggggtttaac tgggagtcgt gcaagcagca gggaggctgg 300
tacaacaggc tcaaggccca ggtcgacgac atcgccaagg ccggcgtcac gcacgtctgg 360
ctgcctccac cctcgcactc cgtctcgcca caaggctaca tgccaggccg cctatacgac 420
ctggacgcgt ccaagtacgg cacggcggcg gagctcaagt ccctgatagc ggcgttccac 480
ggcaggggcg tgcagtgcgt ggcggacatc gtcatcaacc accggtgcgc ggaaaagaag 540
gacgcgcgcg gcgtgtactg catcttcgag ggcgggactc ccgacgaccg cctggactgg 600
ggccccggga tgatctgcag cgacgacacg cagtactcgg acgggacggg gcaccgcgac 660
acgggcgagg ggttcgcggc ggcgcccgac atcgaccacc tcaacccgcg cgtgcagcgg 720
gagctctccg cctggctcaa ctggctcagg tccgacgccg tggggttcga cggctggcgc 780
ctcgacttcg ccaagggcta ctcgccggcc gtcgccagaa tgtacgtgga gagcacgggg 840
ccgccgagct tcgtcgtcgc ggagatatgg aactcgctga gctacagcgg ggacggcaag 900
ccggcgccca accaggacca gtgccggcag gagctgctgg actggacgcg ggccgtcggc 960
gggcccgcca tggcgttcga cttccccacc aagggcctgc tgcaggcggg cgtgcagggg 1020
gagctgtggc ggctgcgcga cagctccggc aacgcggccg gcctgatcgg gtgggcgccc 1080
gagaaggccg tcaccttcgt cgacaaccat gacaccgggt cgacgcagaa gctctggccg 1140
ttcccatccg acaaggtcat gcagggctac gcctacatcc tcacccatcc aggagtcccc 1200
tgcattttct acgaccacat gttcgactgg aacctgaagc aggagatatc cacgctgtct 1260
gccatcaggg cgcggaacgg catccgcgcc gggagcaagc tgcggatcct cgtggcggac 1320
gcggacgcgt acgtggccgt cgtcgacgag aaggtcatgg tgaagatcgg gacaaggtac 1380
ggcgtgagca gcgtggtccc gtcggatttc cacccggcgg cgcacggcaa ggactactgc 1440
gtctgggaga aagcgagcct ccgcgtcccg gcggggcgcc acctctagca gctcagattg 1500
ctcagtcttg tgctgcattg caaacacagc agcacgacac tgcataacgt cttttccttg 1560
a 1561
<210> 2
<211> 519
<212> PRT
<213>Corn (
Zea mays)
<400> 2
Met Ala Ala Thr Met Ala Val Thr Thr Met Val Thr Arg Ser Lys Glu
1 5 10 15
Ser Trp Ser Ser Leu Gln Val Pro Ala Val Ala Phe Pro Trp Lys Pro
20 25 30
Arg Gly Gly Lys Thr Gly Gly Leu Glu Phe Pro Arg Arg Ala Met Phe
35 40 45
Ala Ser Val Gly Leu Asn Val Cys Pro Gly Val Pro Ala Gly Arg Asp
50 55 60
Pro Arg Glu Pro Asp Pro Lys Val Val Arg Ala Ala Cys Gly Leu Val
65 70 75 80
Gln Ala Gln Val Leu Phe Gln Gly Phe Asn Trp Glu Ser Cys Lys Gln
85 90 95
Gln Gly Gly Trp Tyr Asn Arg Leu Lys Ala Gln Val Asp Asp Ile Ala
100 105 110
Lys Ala Gly Val Thr His Val Trp Leu Pro Pro Pro Ser His Ser Val
115 120 125
Ser Pro Gln Gly Tyr Met Pro Gly Arg Leu Tyr Asp Leu Asp Ala Ser
130 135 140
Lys Tyr Gly Thr Ala Ala Glu Leu Lys Ser Leu Ile Ala Ala Phe His
145 150 155 160
Gly Arg Gly Val Gln Cys Val Ala Asp Ile Val Ile Asn His Arg Cys
165 170 175
Ala Glu Lys Lys Asp Ala Arg Gly Val Tyr Cys Ile Phe Glu Gly Gly
180 185 190
Thr Pro Asp Asp Arg Leu Asp Trp Gly Pro Gly Met Ile Cys Ser Asp
195 200 205
Asp Thr Gln Tyr Ser Asp Gly Thr Gly His Arg Asp Thr Gly Glu Gly
210 215 220
Phe Ala Ala Ala Pro Asp Ile Asp His Leu Asn Pro Arg Val Gln Arg
225 230 235 240
Glu Leu Ser Ala Trp Leu Asn Trp Leu Arg Ser Asp Ala Val Gly Phe
245 250 255
Asp Gly Trp Arg Leu Asp Phe Ala Lys Gly Tyr Ser Pro Ala Val Ala
260 265 270
Arg Met Tyr Val Glu Ser Thr Gly Pro Pro Ser Phe Val Val Ala Glu
275 280 285
