CN102229936A - Cloning and applications of chrysanthemum morflorium floral development gene CmCO - Google Patents
Cloning and applications of chrysanthemum morflorium floral development gene CmCO Download PDFInfo
- Publication number
- CN102229936A CN102229936A CN2011101501009A CN201110150100A CN102229936A CN 102229936 A CN102229936 A CN 102229936A CN 2011101501009 A CN2011101501009 A CN 2011101501009A CN 201110150100 A CN201110150100 A CN 201110150100A CN 102229936 A CN102229936 A CN 102229936A
- Authority
- CN
- China
- Prior art keywords
- ser
- chrysanthemum
- ala
- asp
- arg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to cloning and applications of a chrysanthemum morflorium floral development gene CmCO, and relates to the technical field of molecular biology and biotechnology. Cloning processes of the CmCO comprises 1, extracting total RNA from chrysanthemum morflorium spires and carrying out an inverse transcription process on the total RNA to obtain cDNA, 2, designing degenerate primers and carrying out a normal polymerase chain reaction (PCR) process to obtain a PCR product, 3, connecting the PCR product and pMD 18-T vectors to form recombinants, carrying out a conversion of escherichia coli DH5 alpha competent cells by the recombinants and screening the recombinants, 4, carrying out an end 3' and end 5' rapid-amplification process according to a technology of rapid-amplification of end 3' and end 5' to obtain respectively sequences of end 3' and end 5' and carrying out a splicing process to obtain a complete cDNA sequence; and 5, designing specific primers according to the sequences of end 3' and end 5' of cDNA, carrying out a PCR amplification process by chrysanthemum morflorium spire cDNA as a template to obtain a chrysanthemum morflorium floral development gene CmCO. The chrysanthemum morflorium floral development gene CmCO is utilized for ornamental plants and can regulate and control flowering phases of plants.
Description
(1) technical field
The present invention relates to clone, Function Identification and the application of chrysanthemum (Chrysanthemum morflorium) flower development gene C mCO, belong to molecular biology and biological technical field.
(2) background technology
Chrysanthemum is China's tradition famous flower, also is one of the world's four big cut-flowers.Regulate the florescence technology by the photoperiod at present and carry out anniversary production, and year-round provision market.But because chrysanthemum is the short day flowers, for producing the spring take, shading in summer and florescence control measure such as light filling in winter the anniversary, cause the waste of great amount of manpower and material resources and financial resources, restricted the fast development of chrysanthemum industry, cultivated insensitive chrysanthemum new variety of photoperiod by transgenic technology and have important theory and practice significance.
CO is into colored important timing gene, when its product reaches certain thresholding, promotes into colored Expression of Related Genes (Onouchi H et al., 2000) significantly, and CO also may have the effect (Samach A et al., 2000) of direct promotion flower development in addition.The CO gene is that Putterill (1995) adopts the method for map based cloning to be separated to, the encoding zinc finger protein transcription factor, and it plays a crucial role in the photoperiod regulation and control, is to experience the bridge that ambient light is shone and bloomed.Overexpression CO can make Arabidopis thaliana no matter sunshine all can bloom in advance (Putterill J, 19995 of length; Samach A et al., 2000).The CO albumen of high level accumulation has activated the FT expression of gene, thereby the promotion Arabidopis thaliana is bloomed under long day condition (Samach A et al., 2000).Onouchi etc. (2000) find can induce prematurity and forfeiture photoperiod sensitivity by cauliflower mosaic virus 35S promoter constitutive expression CO.Arabidopis thaliana CO mutant shows as the long day delay of blooming, some allos CO genes (as short day plant morning glory PnCO gene and long day plant radish BnCO gene) change in the Arabidopis thaliana CO mutant, can make the CO mutant return to normal phenotype, conservative property and importance (Robert et al., 1998 of the similar gene of plant CO on the flowering time control approach have been shown; Liu et al, 2001).
