CN101200723A - Butterfly orchid PhPI9 gene coded sequence and uses thereof - Google Patents
Butterfly orchid PhPI9 gene coded sequence and uses thereof Download PDFInfo
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Abstract
The present invention belongs to the field of molecular biology and gene engineering technology and concretely provides a coding sequence which expresses MADS-box gene (PhPI9) related to the flower growth inside a butterfly orchid and the application of the coding sequence for changing the form and the growth of a plant flower. The present invention relates to a recombined expression carrier containing the gene and also relates to the transformation plant cell of the expression carrier and the transgenic plant of the gene produced by the transformation cell. The present invention expresses the gene inside the plant, and compared with the wild flower, the flower shape of the obtained transgenic plant has obvious changes that a floral tube and petals split; the petals are redundant; sepals become stamens and little petalody stamens occur.
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field, be specifically related to a kind of MADS-box gene family PI subclass gene PhPI9 that from butterfly orchid, clones, and copy number analysis, gene expression pattern is analyzed, the structure of transgene carrier, Plant Transformation, the evaluation of the acquisition of transfer-gen plant and corresponding physiological property.
Background technology
The process of blooming is the biological key problem of development of plants, and one of them committed step is to be transformed into the floral organ original hase from flower primordium, and then forms the floral organ with specified shape and spatial disposition order.This process is subjected to the control of a series of homeotic genes.Separated this class homeotic gene has from the model animals Arabidopis thaliana: AGAMOUS (AG), APETALA3 (AP3), PISTILATA (PI), APETALA1 (AP1) and APETALA2 (AP2) etc. and sequential analysis shows that the protein N terminal of these coded by said gene comprises a conserved regions, DNA land height homology with human and zymic transcription factor SRF and MCM1 is called the MADS box.Studies show that the related gene coded transcription factor of these flower developments is had an effect each other, determine the position that flower primordium takes place, and make this regional cytodifferentiation become suitable organ type.In addition, because the MADS-box gene family exists ubiquity and high conservative in vegitabilia, significant to studying phanerogamous origin and phylogeny.
Up to the present, the research of the MADS-box gene that participates in the flower development process is focused mostly on dicotyledons, but the effect of MADS box gene in the monocotyledons flower development has received increasing attention equally.This mainly is because monocotyledonous flower has changeable form, and a lot of monocotyledonous flower, fruit, seed have important economic implications.Though first MADS-box gene that is separated in the monocotyledons from orchid, carries out in corn and paddy rice the research of monocotyledons MADS-box gene is many.The orchid family belongs to one of worldwide four especially big sections, old origin, and evolutionary rate and asynchronous is the peak of spermatophyte phylogeny in monocotyledons, representative and typicalness.The flower of butterfly orchid has typical wheel-like structure, substantially form consistent with the structure of dicotyledons, and it has the lip of eggcase again, and the flower type is bigger, for the function of research flower development gene in monocotyledons provides comparatively ideal test materials.Not only can make the research of the relevant gene of butterfly orchid flower development and usefully to replenish but also can open up an approach for the flowers improvement to the application of ABC model in monocotyledons.
Summary of the invention
First purpose of the present invention just provides a kind of new butterfly orchid MADS-box gene PhPI9.
Second purpose of the present invention provides this butterfly orchid gene PhPI9 encoding sequence and is utilizing transgenic technology to make plant produce the application of male-sterile character.
The present invention is material with the butterfly orchid, according to the conservative property of MADS-box gene order, uses RT-PCR and obtained a MADS-box gene relevant with the butterfly orchid flower development, and called after PhPI9, the NCBI-GenBank accession number is AY748818.It is a dna molecular with particular sequence, and its nucleotides sequence is classified SEQ.ID NO.1 as.
The present invention provides a kind of protein molecule of the butterfly orchid PhPI 9 gene of encoding on the other hand, its aminoacid sequence such as SEQ.ID NO.2.
In another aspect of this invention, also provide a kind of method of the PhPI9mRNA of detection expression pattern, its step is as follows:
(1) total RNA (Trizol, commercially available) of extraction butterfly orchid petal.
(2) utilize reverse transcription test kit (commercially available) that total RNA reverse transcription is become cDNA, according to the 1-24 of the sequence conservation of PI class MADS-box, the distinguished sequence design primer of 833-817 carries out PCR then.