Ile Trp Asn Ser Leu Ser Tyr Ser Gly Asp Gly Lys Pro Ala Pro Asn
290 295 300
Gln Asp Gln Cys Arg Gln Glu Leu Leu Asp Trp Thr Arg Ala Val Gly
305 310 315 320
Gly Pro Ala Met Ala Phe Asp Phe Pro Thr Lys Gly Leu Leu Gln Ala
325 330 335
Gly Val Gln Gly Glu Leu Trp Arg Leu Arg Asp Ser Ser Gly Asn Ala
340 345 350
Ala Gly Leu Ile Gly Trp Ala Pro Glu Lys Ala Val Thr Phe Val Asp
355 360 365
Asn His Asp Thr Gly Ser Thr Gln Lys Leu Trp Pro Phe Pro Ser Asp
370 375 380
Lys Val Met Gln Gly Tyr Ala Tyr Ile Leu Thr His Pro Gly Val Pro
385 390 395 400
Cys Ile Phe Tyr Asp His Met Phe Asp Trp Asn Leu Lys Gln Glu Ile
405 410 415
Ser Thr Leu Ser Ala Ile Arg Ala Arg Asn Gly Ile Arg Ala Gly Ser
420 425 430
Lys Leu Arg Ile Leu Val Ala Asp Ala Asp Ala Tyr Val Ala Val Val
435 440 445
Asp Glu Lys Val Met Val Lys Ile Gly Thr Arg Tyr Gly Val Ser Ser
450 455 460
Val Val Pro Ser Asp Phe His Pro Ala Ala His Gly Lys Asp Tyr Cys
465 470 475 480
Val Trp Glu Lys Ala Ser Leu Arg Val Pro Ala Gly Arg His Leu Gln
485 490 495
Leu Arg Leu Leu Ser Leu Val Leu His Cys Lys His Ser Ser Thr Thr
500 505 510
Leu His Asn Val Phe Ser Leu
515
<210> 3
<211> 678
<212> DNA
< 213>synthetic
<400> 3
atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 60
accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120
cacaacaccg tgaagctgaa ggtgacgaag ggcggccccc tgcccttcgc ctgggacatc 180
ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240
gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300
gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360
aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420
atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480
acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540
tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600
atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660
caccacctgt tcctgtag 678
<210> 4
<211> 225
<212> PRT
< 213>synthetic
<400> 4
Met Ala Ser Ser Glu Asn Val Ile Thr Glu Phe Met Arg Phe Lys Val
1 5 10 15
Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu
20 25 30
Gly Glu Gly Arg Pro Tyr Glu Gly His Asn Thr Val Lys Leu Lys Val
35 40 45
Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln
50 55 60
Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro
65 70 75 80
Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val
85 90 95
Met Asn Phe Glu Asp Gly Gly Val Ala Thr Val Thr Gln Asp Ser Ser
100 105 110
Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn
115 120 125
Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu
130 135 140
Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu
145 150 155 160
Thr His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu
165 170 175
Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr
180 185 190
Tyr Tyr Val Asp Ala Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr
195 200 205
Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe
210 215 220
Leu
225
<210> 5
<211> 2771
<212> DNA
<213>Corn (
Zea mays)
<400> 5
agcttgcatg cctgcaggtc gactctagag gatctgcacc ggacactgtc tggtggcata 60