Along with development of molecular biology, utilize genetic engineering means that flowers are improved breeding and also obtained development rapidly in the nearly more than ten years.The genetically engineered of chrysanthemum is started late, early stage research mainly is the influence of various factors to transforming of setting up in effective chrysanthemum regeneration system and the discussion genetic transformation, the gene that changes over to mainly is NPT II (Karle R etc., 1993) and GUS (Boase M R etc., 1998) reporter gene.Later stage is intended to by the research to chrysanthemum orderly improvement proterties then to change goal gene over to, expands the chrysanthemum new variety.The investigator has been cloned into the CO homologous gene and its sequence signature, expression pattern and functional performance has been studied the species surplus 30 at present.
(3) summary of the invention
For utilizing the transgenic technology regulation and control flowering of plant time, the invention provides a kind of chrysanthemum flowers development gene CmCO and the application in ornamental crops thereof, can be used for florescence of plant.
The present invention extracts total RNA from the chrysanthemum spire, reverse transcription obtains cDNA.According to CO gene conservative aminoacid sequence in announced other plant among the GenBank, the design degenerate primer, carry out conventional polymerase chain reaction (Polymerase chain reaction, PCR).The PCR product is connected with the pMD18-T carrier, transformed into escherichia coli DH5 α competent cell, the screening recon carries out sequential analysis.Then by 3 ' and 5 ' terminal rapid amplifying (Rapid-amplification of cDNA ends, RACE) technology obtains 3 respectively ' and 5 ' terminal sequence, and is spliced into complete cDNA sequence.According to 3 of cDNA ' and 5 ' terminal sequence design special primer, be that template is carried out pcr amplification with the cDNA of chrysanthemum spire, obtain the cDNA full length sequence, with this cDNA called after CmCO.
The full-length cDNA of this gene is 1289bp, and wherein open reading frame partly is 1149bp.Push away thus, this gene has 382 amino acid.Its aminoacid sequence is retrieved in GenBank, find and FaCO (the Fragaria ananassa that has delivered, ACJ06578), PsCOL (Pisum sativum, AAX47172), AtCO (Arabidopsis thaliana, NP_197088.1) compare, amino acid identity is respectively 65.8%, 62.0%, 55.6%.Show thus, obtained the gene C mCO that chrysanthemum flowers is grown through above-mentioned clone's step.
This gene order is as follows:
Sequence table
(1) information of SEQ ID NO.1
(a) sequence signature
* length: 1298 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: chrysanthemum (Chrysanthemum morflorium)
(f) sequence description: SEQ ID NO.1
1?ATGTTGAAAC?AAGAGAGTAA?CATTAACGCT?AGTGGAGCGA?ACAGTTGGGC?ACGACTCTGT
61?GACACATGCC?GGTCAGCTCC?CTGCACCGTG?TATTGCCGAG?CAGATTCTGC?CTACTTGTGC
121?GCCGGCTGTG?ATGCCCATGT?TCACGCTGCC?AATCGTGTTG?CTTCCCGCCA?TAAACGCGTC