In another aspect of this invention, also provide a kind of transgenic technology of utilizing that the nucleotide sequence that coding has butterfly orchid MADS-box gene PhPI9 is transformed into tobacco to change the method for its flower shape and growth, its step is as follows:
(1) open reading frame with butterfly orchid MADS-box gene PhPI9 is operably connected to the expression of plants regulating and controlling sequence, forms the plant expression vector that contains butterfly orchid MADS-box gene PhPI9;
(2) change the expression vector in the step (1) over to Agrobacterium, the Agrobacterium that will contain plant expression vector is cultivated altogether with eukaryotic host cell, under 25 ± 2 ℃ of conditions, the dark cultivation after 2 days, by antibiotic-screening, obtain transformant and the regeneration of transgenic plant of butterfly orchid MADS-box family gene PhPI9.The transfer-gen plant that the contains butterfly orchid MADS-box family gene PhPI9 proterties such as showing as cylindrical fireworks and petal cracking of blooming.
In the present invention, term " open reading frame of butterfly orchid PhPI 9 gene " refer to the encode proteic nucleotide sequence of complete butterfly orchid PhPI 9, the i.e. nucleotide sequence and the degenerate sequence thereof of 1-630 position among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the Nucleotide of SEQ ID NO.1 1-630 position, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in the nucleotide sequence homology of 1-630 position be low to moderate about 85% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid etc.
In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other plant cell.
In addition, the quantity that in cell, exists of also available Southern blotting technical Analysis butterfly orchid MADS-box family gene PhPI9.
The proteic Nucleotide full length sequence of butterfly orchid PhPI 9 of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.
In case obtained relevant sequence, just can obtain this sequence in large quantity with recombination method.This normally is cloned into carrier with it, is changing cell over to, is separated to relevant sequence by ordinary method from the host cell after the propagation then.
The present invention also provides a kind of method that is used for test sample butterfly orchid MADS-box gene PhPI9 copy number, and it comprises with SEQ ID NO.1 and hybridizing as probe and sample whether detection probes combination has taken place then; This sample is the butterfly orchid genomic dna, the nucleotide sequence behind digestion with restriction enzyme.
Table 1 is that the homology of the PI-like MADS-box gene nucleotide series (GenBank Accession No.DQ017701) of butterfly orchid MADS-box family gene PhPI9 of the present invention and another orchid Dendrobiumthyrsiflorum compares (FASTA) table.
Table 2 is that the homology of the aminoacid sequence (GenBank Accession No.DQ017701) of butterfly orchid MADS-box family gene PhPI9 Argine Monohydrochloride sequence of the present invention and another orchid ODendrobium thyrsiflorum compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".
Description of drawings:
Fig. 1 identifies for the Southern blot of butterfly orchid PhPI 9 gene: lane1 DraI enzyme is cut, and lane2 HindIII enzyme is cut.
Fig. 2 identifies that for RT-PCR the expression pattern of PhPI9: Actin is contrast .Ped1: three joint position bennets, Ped2: four joint position bennets, Ped3: five joint position bennets, bud: bud, root: root, leaf: leaf, sep: sepal, pet: petal, lip: lip, colum: stamen post .Maker DL 2000 is followed successively by from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 200bp, 100bp.
Fig. 3 is that the PCR of the hygromycin gene of transfer-gen plant identifies (part).PI9 1,2, and 4,7 is PhPI9 transgene tobacco strain system numbering.
Fig. 4 is the part phenotype of transgene tobacco.A is the cylindrical fireworks of transgene tobacco; B is transgene tobacco cylindrical fireworks stamenizations; C is a cylindrical fireworks rimose transgene tobacco; D is the transgene tobacco of petal cracking; E, the transgene tobacco flower of F petal redundancy; G is the transgene tobacco of petal stamenization; H, I are petal and the cylindrical fireworks of unloaded tobacco.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition of being narrated in, or the condition of advising according to manufacturer.
The clone of butterfly orchid PhPI 9 gene
1. get Phalaenopsis leaves and place the freezing preservation of liquid nitrogen immediately.
2.DNA extraction
Get portion of tissue, grind, add the 50ml centrifuge tube of the lysate that fills preheating with mortar, 65 ℃ of insulation 40min are centrifugal.Add RNaseA (0.5 μ g/ml) in the supernatant, 65 ℃ of digestion RNA 1h; Take out albumen one time with isopyknic phenol/chloroform/primary isoamyl alcohol (25/24/1), with ethanol sedimentation DNA, 70% washing with alcohol twice is used TE solution dissolving DNA at last.Detect the quality of DNA with 0.8% sepharose.