ccagacagtc cggtgtgcca gatcagggca cccttcggtt cctttgctcc tttgcttttg 120
aaccctaact ttgatcgttt attggtttgt gttgaacctt tatgcacctg tggaatatat 180
aatctagaac aaactagtta gtccaatcat ttgtgttggg cattcaacca ccaaaattat 240
ttataggaaa aggttaaacc ttatttccct ttcaatctcc ccctttttgg tgattgatgc 300
caacacaaac caaagaaaat atataagtgc agaattgaac tagtttgcat aaggtaagtg 360
cataggttac ttagaattaa atcaatttat acttttactt gatatgcatg gttgctttct 420
tttattttaa cattttggac cacatttgca ccacttgttt tgttttttgc aaatcttttt 480
ggaaattctt tttcaaagtc ttttgcaaat agtcaaaggt atatgaataa gattgtaaga 540
agcattttca agatttgaaa tttctccccc tgtttcaaat gcttttcctt tgactaaaca 600
aaactccccc tgaataaaat tctcctctta gctttcaaga gggttttaaa tagatatcaa 660
ttggaaatat atttagatgc taattttgaa aatataccaa ttgaaaatca acataccaat 720
ttgaaattaa acataccaat ttaaaaaatt tcaaaaagtg gtggtgcggt ccttttgctt 780
tgggcttaat atttctcccc ctttggcatt aatcgccaaa aacggagact ttgtgagcca 840
tttatacttt ctccccattg gtaaatgaaa tatgagtgaa agattatacc aaatttggac 900
agtgatgcgg agtgacggcg aaggataaac gataccgtta gagtggagtg gaagccttgt 960
cttcgccgaa gactccattt ccctttcaat ctacgactta gcatagaaat acacttgaaa 1020
acacattagt cgtagccacg aaagagatat gatcaaaggt atacaaatga gctatgtgtg 1080
taatgtttca atcaaagttt cgagaatcaa gaatatttag ctcattccta agtttgctaa 1140
aggttttatc atctaatggt ttggtaaaga tatcgactaa ttgttctttg gtgctaacat 1200
aagcaatctc gatatcaccc ctttgttggt gatccctcaa aaagtgatac cgaatgtcta 1260
tgtgcttagt gcggctgtgt tcaacgggat tatccgccat gcagatagca ctctcattgt 1320
cacataggag agggactttg ctcaatttgt agccatagtc cctaaggttt tgcctcatcc 1380
aaagtaattg cacacaacaa tgtcctgcgg caatatactt ggcttcggcg gtagaaagag 1440
ctattgagtt ttgtttcttt gaagtccaag acaccaggga tctccctaga aactgacaag 1500
tccctgatgt gctcttccta tcaattttac accctgccca atcggcatct gaatatccta 1560
ttaaatcaaa ggtggatccc ttggggtacc aaagaccaaa tttaggagtg taaactaaat 1620
atctcatgat tcttttcacg gccctaaggt gaacttcctt aggatcggct tggaatcttg 1680
cacacatgca tatagaaagc atactatctg gtcgagatgc acataaatag agtaaagatc 1740
ctatcatcga ccggtatacc ttttggtcta cggatttacc tcccgtgtcg aggtcgagat 1800
gcccattagt tcccatgggt gtcctgatgg gcttggcatc cttcattcca aacttgttga 1860
gtatgtcttg aatgtacttt gtttggctga tgaaggtgcc atcttggagt tgcttgactt 1920
gaaatcctag aaaatatttc aacttcccca tcatagacat ctcgaatttc ggaatcatga 1980
tcctactaaa ctcttcacaa gtagatttgt tagtagaccc aaatataata tcatcaacat 2040
aaatttggca tacaaacaaa acttttgaaa tggttttagt aaagagagta ggatcggctt 2100
tactgactct gaagccatta gtgataagaa aatctcttag gcattcatac catgctgttg 2160
gggcttgctt gagcccataa agcgcctttg agagtttata aacatggtta gggtactcac 2220
tatcttcaaa gccgagaggt tgctcaacat agacctattc accccatttg atcacttttt 2280
tggtccttca ggatctaata gttatgtata atttagagtc tcttgtttaa tggccagata 2340
tttctaatta atctaagaat ttatgatatt ttttaatttt ttatcatgtc tgatgagaat 2400
taacataaag gctcaattgg gtcctgaatt aataatagag tgaaaattaa tccagaggct 2460
ctattagaac cttcaattag taataccaag atatatataa gatagtagag tatagtttaa 2520
atgttggcat tgttcattct ttcttttgtt atttaattta tgctttccac ggtggttagt 2580
