CN102199619B - Transformation method utilizing red fluorescent protein as selection marker of rice transformation - Google Patents

Abstract

The invention provides a transformation method utilizing a red fluorescent protein as a selection marker. The method comprises the following steps of: synthesizing a fluorescent protein (FP) gene and modifying according to the bias of a rice codon, wherein the modified FP gene is used as a selective marker gene of rice transformation and plant regeneration; driving the FP gene by a callus/seed coat-specific promoter, closely connecting with a target gene, and transferring to rice embryonic calli under the mediation of Agrobacterium tumefaciens; screening for three times by a fluorescence microscope in 30 days after coculture; and further verifying an obtained transgenic seedling by utilizing a polymerase chain reaction (PCR) and Southern hybridization. The result shows that the positive rate of the transgenic seedling is up to 80 percent, which proves that FP serving as the selection marker of plant transformation is completely feasible. By the method, the potential hazard of the traditional selection markers such as antibiotic and herbicide resistance genes and the like to environment and food safety is effectively reduced. The invention provides a novel safe plant genetic transformation screening system.

Description

A kind of method for transformation that utilizes red fluorescent protein to be used as the rice conversion selection markers

Technical field

The invention provides a kind of selection markers of the safety that is applied to Plant Transformation newly, and the method for utilizing fluorescent microscope to screen is provided, belong to biological technical field.

Background technology

Utilize transgenic technology the goal gene with good character can be imported in Plant Genome, thereby the inherited character of plant is improved.Yet only have the minority vegetable cell can absorb foreign DNA and be integrated in Plant Genome in conversion process, most cells is unconverted.Therefore, the introducing of goal gene usually need to be given transformant with specific selected marker by means of the selection markers gene, so that identification and evaluation transgenic plant.

The method that traditional evaluation separates transformant is mostly " negative sense screening ", screening-gene commonly used such as antibiotic marker Npt II gene (produce neomycin phosphotransferase, anti-kantlex, G418, paromycin, Liu Suanyan NEOMYCIN SULPHATE), the HPT gene (produces hygromix phosphotransferase, the moisture resistance mycin), the herbicide-resistant gene Bar gene (produces the PPT Transacetylase, anti-Bialaphos or glufosinate) etc., on the substratum that has added selective agent, transformant can be grown, and the growth of no transformed cells is suppressed even and is killed.

Along with the commercialization of transgenic product, the genetically modified Biosafety especially Biosafety of selection markers is subject to extensive concern.The potential hazard of negative sense selection markers gene is: anti-herbicide gene may cause the generation of superweed; Antibiotics resistance gene may escape in environment and propagate, also might by food in enteron aisle horizontal transfer to microorganism, thereby affect the validity of antibiotic therapy; In addition on the one hand, non-transformed cell can excrete poison or growth inhibitor in apoptotic process gradually, and impeding nutritious substance is to the transportation of transformant, thereby affects propagation and the differentiation of transformant.

In view of the various unfavorable factors of negative sense screening-gene, the forward system of selection is arisen at the historic moment.So-called forward screening is for the negative sense screening, and namely on screening culture medium, transgenic cell can effectively utilize the carbon source normal growth in substratum, and non-transformed cell can not utilize the sole carbon source in substratum to be subject to hungry the inhibition.Forward screening-gene such as carbohydrate metabolism enzyme gene xylose isomerase gene (xylA) commonly used, mannose phosphonic isomerase (PMI) and amino acid metabolism enzyme such as L-glutamic acid-1-semialdehyde aminotransferase gene (heml), this screening method does not have toxic substance, do not affect growth and the regeneration of transformant, and a lot of report shows that the forward screening method has higher screening efficiency than the negative sense screening.But, utilizing these carbohydrates to do sole carbon source and may produce the effect that is different from sucrose to the regeneration differentiation of vegetable cell, these all also require further study.