3. Cloning of Entire Gene
Butterfly orchid petal grinding powder in liquid nitrogen with fresh extracts total RNA. primer B with Trizol reagent
265 ' GACTCGAGTCGACATCGAT
173 ' synthetic cDNA first chain, reverse transcription is respectively with FMADS:5 ' ATGGGG/AGC/AGGC/AAAGAGC/AGAGATCAAGA/C3 ' and B
25: 5 ' GACTCGAGTCGACATCGA3 ' carries out PCR as 5 ' and 3 ' primer, and reaction conditions is: 94 ℃ of 30s, and 53 ℃ of 60s, 72 ℃ of 90s carry out 30 circulations altogether.Adopt the RT-PCR method from butterfly orchid cDNA, to amplify a band about 1kb.The PCR product is reclaimed, be connected on the T-carrier, check order as universal primer with SP6 and T7.Sequencing result is committed to NCBI Non-redundant data storehouse, and BLAST result shows corresponding M ADS-box gene order high conservative in this sequence and other species.
Embodiment 2
The sequence information of butterfly orchid PhPI 9 gene and homology analysis
The length of the butterfly orchid PhPI 9 gene that the present invention is new is 833bp, and detailed sequence is seen SEQ ID NO.1.The polypeptide of this genes encoding is made up of 210 amino-acid residues, and molecular weight 25422.40 dalton, pI are 9.37.Detailed sequence is seen SEQID NO.2.
The coding region sequence of butterfly orchid PhPI 9 gene and encoded protein matter sequence thereof are carried out Nucleotide in Non-redundant GenBank+EMBL+DDBJ+PDB and the Non-redundant GenBank CDS translations+PDB+SwissPort+Superdate+PIR database again with blast program and protein homology detects, found that there are certain homology in it and another orchid Dendrobium thyrsiflorum PISTILLATA-like MADS box gene.On nucleotide level, the mRNA encoding sequence (GenBank Accession No.DQ017701) of it and Dendrobium thyrsiflorum PISTILLATA-like MADS box gene has 88% homology (seeing Table 1), on amino acid levels, it and the proteic amino-acid residue of Dendrobium thyrsiflorum PISTILLATA-like MADS box have 95% similarity (seeing Table 2).Therefore there are higher homology in this PhPI9 gene and Dendrobium thyrsiflorumPISTILLATA-like MADS box gene.
Embodiment 3
The copy number analysis of butterfly orchid PhPI 9 gene
With a large amount of extracting butterfly orchid of SDS method genomic dna, get 10 μ g DNA and use Dra I (the DraI restriction enzyme site is arranged in the probe) and Hin d III enzyme to cut respectively, carry out fragment with 0.8% sepharose and separate, DNA is forwarded on the Hybond-N+ nylon membrane, fixing; 3 ' UTR with butterfly orchid PhPI 9 gene is that probe carries out Southern hybridization, identifies its copy number in butterfly orchid, and the result shows that the PhPI9 gene is single copy.
Embodiment 4
The butterfly orchid PhPI 9 gene expression pattern is analyzed
Extract total RNA of root, blade, bennet, bud and the floral organ (calyx, ala, lip and stamen post) of butterfly orchid plant respectively.Measure OD
2601 μ g RNA, reverse transcription are quantitatively taken out in the back. carry out PCR with special primer.Reaction product is separated with 1.0% agarose gel electrophoresis.As can be seen, gene PhPI9 only expresses at the reproductive organ of plant-growth, and does not express in vegetative organ from electrophorogram.Further each organ to bud detects respectively, finds that it has expression in lip.The growth of inferring lip in this gene and the petal has confidential relation, and does not participate in the regulation and control of vegetative organ growth.
Embodiment 5
Butterfly orchid PhPI 9 gene carries out the phenotypic evaluation of eukaryotic expression and transfer-gen plant in tobacco cell
The structure that contains goal gene (butterfly orchid PhPI 9 gene) expression vector
According to the butterfly orchid PhPI 9 gene complete encoding sequence, design and amplify the primer (EQ.IDNO.3) that complete coding is read frame, and on forward and reverse primer, introduce restriction enzyme enzyme recognition site (deciding) respectively, so that construction of expression vector on the carrier of selecting for use.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, will be cloned among the binary expression vector pHB with the butterfly orchid PhPI 9 gene forward, under the prerequisite that guarantees reading frame, it is changed in the Agrobacterium.Utilize leaf disc law technology transformation mode plant tobacco.