ggttacttct gaagggtcca aataatgcat gaagagtttg aggacaagaa gtctgcccta 2640
aaaatagcga tgcaaaggca tggtgtccaa gccatacata tagcgcacta attttatcag 2700
cagaacaatg gtatttatag gtcctagtgc ccaggcaaca agagacacga ataaagcatc 2760
gatcacgaca c 2771
<210> 6
<211> 348
<212> DNA
<213>Corn (
Zea mays)
<400> 6
gatctgacaa agcagcatta gtccgttgat cggtggaaga ccactcgtca gtgttgagtt 60
gaatgtttga tcaataaaat acggcaatgc tgtaagggtt gttttttatg ccattgataa 120
tacactgtac tgttcagttg ttgaactcta tttcttagcc atgccaagtg cttttcttat 180
tttgaataac attacagcaa aaagttgaaa gacaaaaaaa aaaacccccg aacagagtgc 240
tttgggtccc aagctacttt agactgtgtt cggcgttccc cctaaatttc tccccctata 300
tctcactcac ttgtcacatc agcgttctct ttcccctata tctccacg 348
<210> 7
<211> 225
<212> DNA
<213>Corn (
Zea mays)
<400> 7
atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60
ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120
gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180
gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcg 225
<210> 8
<211> 944
<212> DNA
<213>Corn (
Zea mays)
<400> 8
gatccagctt cgcttagttt ttagtttttg gcagaaaaaa tgatcaatgt ttcacaaacc 60
aaatattttt ataacttttg atgaaagaag atcaccacgg tcatatctag gggtggtaac 120
aaattgcgat ctaaatgttt cttcataaaa aataaggctt cttaataaat tttagttcaa 180
aataaatacg aataaagtct gattctaatc tgattcgatc cttaaatttt ataatgcaaa 240
atttagagct cattaccacc tctagtcata tgtctagtct gaggtatatc caaaaagccc 300
tttctctaaa ttccacaccc aactcagatg tttgcaaata aatactccga ctccaaaatg 360
taggtgaagt gcaactttct ccattttata tcaacatttg ttattttttg tttaacattt 420
cacactcaaa actaattaat aaaatacgtg gttgttgaac gtgcgcacat gtctccctta 480
cattatgttt ttttatttat gtattattgt tgttttcctc cgaacaactt gtcaacatat 540
catcattggt ctttaatatt tatgaatatg gaagcctagt tatttacact tggctacaca 600
ctagttgtag ttttgccact tgtctaacat gcaactctag tagttttgcc acttgcctgg 660
catgcaactc tagtattgac acttgtatag catataatgc caatacgaca cctgccttac 720
atgaaacatt atttttgaca cttgtatacc atgcaacatt accattgaca tttgtccata 780
cacattatat caaatatatt gagcgcatgt cacaaactcg atacaaagct ggatgaccct 840
ccctcaccac atctataaaa acccgagcgc tactgtaaat cactcacaac acaacacata 900
tcttttagta acctttcaat aggcgtcccc caagaactag taac 944
<210> 9
<211> 318
<212> DNA
<213>Yam (
Solanum tuberosum)
<400> 9
agacttgtcc atcttctgga ttggccaact taattaatgt atgaaataaa aggatgcaca 60
catagtgaca tgctaatcac tataatgtgg gcatcaaagt tgtgtgttat gtgtaattac 120
tagttatctg aataaaagag aaagagatca tccatatttc ttatcctaaa tgaatgtcac 180
gtgtctttat aattctttga tgaaccagat gcatttcatt aaccaaatcc atatacatat 240
aaatattaat catatataat taatatcaat tgggttagca aaacaaatct agtctaggtg 300
tgttttgcga atgcggcc 318
Claims (3)
1. one kind prevents that foreign gene from floating to the method other plant and the environment from transgenic plant, and this method comprises:
A) nucleotide sequence of one section coding corn alpha-amylase gene, said sequence is connected in pollen specific promoter;
B) with the nucleotide sequence rice transformation in (a);
C) with transformant regeneration plant.