Also have at present some conversion systems, first utilize common selective marker to filter out elementary transfer-gen plant, then utilize the restructuring of transfer-gen plant descendant inheritting that selective marker is separated with goal gene, or utilize the site-specific enzyme marker gene excision to be cultivated the transfer-gen plant of marker-free.These class methods mainly contain three kinds of systems: cotransformation system, double T-DNA border sequence conversion method, differential recombination enzyme conversion system (FLP/ FRTs, Cre/LoxP, R/ RS and GIN system etc.) and Transposon System etc., wherein most widely used is cotransformation method and Cre/lox site-specific recombination system.Yet the rejecting technology of these selection markers still is in the exploratory stage, carrier used is confined to the T-DNA carrier of Agrobacterium mostly, that also mainly studies on model plant such as tobacco and Arabidopis thaliana is comparatively ripe, make these technology be successfully applied to plant breeding, also need a large amount of research work, and set up corresponding marker gene eliminating system for different plants.For example, for the cotransformation method, the heredity of marker gene and goal gene separates and could realize through sexual generation, and this not only can increase breeding time, and can't be applied to vegetative plant variety.And the difficult point of Cre/lox system applies is the time that the Cre recombinase starts and how to start the expression of Cre recombinase and then eliminate sequence between the lox site.Secondly, the scalping method of selection markers needs higher efficient to adapt to large-scale production and application.

Another safe selection markers is fluorescin.The generation of fluorescence is without any need for substrate and cofactor, its expression product also to cell without any toxicity, can not affect normal growth and the function of cell.And, utilize the intensity of fluorescence can tell homozygosity and the heterozygosity of transgenic plant.The fluorescin of first widespread use is green fluorescent protein (GFP), and GFP is used for the selection markers of Plant Transformation and has applied for a patent (US6486382, EP0904371B1).But green protein itself still has a lot of shortcomings, and its emission POP is limited in 440 ~ 529nm, excite with emission spectrum too short, can activated cell in Cucumber generation fluorescence, cause in cell the fluorescence imaging background higher.

Reported first red fluorescent protein drFP583 (DsRed) in 1999, its advantage is apparent: share with GFP series fluorescin, and excite with emission wavelength longlyer, in cell, the imaging background is low, therefore is concerned rapidly.The short several years, a series of researchs to red fluorescent protein, the red fluorescent protein of different wild-types is through the external evolution of some row, obtain the mutant of various different emission, emmission spectrum can cover 574nm to 655nm, greatly enriched the spectrum diversity of fluorescin, for intracellular multi-color marking provides more fluorescence labels.Yet red fluorescent protein also was not in the news as the selection markers of transgenic plant.

Based on green fluorescence protein gene and red fluorescent protein gene, developed a large amount of fluorescence protein gene mutant, their fluorescence color has almost been contained the chromatogram of all visible lights.To the new albumen that the transformation of the green fluorescent protein in original Aequorea victoria jellyfish produces, color all is widely used in life science from indigo plant to the Huang.Mutant research and a series of monomer to red fluorescent protein DsRED are optimized, and have obtained utilizing emitted light versicolor long wavelength's in orange and red spectral region fluorescin, and the monomeric protein of better performances has mCherry, mOrange etc.In addition, a lot of natural versicolor fluorescins have also been found in coral polyp and affinity species thereof, as azarin, orange, grey, brown, brown, yellow etc., enriched greatly fluorescin spectrum diversity, for life science provides more choices.

Summary of the invention

This invention provides a kind of red fluorescent protein FP to be used as the method for selection markers in rice conversion, and the transgenic positive seedling that filters out is through Molecular Identification, and its positive rate is up to 80%.Therefore, this invention provides a kind of safe and reliable selection markers for Plant Transformation.

The advantage of this invention is, the selection markers that use (1) is all safe to vegetable cell itself and to environment; (2) use the positive transgenic seedling that this selection markers filters out to prove that through Molecular Identification its reliability is high; (3) screening process is finished drilling at fluorescent microscope and is completed, and has intuitively, characteristics easily.

The present invention realizes by following scheme: at first, and synthetic FP gene, and be placed in callus/kind of skin specific promoter END2 downstream, and closely connect with goal gene, be implemented in conversion carrier; Utilize agriculture bacillus mediated method to change the carrier that builds over to acceptor material, and utilize fluorescent microscope to carry out three screenings in common cultivation in rear 30 days, the stable fluorophore that obtains at last enters differential period; At last, the transgenosis seedling that obtains is further identified on molecular level.

