CN102229976A - Method for simply and rapidly identifying transgenic seeds and estimating copy numbers - Google Patents
Method for simply and rapidly identifying transgenic seeds and estimating copy numbers Download PDFInfo
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- CN102229976A CN102229976A CN2010105665268A CN201010566526A CN102229976A CN 102229976 A CN102229976 A CN 102229976A CN 2010105665268 A CN2010105665268 A CN 2010105665268A CN 201010566526 A CN201010566526 A CN 201010566526A CN 102229976 A CN102229976 A CN 102229976A
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Abstract
The invention provides a method for simply and rapidly identifying transgenic seeds and estimating copy numbers. According to the invention, the red fluorescent protein (FP) gene (driven by callus/seed coat-specific promoters) is closely connected in series with the target gene and then introduced into the rice male sterile mutant ms26 by means of Agrobacterium tumefaciens-mediated transformation. The transgenic plants are self-crossed to generate the following two types of seeds: non-fluorescent seeds free of transgenic components and fluorescent seeds containing transgenic components. The two types of seeds can be directly separated with the naked eye or with the aid of a fluorescence microscopy, and the separation method is simple and has high accuracy up to 100%. Further, according to the proportions of the fluorescent seeds and the non-fluorescent seeds, the copy numbers of the exogenous gene in the transgenic plant line can be estimated. Since the method provided by the invention does not need the molecular detection of the germinated seeds and young seedlings, the identified seeds can be conveniently applied to the late-stage scientific researches or production.
Description
Technical field
The invention belongs to biological technical field and field of plant variety breeding technology.The invention provides the method for a kind of simple and effective Rapid identification transgenic seed and non-transgenic seed, this method does not need seed germination, can easily transgenosis and non-transgenic two class seed zones separately also be used for scientific research or production easily.
Background technology
Transgenic technology has represented huge application potential at aspects such as the quality-improvings of farm crop, resistance enhancings.Yet in genetically modified T0 generation, is the transgenosis heterozygote often, and its selfing produces three kinds of genotypic seeds of 1:2:1, and 1/4th are the non-transgenic seed that isozygotys, and 1/4th are the transgenic seed that isozygotys, and 1/2nd is the heterozygosis transgenic seed.In traditional method for transformation, at fubaritic transgenic seed of seed stage and non-transgenic seed, so will be according to each separately sowing of strain system, the target gene phenotype is tangible, can transgenic seedling and the discriminating of non-transgenic seedling be come by its phenotype, the target gene phenotype is unconspicuous, can only identify by molecular biology method such as round pcr, workload is very big, and expense is also very high.Another method because tradition uses weedicide or antibiotics resistance marker gene to be screening-gene in transforming mostly, so at T1 for using seedling stage weedicide or microbiotic etc. that the non-transgenic seedling is killed.This method has a lot of problems, and the first, the different steps of seedling development, different to weedicide with antibiotic resistance level, the bad grasp of the dosage of selective agent, high transgenic seedling may being killed low may make the non-transgenic seedling mix wherein.Second, weedicide and microbiotic have been subjected to increasing concern to the potential risk of environment and food safety, therefore people have selected key enzyme such as PMI, xylA etc. in safer selection markers such as the sugar metabolism process for use as far as possible in the conversion process, but the transgenic plant of this class selection markers are in big field screening difficulty relatively.
If mixed the non-transgenic seed in the transgenic seed also is a very serious problem, can directly influence peasant's production imcome.For example, it is reported that the transgenic cotton flower seed on the India market has 46% all to be mixed with the non-transgenic cotton seed, has caused painful financial loss to the peasant, therefore a lot of cotton growers have born heavy debt and have committed suiside.
Therefore, if can identify transgenic seed and non-transgenic seed, can bring huge facility to next step scientific effort and the practical application in the production at seed stage.At present, also do not have a kind of fast, stable, safe method can finish this work.This invention will be filled up this blank.We use fluorescin to do selection markers in the conversion process, fluorescin is a kind of safe selection markers, the generation of fluorescence is without any need for substrate and cofactor, its expression product also pair cell without any toxicity, can not influence the normal growth and the function of cell, at seed stage, utilize fluorescent microscope or only just can identify with the naked eye whether seed has transgene component, thereby easily transgenosis and non-transgenic two class seed zones are separated.
Summary of the invention
Transgenic plant T0 is for being the transgenosis heterozygote often, and selfing produces three kinds of genotypic seeds of 1:2:1.From conventional art, can't distinguish transgenosis and non-transgenic seed at seed stage, brought inconvenience therefore for next step work.The present invention utilizes the red fluorescent protein gene to do selection markers, and whether seed that transfer-gen plant is tied utilizes fluorescent microscope even only with the naked eye just can identify is transgenic seed, solved this difficult problem effectively.