Utilize leaf dish method transformation of tobacco
1, with the positive colony on the sterilization toothpick picking YEB selection substratum, is inoculated in 2mlYEB liquid (Rifampin 40mg/L, kantlex 50mg/L, gentamicin 100mg/L), 28 ℃ of 200rpm shaking culture 24-36h;
2, the centrifugal 10min of 4000g under the room temperature;
3, abandon supernatant, thalline is resuspended with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes OD
600About=0.5;
4, get the aseptic blade of the tobacco about two weeks of growth, remove its rib, it is cut into about 1 square centimeter of square vanelets;
5, blade is put into the bacterium liquid for preparing, soaked 2-5min, on aseptic filter paper, blot bacterium liquid;
6, the blade that will infect is put on the MS substratum, 28 ℃ of dark 48h that cultivate;
7, blade is forwarded on the callus substratum (MS+6-BA 1.0mg/L+NAA 0.1mg/L+ Totomycin 50mg/L+cef250mg/L), 25-28 ℃ of illumination is cultivated down, sees that callus formed in 7-15 days;
8, visible differentiation bud grows after about 20 days, treat that bud is grown up after, downcut, place (1/2MS+NAA (0.5mg/L+ Totomycin 50mg/L+cef250mg/L) on the root media;
9, treat well developed root system after, plant is taken out, clean the solid medium that adheres to sterilized water, move in the soil, cultivate in the greenhouse.
The Molecular Identification of transfer-gen plant
With the SDS method genomic dna of extracting part transfer-gen plant in a small amount, with the tobacco that changes the pHB empty carrier in contrast,, carry out PCR and detect (partial results) according to hygromycin gene sequences Design primer on the carrier.The result shows that the expression plasmid that carries the external source butterfly orchid PhPI 9 gene has been inserted in the tobacco gene group.
Transfer-gen plant carries out phenotypic evaluation
Petal and the cylindrical fireworks of comparing transgene tobacco with the flower of wild-type tobacco ftracture more, petal overlapping (redundancy).And the petalody stamen appears in indivedual tobaccos and occur having on the cylindrical fireworks stamen structure to produce on a small quantity.Supposition external source PhPI9 gene has been realized overexpression in the host tobacco, influenced the growth of transfer-gen plant petal and stamen.Test-results shows that the butterfly orchid PhPI 9 gene that obtains among the embodiment 1 is the gene that function is arranged, and can influence the growth of plant petal.
Table 1
The homology of the aminoacid sequence of butterfly orchid MADS-box family gene PhPI9 nucleotide sequence and another orchid Dendrobium thyrsiflorum relatively
gb|DQ017701.1|Dendrobium?thyrsiflorum?PISTILLATA-like?MADS?box?protein(PI)
mRNA，partial?cds?Length＝859?Score＝880bits(476)，Expect＝0.0；Identities＝665/753(88％)，
Gaps＝26/753(3％) Strand＝Plus/Plus