Concrete steps comprise: 1, and construction of expression vector; 2, Genetic Transformation in Higher Plants; 3, molecular biology identification.

Description of drawings:

Fig. 1 has shown plant expression vector pSPT18 structural representation in embodiment 1.

Fig. 2 has shown in embodiment 2 and has cultivated altogether the rear 10 days callus of phosphor dot redly with Agrobacterium.

Fig. 3 has shown in embodiment 2 and has cultivated altogether the rear 20 days callus of phosphor dot redly with Agrobacterium.

Fig. 4 has shown the callus of cultivating altogether the fluorophore redly of rear 30 days in embodiment 2 with Agrobacterium.

Fig. 5 has shown the fluorophore callus before differentiation in embodiment 2.

Fig. 6 has shown the regeneration plant after Agrobacterium-mediated Transformation in embodiment 2 (left side is the regeneration plant of differential period, and the right is the regeneration plant that changes root media over to).

Fig. 7 has shown that the PCR of part plant in embodiment 3 identifies (M:DNA molecular weight standard;-: negative/negative control; +: just/positive control).

Fig. 8 has shown that the Southern blot of part transfer-gen plant in embodiment 4 identifies (C: negative control, M: molecular weight standard; Plasmid: to transform plasmid as positive control).

Embodiment:

Hereinafter specifically describe technical scheme of the present invention with accompanying drawing in conjunction with the embodiments, but be not limited to this.

Being specifically related to two genes in the embodiment of the present invention, is respectively FP and OsCYP704B2.FP is with giving a report/the selection markers gene, derive from reef coral (Discosoma sp.), its codified red fluorescent protein, can be inspired red fluorescence, be used for the screening of genetically modified callus and seed, its nucleotide sequence and aminoacid sequence are seen respectively SEQ ID NO.1 and SEQ ID NO.2; OsCYP704B2 is the paddy rice native gene, and catalysis lipid acid hydroxylation participates in the synthetic extine that reaches of flower pesticide cutin haplont and forms, and the OsCYP704B2 gene changes ms26 male-sterile mutation body over to can make its fertility restorer.Its nucleotide sequence and aminoacid sequence are seen respectively SEQ ID NO.3 and SEQ ID NO.4.

Embodiment 1: construction of expression vector

Transform the expression vector name of using in the present invention and be called pSPT18(Fig. 1).This carrier is artificial constructed on the pPZP basis.2 expression casettes are arranged on expression vector: the one, OsCYP704B2 expression casette, this expression cassette is made of OsCYP704B2 gene and self endogenesis promoter and terminator.For the endogenous OsCYP704B2 gene of distinguishing paddy rice itself and the OsCYP704B2 gene that is transformed into paddy rice by expression vector, be converted into paddy rice after the sudden change of 3 single nucleotide mutation sites of introducing (SNP) in wild-type allele OsCYP704B2 in, these 3 SNP at the 1468th, 1470 and 1473 bit base places, are from G and sport C respectively.These change does not all affect coded aminoacid sequence.OsCYP704B2 gene nucleotide series after modification sees that shown in SEQ ID NO.3, wherein SNP is marked by square frame; The 2nd, report/selection markers gene FP gene (seeing SEQ ID NO.1) expression cassette, this expression cassette is by driving (its nucleotide sequence is seen SEQ ID NO.5) from the callus of corn/seed specific promoters END2, and stopped transcribing by the terminator PIN II from potato, the nucleotide sequence of PIN II is seen SEQ ID NO.6.

Do not contain antibiotics resistance marker gene and herbicide screening marker gene in carrier in the T-DNA section.

Embodiment 2: rice conversion

Passed through following steps:

One, seed disinfection

1, the rice paddy seed that removes crust soaked 2-3 minute in 95% ethanol;

2, rinsing is 15 minutes in containing 40% chlorine bleach liquor of tween (40ul, 20%Tween/100ml), changes one time rinsing liquid, continues rinsing 15 minutes;

3, rinsing is 15 minutes in not containing 40% chlorine bleach liquor of tween, changes rinsing liquid, continues rinsing 15 minutes;

4, with sterilized water with seed rinsing 4 times.