2. in claims 1, pollen specific promoter is the PG47 promotor.
3. the transfer-gen plant that obtains in claims 1 comprises the corn alpha-amylase gene by the pollen-specific promoters driven, and with the fluorescent screening marker gene
FPClosely connect.
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| CN 201010563555 CN102477443B (en) | 2010-11-29 | 2010-11-29 | Method for effectively reducing gene drift of transgene plant through pollen mediation |
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| CN103834684A (en) * | 2013-03-29 | 2014-06-04 | 湖南杂交水稻研究中心 | Method for mechanically producing seed by using female sterile hybrid rice |
| CN104004775A (en) * | 2013-02-26 | 2014-08-27 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Fertility regulation gene and its application |
| WO2014131342A1 (en) * | 2013-02-26 | 2014-09-04 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | New construct for regulating fertility of wheat and use thereof |
| WO2014187311A1 (en) * | 2013-05-23 | 2014-11-27 | 深圳市作物分子设计育种研究院 | Plant pollen specificity-inactivating carrier and use thereof |
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| WO2009103049A2 (en) * | 2008-02-14 | 2009-08-20 | Pioneer Hi-Bred International, Inc. | Plant genomic dna flanking spt event and methods for identifying spt event |
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| CN104379751A (en) * | 2013-02-26 | 2015-02-25 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | New construct for regulating fertility of wheat and use thereof |
| CN104004775B (en) * | 2013-02-26 | 2018-08-28 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | One sterility changing gene and its application |
| CN104004775A (en) * | 2013-02-26 | 2014-08-27 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Fertility regulation gene and its application |
| WO2014131342A1 (en) * | 2013-02-26 | 2014-09-04 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | New construct for regulating fertility of wheat and use thereof |
| WO2014154141A1 (en) * | 2013-03-29 | 2014-10-02 | 湖南杂交水稻研究中心 | Mechanized seed production method using female-sterile hybrid rice plants |
| CN103834684B (en) * | 2013-03-29 | 2015-02-18 | 湖南桃花源种业有限责任公司 | Method for mechanically producing seed by using female sterile hybrid rice |
| CN103834684A (en) * | 2013-03-29 | 2014-06-04 | 湖南杂交水稻研究中心 | Method for mechanically producing seed by using female sterile hybrid rice |
| WO2014187311A1 (en) * | 2013-05-23 | 2014-11-27 | 深圳市作物分子设计育种研究院 | Plant pollen specificity-inactivating carrier and use thereof |
| CN103667211A (en) * | 2013-12-31 | 2014-03-26 | 北京大北农科技集团股份有限公司 | Protein influencing male fertility and encoding gene and application thereof |
| CN103667211B (en) * | 2013-12-31 | 2016-01-20 | 北京大北农科技集团股份有限公司 | Affect the protein of male fertility, its encoding gene and purposes |
| CN105063083A (en) * | 2015-07-16 | 2015-11-18 | 湖南杂交水稻研究中心 | Method for creating rice engineering maintainer lines preventive against gene flow and application of rice engineering maintainer lines |
| CN105063083B (en) * | 2015-07-16 | 2018-07-06 | 湖南杂交水稻研究中心 | Prevent method for creating and its application of the Rice Engineering maintainer of genetic drift |
| CN106480077A (en) * | 2016-12-02 | 2017-03-08 | 海南波莲水稻基因科技有限公司 | Millet alpha amylase and its encoding gene and application |
| CN111269914A (en) * | 2020-02-18 | 2020-06-12 | 湖南杂交水稻研究中心 | DNA molecule and method for effectively preventing pollen of transgenic plant from escaping |
| CN116926109A (en) * | 2023-04-20 | 2023-10-24 | 中国农业科学院作物科学研究所 | Plant programmed pollen self-cleaning CRISPR/Cas gene editing method |
| CN116926109B (en) * | 2023-04-20 | 2024-05-14 | 中国农业科学院作物科学研究所 | Plant programmed pollen self-cleaning CRISPR/Cas gene editing method |
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