Query?29 GGATCGAGAACTCAACCAACCGGCAAGTGACCTTCTCGAAGAGGCGGAATGGAATCATGA 88
||||||||||||||||?|||||?|||||||||||||||||||||?|||||||||||||||
Sbjct?2 GGATCGAGAACTCAACGAACCGCCAAGTGACCTTCTCGAAGAGGTGGAATGGAATCATGA 61
Query?89 AGAAGGCGAAGGAGATCAGCGTGCTCTGCGACGCCCAGGTTTCGCTTGTCATCTTTTCCA 148
|||||||?|||||||||||||||||?||||||||||||||?||||||||?|||||?||||
Sbjct?62 AGAAGGCCAAGGAGATCAGCGTGCTATGCGACGCCCAGGTCTCGCTTGTTATCTTCTCCA 121
Query?149 GCCTTGGAAAGATGTTTGAGTATTGTAGCCCATCCACCACGCT-GTCGAAGATGCTGGAG 207
|||||||||||||||||||||||||||||||?||||||||?||?||||||||||||||||
Sbjct?122 GCCTTGGAAAGATGTTTGAGTATTGTAGCCCCTCCACCAC-CTTGTCGAAGATGCTGGAG 180
Query?208 AAATACCAGCAGAACTCTGGG-AAGAAGCTCTGGGACGCCAAGCACGAGAACTTGAGCGC 266
|||||||||||?|||||?|||?|||||?|||||||||||?||?|||||?|||||||||||
Sbjct?181 AAATACCAGCAAAACTC-GGGCAAGAAACTCTGGGACGCAAAACACGAGAACTTGAGCGC 239
Query?267 GGAGATTGATCGTATCAAGAAGGAAAATGATAATATGCAGATCGAACTCAGGCATTTGAA 326
||||||?|||||?|||||||||||?|||||||||||||||||||||||||||||||||||
Sbjct?240 GGAGATCGATCGGATCAAGAAGGAGAATGATAATATGCAGATCGAACTCAGGCATTTGAA 299
Query?327 AGGGGAGGATCTGAACTCTCTTAACCCAAAAGAGCTTATTCCGATTGAGGAAGCCCTGCA 386
||||||||||||||||||||||||||||||||||||?||?|||||?||||||||?||?||
Sbjct?300 AGGGGAGGATCTGAACTCTCTTAACCCAAAAGAGCTCATCCCGATCGAGGAAGCGCTCCA 359
Query?387 GAATGGTCTCACTAGCGTTCGGGATAAACAAATGGACTACTTGAAGATGCTTAAAAAGAA 446
|||||||||?|?|?|?|||||?||||||||?|||||||?||||||||||||?||||||||
Sbjct?360 GAATGGTCTTAGTGGTGTTCGAGATAAACAGATGGACTTCTTGAAGATGCTAAAAAAGAA 419
Query?447 TGAAAGGATGCTTGAAGATGAAAATAAAAGGCTCACATACCTATTGCACCAACAACAAAT 506
||||||?|||||||||||?|||||||||||||||||||||||||||||?||?||?|||?|
Sbjct?420 TGAAAGAATGCTTGAAGAGGAAAATAAAAGGCTCACATACCTATTGCATCATCAGCAACT 479
Query?507 GGCAATGGAAGGGAGTATGAGAGAACTCGACATTGGCTATCATCATAAAGATCGCGAGTA 566
||||||||||||||?|||||||||||?|||||?||||||||?|||||||||?|?|||||
Sbjct?480 AGCAATGGAAGGGAGCATGAGAGAACTGGACATCGGCTATCATCAGAAAGATAGGGAGTA 539
Query?567 TGCGGCTCAGATGCCAATGACTTTTCGTGTCCAACCCATACAGCCCA-ACTTGCAGGGAA 625
|||?||||||||||||?||||?||||||||?||?|||||?|||||?|?|?||||||||||
Sbjct?540 TGCAGCTCAGATGCCATTGACCTTTCGTGTGCAGCCCATTCAGCC-AGACTTGCAGGGAA 598
Query?626 ATAAGTAACTGTGTTAGCCTACTGCTTTCCTGTT--GTTTAAATGAATTATTA-TATTAA 682
|||||||||?|?|||||||||||?|||||||?|| |||?|||||||||?|||?|||||
Sbjct?599 ATAAGTAACAGCGTTAGCCTACTACTTTCCTTTTTAGTT-AAATGAATT-TTAATATTAG 656
Query?683 CTTTTG-----GCAGTT-C-TGTGAGAATATGAA-AACTT-ATATGGCTAATT-ATCAGA 732
||||| ||||||?|?|?|?||||||||||?|||||?| || | ||?||?|||
Sbjct?657?CTTTTTAATTAGCAGTTTCAT-T-AGAATATGAAGAACTTCAG-TGA-TG-TTCATGAGA 711
Query?733?TATGTGCTTACTAGTGATATTCATATTGTAACT 765
|||||||?|?|||||||||||?||||||?||||
Sbjct?712?TATGTGCGTGCTAGTGATATTGATATTGAAACT 744
Table 2
Butterfly orchid MADS-box family gene PhPI9 Argine Monohydrochloride sequence and another orchid Dendrobium
The homology of the aminoacid sequence of thyrsiflorum relatively