Two, callus of induce

32 ℃ of illumination box evoked callus 11 days.

Three, Agrobacterium-mediated Transformation

1, picking list bacterium colony is connected to the Agrobacterium substratum (interpolation microbiotic) of the fresh preparation of 25ml, and 28 ℃ of 250-300 RPM shake training and spend the night, the Agrobacterium to 0.3 that spreads cultivation＜OD550＜1.0;

2, with centrifugal 5 minutes of 6000Xg (4 ℃), soft resuspended Agrobacterium is added 100 μ M AS to OD550=0.1(with bacterium liquid, and the mother liquid concentration that AS is dissolved in DMSO is 100mmol/L);

3, infected pre-incubated callus 5-10 minute, gently overturn at regular intervals or the wave and culture bottle;

4, take out agrobacterium liquid, callus dries in the shade on filter paper.

, callus being placed on the common culture medium flat plate that is covered with filter paper, every ware placement amount is there to be the gap to be as the criterion between callus;

6, in 21-25 ℃ of incubator dark culturing 72 hours.

, after ordinary method rinsing callus, dry in the shade and be transferred to screening culture medium, in 28 ℃ of dark culturing.

Four, fluorescent screening

1, cultivate altogether and finish rear 4 days, can observe transformation efficiency.Cultivate altogether and finish rear the 10th day, abandon non-blooming callus, fluorescigenic callus is cut for the first time, fluorescigenic cell mass is cut into the 0.1-0.2mm size, be transferred to new screening culture medium.

, cultivate altogether and finish rear the 20th day, cut for the second time, be divided into the independent cell mass that transforms as far as possible, size is 0.1-0.2mm, is transferred to new screening culture medium.

, cultivate altogether and finish rear the 30th day, to cut for the third time, the cutting standard is that each cell mass size is 0.1-0.2mm, goes to new screening culture medium.

Five, differentiation

1, the callus of cutting after three times is grown on screening culture medium〉during 0.2mm, transfer to pre-division culture medium, lower 28 ℃ of dark condition was cultivated 7-10 days.

2, callus is transferred to division culture medium, 2-3 week is visible young shoot afterwards.

, when seedling grows to 7-8cm, transfer to root media, hardening, transplanting after 7 days.

Embodiment 3: the PCR of transfer-gen plant detects

Get the transgenic rice plant blade in embodiment 4, extract total DNA.Take the FP gene order as template design primer, T0 is carried 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 7 is part transfer-gen plant pcr amplification result, illustrates that foreign gene has been integrated in paddy rice acceptor gene group.

Embodiment 4: the Southern Blot of transfer-gen plant detects

T0 is carried out Southern Blot for the transgenic paddy rice genomic dna detect, to identify the integration of foreign gene in the rice conversion acceptor.Get that in embodiment 4, T0 for the transgenic rice plant blade, extracts total DNA.526bp nucleotide fragments in the FP gene order is as probe, selects the Xba I restriction enzyme of NEB company to carry out digestion reaction.The endonuclease reaction system is 200ul: genomic dna 15ug, NE Buffer 20ul, BSA 2ul, Xba I 4ul(20 U/ul) and with deionized water, system is complemented to 200 ul.37 ℃ of incubations 6 hours.Southern Blot detected result shows, foreign gene has been integrated in the acceptor rice genome.

Sequence table

＜110〉Beijing Weiming Kaituo Crops Design Center Ltd

The Beijing Kaituo Dna Biotech Research Center Co., Ltd

＜120〉a kind of method of utilizing red fluorescent protein to be used as the rice conversion selection markers

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Thr Phe Asp Leu Val Glu Asp His Pro Val Lys Tyr Arg Met Met Thr

515 520 525

Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Thr Ser Val

530 535 540

<210> 5

<211> 944

<212> DNA

＜213〉corn (Zea mays)

<400> 5

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> 6

<211> 318

<212> DNA

＜213〉potato (Solanum tuberosum)

<400> 6

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 method of utilizing red fluorescent protein to be used as the rice conversion selection markers

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 678

<212> DNA

＜213〉synthetic

<400> 1

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> 2

<211> 225

<212> PRT

＜213〉synthetic

<400> 2

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> 3

<211> 3808

<212> DNA

＜213〉paddy rice ( Oryza sativa)