The invention has the advantages that, need not seed germination, also need not by means such as molecular biology, just can simply identify transgenic seed and non-transgenic seed, accuracy rate is up to 100%, simultaneously, according to the ratio of fluorescent seeds and non-fluorescent seeds, can estimate the copy number of foreign gene in Plant Genome.
The present invention realizes successively through the following steps:
1) vector construction: the selection markers gene fusion in callus and kind skin specificity promoter downstream, is used to transform individual callus and plants the skin screening.Goal gene and selection markers gene close linkage are implemented in plasmid vector T-DNA district.
2) transform: utilize agrobacterium mediation method to transform plant, utilize the selection markers gene pairs to transform individuality and screen, and then be divided into regeneration plant.
3) solid: positive transfer-gen plant selfing produces fluorescent seeds and non-fluorescent seeds.
4) identify: utilize naked eyes or identify fluorescent seeds (transgenic seed) and non-fluorescent seeds (non-transgenic seed) by fluorescent microscope.
5) further detect: the seed germination with separating, grow up to seedling, utilize PCR, Southern blot equimolecular biological method further detects.
6) estimation copy number: according to the ratio of fluorescent seeds and non-fluorescent seeds, the copy number of estimation foreign gene in transgenic line.
Plant leaf total DNA extraction technology involved in the present invention, round pcr, Southern blot technology, plant genetic transformation technology etc. are known technology, wherein from the nucleotide sequence of the callus of corn and seed specific promoters END2,
FPGene coded sequence reaches from the sequence of the terminator PIN II terminator of potato and all can consult in ncbi database.
Description of drawings
Fig. 1 has shown plant expression vector pSPT51 structural representation among the embodiment 1.
Fig. 2 has shown the rice callus tissue of expressing red fluorescent protein among the embodiment 2 after Agrobacterium-mediated Transformation.
Fig. 3 has shown the regeneration plant that obtains among the embodiment 2 after Agrobacterium-mediated Transformation.
Fig. 4 has shown that the PCR of part plant among the embodiment 3 identifies
(The 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 (C: negative control, M: molecular weight standard; Plasmid: to transform plasmid) as positive control.
Fig. 6 has shown the transfer-gen plant that big Tanaka is observed visually among the embodiment 5, solid red fluorescence seed and the no fluorescent seeds of comprising of institute.
Fig. 7 shows and fluoresces among the embodiment 5 and not fluorescent brown rice.
Embodiment
Hereinafter specifically describe technical scheme of the present invention with accompanying drawing in conjunction with the embodiments, but be not limited thereto.
Be specifically related to two genes in the embodiment of the invention, be respectively
FPWith
OsCYP704B2 FPWith giving a report/the selection markers gene, derive from the reef coral (
Discosoma sp.), its codified red fluorescent protein can be inspired red fluorescence, is used for the screening of transgenic callus and seed, and its nucleotide sequence and aminoacid sequence are seen SEQ ID NO.1 and SEQ ID NO.2 respectively;
OsCYP704B2Be the paddy rice native gene, catalysis lipid acid hydroxylation participates in the synthetic and extine type one-tenth of flower pesticide cutin haplont,
OsCYP704B2Gene changes ms26 male sterile mutant over to can make its fertility restorer.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 pSPT51(Fig. 1).This carrier is artificial constructed on the pPZP basis.2 expression casettes are arranged on the expression vector: the one,
OsCYP704B2Expression casette, this expression cassette by
OsCYP704B2Gene and self endogenesis promoter and terminator constitute.In order to distinguish the endogenous of paddy rice itself
OsCYP704B2Gene and be transformed into paddy rice by expression vector
OsCYP704B2Gene is at wild-type allele
OsCYP704B2The middle introducing is converted in the paddy rice after 3 single nucleotide mutation sites (SNP) suddenly change, and these 3 SNP sport C respectively at the 1468th, 1470 and 1473 bit base places by G.These change does not all influence coded aminoacid sequence.After the modification
OsCYP704B2Gene nucleotide series sees that wherein SNP is marked by square frame shown in the SEQ ID NO.3; The 2nd, report/selection markers gene
FPGene (SEQ ID NO.1) expression cassette, this gene is seen SEQ ID NO.5 by callus/its nucleotide sequence of seed specific promoters END2(from corn) drive, and by stopping transcribing from the terminator PIN II of potato, the nucleotide sequence of PIN II is seen SEQ ID NO.6.
Do not contain the antibiotics resistance marker gene in the T-DNA section, do not contain the herbicide screening marker gene yet.
Embodiment 2: rice transformation ms26 male sterile mutant material obtains transgenic rice plant
Expression vector among the embodiment 1 is imported among the agrobacterium strains AGLO with electric shocking method, be used for the callus of rice transformation ms26 male sterile material.Utilize red fluorescent protein that transformed calli is screened, accompanying drawing 2 is the callus of expression alien gene, the transformed plant (accompanying drawing 3) of the expression alien gene of further its differentiation being regenerated.