gb|AAY86363.1|PISTILLATA-like?MADS?box?protein[Dendrobium?thyrsiflorum]Length＝201?Score＝384bits(985)，Expect＝5e-105?Identities＝191/201(95％)，Positives＝197/201(98％)，Gaps＝0/201(0％)Frame＝+1
Query?28 RIENSTNRQVTFSKRRNGIMKKAKEISVLCDAQVSLVIFSSLGKMFEYCSPSTTLSKMLE?207
RIENSTNRQVTFSKR?NGIMKKAKEISVLCDAQVSLVIFSSLGKMFEYCSPSTTLSKMLE
Sbjct?1 RIENSTNRQVTFSKRWNGIMKKAKEISVLCDAQVSLVIFSSLGKMFEYCSPSTTLSKMLE?60
Query?208?KYQQNSGKKLWDAKHENLSAEIDRIKKENDNMQIELRHLKGEDLNSLNPKELIPIEEALQ?387
KYQQNSGKKLWDAKHENLSAEIDRIKKENDNMQIELRHLKGEDLNSLNPKELIPIEEALQ
Sbjct?61 KYQQNSGKKLWDAKHENLSAEIDRIKKENDNMQIELRHLKGEDLNSLNPKELIPIEEALQ?120
Query?388?NGLTSVRDKQMDYLKMLKKNERMLEDENKRLTYLLHQQQMAMEGSMRELDIGYHHKDREY?567
NGL+?VRDKQMD+LKMLKKNERMLE+ENKRLTYLLH?QQ+AMEGSMRELDIGYH?KDREY
Sbjct?121?NGLSGVRDKQMDFLKMLKKNERMLEEENKRLTYLLHHQQLAMEGSMRELDIGYHQKDREY?180
Query?568?AAQMPMTFRVQPIQPNLQGNK?630
AAQMP+TFRVQPIQP+LQGNK
Sbjct?181?AAQMPLTFRVQPIQPDLQGNK?201
Query is a butterfly orchid MADS-box family gene PhPI9 aminoacid sequence
Sbjct is a Dendrobium thyrsiflorum PI-like MADS boxI aminopeptidase gene acid sequence (GenBankAccession No.DQ017701)
Sequence table
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 833bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: nucleic acid
(iii) sequence description: SEQ ID NO.1
1?atggggaggg?gcaagataga?gatcaagagg?atcgagaact?caaccaaccg?gcaagtgacc
61?ttctcgaaga?ggcggaatgg?aatcatgaag?aaggcgaagg?agatcagcgt?gctctgcgac
121?gcccaggttt?cgcttgtcat?cttttccagc?cttggaaaga?tgtttgagta?ttgtagccca
181?tccaccacgc?tgtcgaagat?gctggagaaa?taccagcaga?actctgggaa?gaagctctgg
241?gacgccaagc?acgagaactt?gagcgcggag?attgatcgta?tcaagaagga?aaatgataat
301?atgcagatcg?aactcaggca?tttgaaaggg?gaggatctga?actctcttaa?cccaaaagag
361?cttattccga?ttgaggaagc?cctgcagaat?ggtctcacta?gcgttcggga?taaacaaatg
421?gactacttga?agatgcttaa?aaagaatgaa?aggatgcttg?aagatgaaaa?taaaaggctc
481?acatacctat?tgcaccaaca?acaaatggca?atggaaggga?gtatgagaga?actcgacatt
541?ggctatcatc?ataaagatcg?cgagtatgcg?gctcagatgc?caatgacttt?tcgtgtccaa
601?cccatacagc?ccaacttgca?gggaaataag?taactgtgtt?agcctactgc?tttcctgttg
661?tttaaatgaa?ttattatatt?aacttttggc?agttctgtga?gaatatgaaa?acttatatgg
721?ctaattatca?gatatgtgct?tactagtgat?attcatattg?taactctcca?aactcattag
781?taactatggc?taaatatttt?tatgttctag?tcaaaaaaaa?aaaaaaaaaa?aaa
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 210 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: polypeptide
(iii) sequence description: SEQ ID NO.2
1?mgrgkieikr?ienstnrqvt?fskrrngimk?kakeisvlcd?aqvslvifss?lgkmfeycsp
61?sttlskmlek?yqqnsgkklw?dakhenlsae?idrikkendn?mqielrhlkg?edlnslnpke
121?lipieealqn?gltsvrdkqm?dylkmlkkne?rmledenkrl?tyllhqqqma?megsmreldi
181?gyhhkdreya?aqmpmtfrvq?piqpnlqgnk
(3) information of SEQ ID NO.3
FMADS：5’ATGGGG/AGC/AGGC/AAAGAGC/AGAGATCAAGA/C3’
B
25：5’GACTCGAGTCGACATCGA3’
Claims (6)
1. an isolated butterfly orchid PhPI 9 gene is characterized in that its nucleotides sequence is classified as shown in the SEQ ID NO.1 in order to have the dna molecular of specific sequence.