<400> 3

aggtggaaga caaggtggtg aggattggga gggctaccta tggcagggta gtgaagaggc 60

aggcaatgag agctctcttc agacttacat tggatgctga cagtaacaaa agcctgtagg 120

ttttgatact cttgattgat tgtttattta gttacctagt atcttcagta acagatgaga 180

gatttattca gcaaatgctc cggtttgctc gaaggttgta ataagagtgt gggcaagaat 240

caaggtcaat ccataagagc actattttca tgctcttctg atcttggttt cagacttgtt 300

tcagtgttga cattggttat ttctcaattc attcgagtat ttgttgttac atcacaaagg 360

ataagttcta tagaaaaaat cttccttttc aagtgatgtt ctttaatttt ctgtagaatt 420

gtgccctgca atttctcaaa tctttgatag atggcttatt tgtattgact ggaaaagaaa 480

ttagttgtca ataactagaa gctttagaga tgcaaagtat tggatatatc ttggcaatag 540

tattttatat tgcttgttta tgtgagaatg ttttaactag atggcaactg atttctggga 600

caaaatcgct tctacaatag cattttatgg aactcgtact cgtcgatagc atttcttgga 660

tttgggtgtt tgtaaatggc atttcttgga ttttctcttc attaaaatag cctattcaga 720

tgaagtagaa ttcaggtgaa gtagaaacca actactttgg gttcacaatt tatatttctt 780

ttgaggatac cccatttcat tttagttgtc atcaaagact agacaatatc gacagaaaat 840

ggtaagcctg gtttcagttg gtgacaattt aacagaattc agatggatat ggttctgata 900

ttagaaggtg gcataccttt agtcgctgca aacgcttcag ttatctgaac aaaacaacga 960

acttggctga gcaggggaaa aaaatactgt agcattcatt ttgtgtttac atgagtaacg 1020

attcttttct aggtggacag atcacaaaaa gaaaactaaa gctaagatcc aactcctaag 1080

ggtgttaggt tagggacacc atatgaatga gacaatctta attcttggtc acacaaagat 1140

tgtctcaagg ttggtagcat cagtgcccaa tatatcacct aactatgcca tccaaaatgc 1200

tacatagcat ctcttgtaga ctgaaccctt catgaagagc cccatggagg aagctcatgc 1260

aatgccagtg acatcattct tcccagtagc aggaatccac aagctcatag ctatcttcct 1320

tgttgtcctc tatggatctt ggtccacaag tggagcctga ggaaccagaa agggccaaga 1380

tcatggccaa tcatcggcgc gacagtggag caactgaaga actaccacag gatgcatgac 1440

tggcttgtcg agtacttgtc gaaggaccgc accgtgaccg tcgacatgcc tttcacctcc 1500

tacacctaca ttgccgaccc ggtgaacgtc gagcatgtcc tgaagaccaa cttcaccaat 1560

taccccaagg taaaagaacc ataggatctt cagtgtactg taaaatgtgc cttgcacagt 1620

actaacactg acacaaaaaa tgtctgaaaa tatgcagggt gaagtgtaca ggtcttacat 1680

ggatgtgctg ctcggtgatg gcatattcaa tgccgacggc gagatgtgga ggaagcaaag 1740

gaagacggcg agcttcgagt ttgcctccaa gaacttgaga gacttcagca ctgtggtgtt 1800

cagggagtac tccctgaagc tatcaagcat tctgagccaa gcatgcaagg ccggcagagt 1860

tgtagacatg caggtaacca actgaattcc ttgcctaata cctaaacatt tcttgagaaa 1920