181?AGGGTCTGCG?AGGCGTGTGA?GCGTGCTCCA?GCTGCTTTTT?TATGTAAGGC?AGATGCAGCC
241?TCTCTTTGCA?CCGCCTGTGA?TGCAGATATT?CACTCTGCAA?ACCCTCTTGC?ACGACGCCAC
301?CAACGTGTCC?CGGTTATTCC?TATTTCAGGG?TCTACGTATG?AATCTCAAGG?CAGGTTTTTT
361?CCCCAAGGGT?CAGACGGAAC?TGTTAATAAA?GAAGAAGAAG?ACGAAGCAGC?GTCGTGGCTG
421?CTATTCGATA?CTCCTGCTAA?AAACAACCAA?AACCAAGAAT?ATACGAACGA?GTTTTTGTTT
481?AATGGAGAGG?GGGGTGTAGA?TGAGTATTTG?GATCTTGTGG?ACTACAATTC?TTGTCAAGAT
541?ACTCAGTTTA?GTGATGATCA?TAAATGCAAT?AATTTGCAAT?TTAATGATGA?CTATAAGTAT
601?ACGAATGATG?TTACTAATTA?TAGTAAGGAT?ATGCGGAAGT?ATGGCAGAGG?TGATGCAGAT
661?AGCGTAGTGC?CAGTTGGAGG?TGGAGAGGCT?AAGAAGGAGC?ATCAAATTTA?TGATCTTAAC
721?TTCCAACATC?AAAAATTTCA?ATTGGGATGT?GATTATGAGG?CTTCAAATGG?TGGCTACAGC
781?TACCCTGCTT?CACGTGGTCA?TAGTGTTTCT?ATGTCATCAC?TGGATGTTGG?AGTAGTACCA
841?GAATCTTCAA?TCTCAAGTTC?AAGATCTTCA?AAAGGGACAA?CTGACTTATT?CTCAGGTACT
901?TCAATTCAGA?TGCCAACGCA?ACTTACTCCG?TTAGACAGAG?AGGCAAGAGT?CTTGAGTTAC
961?AGGGAAAAGA?AGAAAACAAG?AAAATTTGAG?AAAACGATTA?GGTACGCATC?TAGAAAAGCG
1021?TATGCAGAAA?CAAGGCCTAG?GATCAAAGGC?CGTTTCTCAA?AGCGAACAAA?TGTTGATGTC
1081?GAGGTGGATC?AAATGTTTTC?GACAACATTA?ATGACAGAAG?GCGGATACTG?TATTGTCCCT
1141?TCGTTTTAAT?GAATGGTTAG?GAGCAATAGA?TGCCAATATT?GTAACTGGTT?TTTTTCATTC
1201?AAGTCATGAG?GTTATTATAG?TAAATCTCGT?AACTGTATGT?TCAACGCTTC?TCATTTCCTT
1261?AAATGCAAGC?AAACTTTCCA?GTTTTGGTT
(2) information of SEQ IN NO.2
(a) sequence signature
* length: 382 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
Sequence description
Met?Leu?Lys?Gln?Glu?Ser?Asn?Ile?Asn?Ala?Ser?Gly?Ala?Asn?Ser
1 5 10 15
Trp?Ala?Arg?Leu?Cys?Asp?Thr?Cys?Arg?Ser?Ala?Pro?Cys?Thr?Val
20 25 30
Tyr?Cys?Arg?Ala?Asp?Ser?Ala?Tyr?Leu?Cys?Ala?Gly?Cys?Asp?Ala
35 40 45
His?Val?His?Ala?Ala?Asn?Arg?Val?Ala?Ser?Arg?His?Lys?Arg?Val
50 55 60
Arg?Val?Cys?Glu?Ala?Cys?Glu?Arg?Ala?Pro?Ala?Ala?Phe?Leu?Cys
65 70 75
Lys?Ala?Asp?Ala?Ala?Ser?Leu?Cys?Thr?Ala?Cys?Asp?Ala?Asp?Ile
80 85 90
His?Ser?Ala?Asn?Pro?Leu?Ala?Arg?Arg?His?Gln?Arg?Val?Pro?Val
95 100 105
Ile?Pro?Ile?Ser?Gly?Ser?Thr?Tyr?Glu?Ser?Gln?Gly?Arg?Phe?Phe
110 115 120
Pro?Gln?Gly?Ser?Asp?Gly?Thr?Val?Asn?Lys?Glu?Glu?Glu?Asp?Glu
125 130 135
Ala?Ala?Ser?Trp?Leu?Leu?Phe?Asp?Thr?Pro?Ala?Lys?Asn?Asn?Gln
140 145 150
Asn?Gln?Glu?Tyr?Thr?Asn?Glu?Phe?Leu?Phe?Asn?Gly?Glu?Gly?Gly
155 160 165
Val?Asp?Glu?Tyr?Leu?Asp?Leu?Val?Asp?Tyr?Asn?Ser?Cys?Gln?Asp
170 175 180
Thr?Gln?Phe?Ser?Asp?Asp?His?Lys?Cys?Asn?Asn?Leu?Gln?Phe?Asn
185 190 195
Asp?Asp?Tyr?Lys?Tyr?Thr?Asn?Asp?Val?Thr?Asn?Tyr?Ser?Lys?Asp
200 205 210
Met?Arg?Lys?Tyr?Gly?Arg?Gly?Asp?Ala?Asp?Ser?Val?Val?Pro?Val
215 220 225
Gly?Gly?Gly?Glu?Ala?Lys?Lys?Glu?His?Gln?Ile?Tyr?Asp?Leu?Asn
230 235 240
Phe?Gln?His?Gln?Lys?Phe?Gln?Leu?Gly?Cys?Asp?Tyr?Glu?Ala?Ser
245 250 255
Asn?Gly?Gly?Tyr?Ser?Tyr?Pro?Ala?Ser?Arg?Gly?His?Ser?Val?Ser
260 265 270
Met?Ser?Ser?Leu?Asp?Val?Gly?Val?Val?Pro?Glu?Ser?Ser?Ile?Ser
275 280 285
Ser?Ser?Arg?Ser?Ser?Lys?Gly?Thr?Thr?Asp?Leu?Phe?Ser?Gly?Thr
290 295 300
Ser?Ile?Gln?Met?Pro?Thr?Gln?Leu?Thr?Pro?Leu?Asp?Arg?Glu?Ala
305 310 315
Arg?Val?Leu?Ser?Tyr?Arg?Glu?Lys?Lys?Lys?Thr?Arg?Lys?Phe?Glu
320 325 330