2. the protein molecule of the described butterfly orchid PhPI 9 gene of claim 1 of encoding is characterized in that having the aminoacid sequence shown in the SEQID NO.2.
3. one kind is utilized transgenic technology to change the nucleotide sequence that coding has butterfly orchid MADS-box gene PhPI9 over to tobacco to change the method for colored type and growth, it is characterized in that the concrete operations step is:
(1) coding is had the exercisable expression of plants regulating and controlling sequence that is connected in of open reading frame of butterfly orchid MADS-box gene PhPI9, form and contain from the plant expression vector of the nucleotide sequence of Nucleotide 1-630 position;
(2) change the expression vector in the step (1) over to Agrobacterium; The Agrobacterium that will contain plant expression vector is cultivated altogether with eukaryotic host cell, under 25 ± 2 ℃ of conditions, secretly cultivate 2 days after, by antibiotic-screening, obtain transformant and the regeneration of transgenic plant of butterfly orchid MADS-box family gene PhPI9.
4. a method that is used for test sample butterfly orchid MADS-box gene PhPI9 copy number is characterized in that it comprises usefulness SEQ ID NO.1 as probe and sample hybridization, and whether detection probes combination has taken place then; This sample is the butterfly orchid genomic dna, the nucleotide sequence behind digestion with restriction enzyme.
5. be used to identify the nucleic acid molecule of transgene tobacco, it is characterized in that it comprises the reverse complementary sequence of 1-24 successive Nucleotide among the SEQ ID NO.1 or 614-630 continuous nucleotide.
6. one kind is used for the method whether test sample exists hygromycin gene nucleotide sequence in the expression vector, it is characterized in that it comprises carrying out PCR with described nucleotide sequence of claim 5 and sample reacts, and detects whether amplify the purpose fragment then; Sample is the transgene tobacco genomic dna.
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| CN2007100471851A CN101200723B (en) | 2007-10-18 | 2007-10-18 | Butterfly orchid PhPI9 gene coded sequence and uses thereof |
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| CN2007100471851A CN101200723B (en) | 2007-10-18 | 2007-10-18 | Butterfly orchid PhPI9 gene coded sequence and uses thereof |
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| CN101200723A true CN101200723A (en) | 2008-06-18 |
| CN101200723B CN101200723B (en) | 2010-12-15 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134568B (en) * | 2010-01-25 | 2012-12-26 | 广西大学 | Promoter for MADS-box gene in maize and application thereof |
| CN109055392A (en) * | 2018-08-01 | 2018-12-21 | 广东省农业科学院环境园艺研究所 | State's orchid allelotaxis controlling gene and its coding albumen and application |
| CN115927390A (en) * | 2022-12-27 | 2023-04-07 | 华南农业大学 | Cymbidium sinense organ development gene CsPI1 and coding protein and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100366744C (en) * | 2005-09-15 | 2008-02-06 | 复旦大学 | Butterfly orchid pPI15 encoding sequence and its uses |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134568B (en) * | 2010-01-25 | 2012-12-26 | 广西大学 | Promoter for MADS-box gene in maize and application thereof |
| CN109055392A (en) * | 2018-08-01 | 2018-12-21 | 广东省农业科学院环境园艺研究所 | State's orchid allelotaxis controlling gene and its coding albumen and application |
| CN109055392B (en) * | 2018-08-01 | 2021-09-10 | 广东省农业科学院环境园艺研究所 | Orchis orchid organ development regulation gene and its coded protein and application |
| CN115927390A (en) * | 2022-12-27 | 2023-04-07 | 华南农业大学 | Cymbidium sinense organ development gene CsPI1 and coding protein and application thereof |
| CN115927390B (en) * | 2022-12-27 | 2024-02-13 | 华南农业大学 | Cymbidium organ development gene CsPI1 and encoding protein and application thereof |
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| Publication number | Publication date |
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| CN101200723B (en) | 2010-12-15 |
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