ccaaattgtt cagaattctg atgcaagaac taaccaaaat tcaggaattg ttcatgagga 1980

tgacactgga ctcgatctgc aaggtcgggt ttggggttga gatcgggacg ctgtcacctg 2040

atctcccgga gaacagcttt gcccaggcat tcgacgctgc caacatcatc gtcacgctgc 2100

ggttcatcga tcctctgtgg cgtctgaaga agttcttgca cgtcggatca gaggctctcc 2160

tcgagcagag catgaagctg gttgatgact tcacctacag cgtgatccgc cgccgcaagg 2220

ctgagatctt gcaggctcga gccagcggca agcaagagaa ggtgatcctt cctctcttgc 2280

tcaaagaatc agtagaactg aactgacatg gtaatggtga tgatcagatc ggaaaaggtt 2340

ttgtttcttg atatcgttga tttgtaatgg cgagcagatc aagcacgaca tactgtcgcg 2400

gttcatcgag ctcggggagg ccggcggcga cgaggggggc ggcagcttcg gggacgacaa 2460

gagcctccgc gacgtggtgc tcaacttcgt gatcgccggg cgtgacacga cggcgacgac 2520

gctgtcgtgg ttcacgtaca tggcgatgac gcacccggcc gtcgccgaca agctccggcg 2580

cgagctggcc gcgttcgagg atgagcgcgc gcgcgaggag ggcgtcgcgc tcgccgacgc 2640

cgccggcgag gcgtcgttcg cggcgcgcgt ggcgcagttc gcgtcgctgc tgagctacga 2700

cgcggtgggg aagctggtgt acctgcacgc gtgcgtgacg gagacgctcc gcctctaccc 2760

ggcggtgccg caggacccca aggggatcgt ggaggacgac gtgctccccg acggcaccaa 2820

ggtgcgcgcc ggcgggatgg tgacgtacgt gccctactcc atggggagga tggagtacaa 2880

ctggggcccc gacgcggcga gcttccggcc ggagcggtgg ctcagcggcg acggcggcgc 2940

gttccggaac gcgtcgccgt tcaagttcac cgcgttccag gccgggccgc ggatctgcct 3000

cggcaaggac tccgcctacc tccagatgaa gatggcgctc gccatcctct tccgcttcta 3060

caccttcgac ctcgtcgagg accaccccgt caagtaccgg atgatgacca tcctctccat 3120

ggctcacggc ctcaaggtcc gcgtctccac ctccgtctga cccccgccgc cgctcgccgg 3180

cagccgcgcc gccgccgccc gtatcgctta ccggagtagt aaataagcct atgtaatctg 3240

gtttgaattt gaaatttgaa tgtaccatgt ttgattctag gatttgttgg tcctagaccc 3300

tgcttgaaac ggtgcgaatt tcatctaaat ggttgagaaa ttttatcgaa agctgttcca 3360

ttctacgcta caaatggtgg gactggattt aaacattggc gacgtggaca aggccgtatc 3420

accatgtttg cacattttta aacctgtaat ctggtttgaa tttgaatgta ccatgacacc 3480

atgtttgcaa aactttacat gaatgtttga gaaaaaatat ggagaactgt tcaattagta 3540

tgcgtttaaa atgggactgg atttaaacat tggcgacgtg gacaaggcta gtggactgag 3600

actctgagat gttgcggaag tcggggacgc agcggcggca gccgccggcg tggcggcggt 3660

gccggagcct gcgacacatc aagcagatgc acgcggtgat ggcgctccgg ggcttcctct 3720

ccgatccctc cgagctccgc gagctccttt tcgcctccgc cgtcgcggtc cgcggcgcca 3780

tcgcgcacgc ctacctcgtg ttcgacca 3808

<210> 4

<211> 544

<212> PRT

＜213〉paddy rice ( Oryza sativa)