Lys?Thr?Ile?Arg?Tyr?Ala?Ser?Arg?Lys?Ala?Tyr?Ala?Glu?Thr?Arg
335 340 345
Pro?Arg?Ile?Lys?Gly?Arg?Phe?Ser?Lys?Arg?Thr?Asn?Val?Asp?Val
350 355 360
Glu?Val?Asp?Gln?Met?Phe?Ser?Thr?Thr?Leu?Met?Thr?Glu?Gly?Gly
365 370 375
Tyr?Cys?Ile?Val?Pro?Ser?Phe
380
The present invention also provides chrysanthemum flowers development gene CmCO application in ornamental crops, can regulate and control the florescence of plant.Step is:
(a) utilize chrysanthemum flowers development gene CmCO, the cDNA sequence is placed under the CaMV 35S promoter, make up plant expression vector.
(b) expression vector is changed over to Agrobacterium LBA4404 competent cell.
(c) transformant that utilizes (b) to obtain transforms plant, obtains transfer-gen plant.
The present invention isolates flower development gene (CmCO) from chrysanthemum, with this cDNA sequence construct on eucaryon conversion carrier pROK3 by composing type CaMV 35S strong promoter control, make up the sense expression vector of CmCO, adopted agrobacterium mediation method to transform chrysanthemum.The transgenosis chrysanthemum that obtains is carried out the flowering time analysis revealed, and the overexpression of CmCO can be bloomed in advance in the transgenosis chrysanthemum.With respect to the non-transgenic tobacco, transfer-gen plant has the photoperiod insensitivity.Other ornamental plant crops of this gene transformation can regulation florescence, cultivates photoperiod non-sensitive type new variety, improves quality, reduces work and drops into, and has great economic worth and social value.
(4) description of drawings
The cDNA agarose gel electrophoresis picture of Fig. 1 .PCR amplification chrysanthemum flowers development gene CmCO
M represents molecular weight marker, and 1-4 represents CmCO cDNA band
Fig. 2. the comparative result of the CONSTANS aminoacid sequence of CmCO aminoacid sequence and several other plants in the chrysanthemum.Wherein identical amino-acid residue is represented with black matrix.Their number of registration and source of species thereof in GenBank are respectively: FaCO (Fragaria ananassa, ACJ06578), PsCOL (Pisum sativum, AAX47172), AtCO (Arabidopsis thaliana, NP_197088.1).
Fig. 3 .CmCO gene is at the different flower bud differentiation period expression analysis of chrysanthemum
CmCO: this expression of gene trend, Actin: internal control gene
Fig. 4. the construction procedures of chrysanthemum sense expression vector.
Fig. 5. the PCR of agrobacterium tumefaciens bacterium colony detects
M represents molecular weight marker, and 2 and 3 represent pROK3-CO PCR product
By freeze-thaw method expression vector pROK3-CO is changed among the Agrobacterium LBA4404, be applied on the YEP flat board that contains Km and Carb, cultivate 2-3d down for 28 ℃, 2 of picking list bacterium colonies carry out PCR at random.Carry out agarose gel electrophoresis, the result shows that 2 single bacterium colonies all have the band about 1298bp, illustrates that just carrier pROK3-CO successfully changes among the Agrobacterium LBA4404.