Embodiment 3: the PCR of transfer-gen plant detects
Get the transgenic rice plant blade among the embodiment 2, extract total DNA.With
FPGene 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, illustrates 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(20 U/ul) and with deionized water system is complemented to 200 ul.37 ℃ of incubations 6 hours.Southern Blot detected result shows that foreign gene has been integrated in the acceptor rice genome.
Embodiment 5: identify transgenic seed and non-transgenic seed
At transgenic paddy rice T
1For the seed of gathering in the crops on the plant (is T
2For seed) in, express
FPThe brown rice of gene (removing the seed of clever shell) can be inspired red fluorescence (left-half of accompanying drawing 7), but not transgenic seed does not have fluorescence (right half part of accompanying drawing 7).
Sequence table
＜110〉Beijing Weiming Kaituo Crops Design Center Ltd
The Beijing Kaituo Dna Biotech Research Center Co., Ltd
＜120〉method of a kind of simple Rapid identification transgenic seed and estimation copy number
<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
Sequence table
＜110〉Beijing Weiming Kaituo Crops Design Center Ltd
The Beijing Kaituo Dna Biotech Research Center Co., Ltd
＜120〉method of a kind of simple Rapid identification transgenic seed and estimation copy number
<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 (6)
1. identify the method that whether contains the transgenosis composition in the filial generation for one kind, this method comprises:
A) one section Nucleotide comprises the fluorescent screening flag sequence, and is driven by callus and kind skin specific promoter;
B) the selection markers gene transformation is to paddy rice ms26 male sterile mutant callus, and microbiotic or herbicide screening gene are not contained in the T-DNA zone of conversion carrier;
C) transformant of screening band fluorescence;
D) transformant regeneration plant.
2. claims 1 described fluorescent seeds.
3. described in claims 1, the selection markers gene be selected from a kind of fluorescin (
FP) gene.
4. be the fluorescent screening flag sequence in claims 1 with by three SNP modifieds
MS26Sequence closely connects.
5. claims 3 are described
FPGene order is SEQ ID NO:1.
6. the transformed plant in claims 1 comprises modified
MS26Sequence, this sequence with drive by callus/kind of skin specificity promoter END2 under
FPThe gene close linkage.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105665268A CN102229976A (en) | 2010-11-30 | 2010-11-30 | Method for simply and rapidly identifying transgenic seeds and estimating copy numbers |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105665268A CN102229976A (en) | 2010-11-30 | 2010-11-30 | Method for simply and rapidly identifying transgenic seeds and estimating copy numbers |
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|---|---|
| CN102229976A true CN102229976A (en) | 2011-11-02 |
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ID=44842581
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105665268A Pending CN102229976A (en) | 2010-11-30 | 2010-11-30 | Method for simply and rapidly identifying transgenic seeds and estimating copy numbers |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102870670A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice |
| WO2013066423A3 (en) * | 2011-06-21 | 2013-09-12 | Pioneer Hi-Bred International, Inc. | Methods and compositions for producing male sterile plants |
| CN103805630A (en) * | 2012-11-12 | 2014-05-21 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Novel plant fertility regulation structure and application thereof |
| CN103926224A (en) * | 2014-03-26 | 2014-07-16 | 北京市心肺血管疾病研究所 | Method for assisting in detecting whether transgenic plant components are contained in food |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013066423A3 (en) * | 2011-06-21 | 2013-09-12 | Pioneer Hi-Bred International, Inc. | Methods and compositions for producing male sterile plants |
| US9574208B2 (en) | 2011-06-21 | 2017-02-21 | Ei Du Pont De Nemours And Company | Methods and compositions for producing male sterile plants |
| CN102870670A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice |
| CN103805630A (en) * | 2012-11-12 | 2014-05-21 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Novel plant fertility regulation structure and application thereof |
| 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 |
| 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 |
| CN103926224A (en) * | 2014-03-26 | 2014-07-16 | 北京市心肺血管疾病研究所 | Method for assisting in detecting whether transgenic plant components are contained in food |
| CN103926224B (en) * | 2014-03-26 | 2016-04-13 | 北京市心肺血管疾病研究所 | The method of composition of transgenic plants whether is contained in a kind of auxiliary detection food |
| CN109892218A (en) * | 2019-03-07 | 2019-06-18 | 中国水稻研究所 | A method of rapidly and efficiently screen in paddy gene editor strain without transgene component plant |
| CN109892218B (en) * | 2019-03-07 | 2021-07-13 | 中国水稻研究所 | Method for rapidly and efficiently screening plants without transgenic components in rice gene editing strain |
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