<400> 4

Met Lys Ser Pro Met Glu Glu Ala His Ala Met Pro Val Thr Ser Phe

1 5 10 15

Phe Pro Val Ala Gly Ile His Lys Leu Ile Ala Ile Phe Leu Val Val

20 25 30

Leu Ser Trp Ile Leu Val His Lys Trp Ser Leu Arg Asn Gln Lys Gly

35 40 45

Pro Arg Ser Trp Pro Ile Ile Gly Ala Thr Val Glu Gln Leu Lys Asn

50 55 60

Tyr His Arg Met His Asp Trp Leu Val Glu Tyr Leu Ser Lys Asp Arg

65 70 75 80

Thr Val Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile Ala Asp

85 90 95

Pro Val Asn Val Glu His Val Leu Lys Thr Asn Phe Thr Asn Tyr Pro

100 105 110

Lys Gly Glu Val Tyr Arg Ser Tyr Met Asp Val Leu Leu Gly Asp Gly

115 120 125

Ile Phe Asn Ala Asp Gly Glu Met Trp Arg Lys Gln Arg Lys Thr Ala

130 135 140

Ser Phe Glu Phe Ala Ser Lys Asn Leu Arg Asp Phe Ser Thr Val Val

145 150 155 160

Phe Arg Glu Tyr Ser Leu Lys Leu Ser Ser Ile Leu Ser Gln Ala Cys

165 170 175

Lys Ala Gly Arg Val Val Asp Met Gln Glu Leu Phe Met Arg Met Thr

180 185 190

Leu Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr Leu

195 200 205

Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp Ala Ala

210 215 220

Asn Ile Ile Val Thr Leu Arg Phe Ile Asp Pro Leu Trp Arg Leu Lys

225 230 235 240

Lys Phe Leu His Val Gly Ser Glu Ala Leu Leu Glu Gln Ser Met Lys

245 250 255

Leu Val Asp Asp Phe Thr Tyr Ser Val Ile Arg Arg Arg Lys Ala Glu

260 265 270

Ile Leu Gln Ala Arg Ala Ser Gly Lys Gln Glu Lys Ile Lys His Asp

275 280 285

Ile Leu Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Gly Asp Glu Gly

290 295 300

Gly Gly Ser Phe Gly Asp Asp Lys Ser Leu Arg Asp Val Val Leu Asn

305 310 315 320

Phe Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser Trp Phe

325 330 335

Thr Tyr Met Ala Met Thr His Pro Ala Val Ala Asp Lys Leu Arg Arg

340 345 350

Glu Leu Ala Ala Phe Glu Asp Glu Arg Ala Arg Glu Glu Gly Val Ala

355 360 365

Leu Ala Asp Ala Ala Gly Glu Ala Ser Phe Ala Ala Arg Val Ala Gln

370 375 380

Phe Ala Ser Leu Leu Ser Tyr Asp Ala Val Gly Lys Leu Val Tyr Leu

385 390 395 400

His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala Val Pro Gln

405 410 415

Asp Pro Lys Gly Ile Val Glu Asp Asp Val Leu Pro Asp Gly Thr Lys

420 425 430

Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr Ser Met Gly Arg

435 440 445

Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala Ser Phe Arg Pro Glu Arg

450 455 460

Trp Leu Ser Gly Asp Gly Gly Ala Phe Arg Asn Ala Ser Pro Phe Lys

465 470 475 480

Phe Thr Ala Phe Gln Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Ser

485 490 495

Ala Tyr Leu Gln Met Lys Met Ala Leu Ala Ile Leu Phe Arg Phe Tyr

500 505 510

Thr Phe Asp Leu Val Glu Asp His Pro Val Lys Tyr Arg Met Met Thr

515 520 525

Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Thr Ser Val

530 535 540

<210> 5

<211> 944

<212> DNA

＜213〉corn ( Zea mays)

<400> 5

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> 6

<211> 318

<212> DNA

＜213〉potato ( Solanum tuberosum)

<400> 6

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 (2)

1. the plant genetic transformation method of an antibiotic-free and weedicide marker gene, the method comprises:

A) one section nucleotide fragments comprises fluorescin selection markers gene order, and described sequence closely is connected in callus and plants the skin specificity promoter;

B) with claim 1.a) described selection markers gene changes paddy rice over to Ms26In the callus of male-sterile mutation body, the T-DNA zone of conversion carrier does not contain microbiotic and herbicide screening gene;

C) transformant of fluorescence is expressed in screening;

D) Calli Differentiation that filters out is become transgenic seedling;

It is characterized in that the nucleotide sequence of described fluorescin selection markers gene as shown in SEQ ID NO:1, the nucleotide sequence of described callus and kind skin specificity promoter is as shown in SEQ ID NO:5.

2. plant genetic transformation method claimed in claim 1, wherein said fluorescin selection markers gene with include 3 SNP's MS26Sequence closely connects, and wherein saidly contains 3 SNP's MS26Nucleotide sequence as shown in SEQ ID NO:3.

Images