(5) concrete invention embodiment
Embodiment 1: the acquisition of Folium chrysanthemi film clips development gene CmCO
1. the cultivation of vegetable material: get chrysanthemum kind ' refreshing horse ' (Chrysanthemum morflorium ' Jinba ') cuttage seeding and be colonizated in (1 strain of 1 basin) in the flowerpot, after process 35d nourishes and grows under the long day condition, (8h/16h SD) handles 15d, relative humidity 65% to short day; Intensity of illumination 110 μ molm-2s-1.
2. the extraction of total RNA: remove to handle mature leaf, utilize TRIZOL method (Invitrogen) to extract total RNA.
3. get the total RNA of 2 micrograms, become strand cDNA with MMLV Reverse Transcriptase (Promega) reverse transcription.
4. the cDNA with the chrysanthemum blade that obtained is a template, uses upstream primer
5 '-ATGTTGAAACAAGAGAGTAAC-3 ' (SEQ ID NO:3) and downstream primer
5 '-AACCAAAACTGGAAAGTTTGC-3 ' (SEQ ID NO:4) is PCR.The PCR system
(TIANGEN:2 * Taq PCR MasterMix): ddH
2O 9.5 μ l; 2 * MasterMix, 12.5 μ l; Each 1 μ l of upstream and downstream primer (10 μ M); Template 1 μ l.Reaction conditions: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 30 seconds, 72 ℃ were extended 33 circulations 1 minute; 72 ℃ were extended 10 minutes.After the fragment glue recovery with amplification, be connected among the cloning vector pMDl8-T.
5. sequencing: originally be operated in the Beijing Liuhe Huada Genomics Technology Co., Ltd and carry out.Obtain the cDNA fragment of a 1289bp.
6. homology retrieval: utilize BLAST software that the aminoacid sequence of the cDNA fragment coding of above-mentioned acquisition and the sequence in the gene library are compared, find that it comprises 2 complete B-box and CCT conservative domain district, determines that it belongs to the CONSTANS family gene.
Embodiment 2: Folium chrysanthemi film clips development gene CmCO, and following sequence:
(1) information of SEQ ID NO.1
(g) sequence signature
* length: 1298 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(h) molecule type: cDNA
(i) suppose: not
(j) antisense: not
(k) initial source: chrysanthemum (Chrysanthemum morflorium)
(l) sequence description: SEQ ID NO.1
1?ATGTTGAAAC?AAGAGAGTAA?CATTAACGCT?AGTGGAGCGA?ACAGTTGGGC?ACGACTCTGT
61?GACACATGCC?GGTCAGCTCC?CTGCACCGTG?TATTGCCGAG?CAGATTCTGC?CTACTTGTGC
121?GCCGGCTGTG?ATGCCCATGT?TCACGCTGCC?AATCGTGTTG?CTTCCCGCCA?TAAACGCGTC
181?AGGGTCTGCG?AGGCGTGTGA?GCGTGCTCCA?GCTGCTTTTT?TATGTAAGGC?AGATGCAGCC
241?TCTCTTTGCA?CCGCCTGTGA?TGCAGATATT?CACTCTGCAA?ACCCTCTTGC?ACGACGCCAC
301?CAACGTGTCC?CGGTTATTCC?TATTTCAGGG?TCTACGTATG?AATCTCAAGG?CAGGTTTTTT
361?CCCCAAGGGT?CAGACGGAAC?TGTTAATAAA?GAAGAAGAAG?ACGAAGCAGC?GTCGTGGCTG
421?CTATTCGATA?CTCCTGCTAA?AAACAACCAA?AACCAAGAAT?ATACGAACGA?GTTTTTGTTT
481?AATGGAGAGG?GGGGTGTAGA?TGAGTATTTG?GATCTTGTGG?ACTACAATTC?TTGTCAAGAT
541?ACTCAGTTTA?GTGATGATCA?TAAATGCAAT?AATTTGCAAT?TTAATGATGA?CTATAAGTAT
601?ACGAATGATG?TTACTAATTA?TAGTAAGGAT?ATGCGGAAGT?ATGGCAGAGG?TGATGCAGAT
661?AGCGTAGTGC?CAGTTGGAGG?TGGAGAGGCT?AAGAAGGAGC?ATCAAATTTA?TGATCTTAAC
721?TTCCAACATC?AAAAATTTCA?ATTGGGATGT?GATTATGAGG?CTTCAAATGG?TGGCTACAGC
781?TACCCTGCTT?CACGTGGTCA?TAGTGTTTCT?ATGTCATCAC?TGGATGTTGG?AGTAGTACCA
841?GAATCTTCAA?TCTCAAGTTC?AAGATCTTCA?AAAGGGACAA?CTGACTTATT?CTCAGGTACT
901?TCAATTCAGA?TGCCAACGCA?ACTTACTCCG?TTAGACAGAG?AGGCAAGAGT?CTTGAGTTAC
961?AGGGAAAAGA?AGAAAACAAG?AAAATTTGAG?AAAACGATTA?GGTACGCATC?TAGAAAAGCG
1021?TATGCAGAAA?CAAGGCCTAG?GATCAAAGGC?CGTTTCTCAA?AGCGAACAAA?TGTTGATGTC
1081?GAGGTGGATC?AAATGTTTTC?GACAACATTA?ATGACAGAAG?GCGGATACTG?TATTGTCCCT
1141?TCGTTTTAAT?GAATGGTTAG?GAGCAATAGA?TGCCAATATT?GTAACTGGTT?TTTTTCATTC
1201?AAGTCATGAG?GTTATTATAG?TAAATCTCGT?AACTGTATGT?TCAACGCTTC?TCATTTCCTT
1261?AAATGCAAGC?AAACTTTCCA?GTTTTGGTT
(2) information of SEQ IN NO.2
(a) sequence signature
* length: 382 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
Sequence description
Met?Leu?Lys?Gln?Glu?Ser?Asn?Ile?Asn?Ala?Ser?Gly?Ala?Asn?Ser
1 5 10 15
Trp?Ala?Arg?Leu?Cys?Asp?Thr?Cys?Arg?Ser?Ala?Pro?Cys?Thr?Val
20 25 30
Tyr?Cys?Arg?Ala?Asp?Ser?Ala?Tyr?Leu?Cys?Ala?Gly?Cys?Asp?Ala
35 40 45
His?Val?His?Ala?Ala?Asn?Arg?Val?Ala?Ser?Arg?His?Lys?Arg?Val
50 55 60
Arg?Val?Cys?Glu?Ala?Cys?Glu?Arg?Ala?Pro?Ala?Ala?Phe?Leu?Cys
65 70 75
Lys?Ala?Asp?Ala?Ala?Ser?Leu?Cys?Thr?Ala?Cys?Asp?Ala?Asp?Ile
80 85 90
His?Ser?Ala?Asn?Pro?Leu?Ala?Arg?Arg?His?Gln?Arg?Val?Pro?Val
95 100 105
Ile?Pro?Ile?Ser?Gly?Ser?Thr?Tyr?Glu?Ser?Gln?Gly?Arg?Phe?Phe
110 115 120
Pro?Gln?Gly?Ser?Asp?Gly?Thr?Val?Asn?Lys?Glu?Glu?Glu?Asp?Glu
125 130 135
Ala?Ala?Ser?Trp?Leu?Leu?Phe?Asp?Thr?Pro?Ala?Lys?Asn?Asn?Gln
140 145 150
Asn?Gln?Glu?Tyr?Thr?Asn?Glu?Phe?Leu?Phe?Asn?Gly?Glu?Gly?Gly
155 160 165
Val?Asp?Glu?Tyr?Leu?Asp?Leu?Val?Asp?Tyr?Asn?Ser?Cys?Gln?Asp
170 175 180
Thr?Gln?Phe?Ser?Asp?Asp?His?Lys?Cys?Asn?Asn?Leu?Gln?Phe?Asn
185 190 195
Asp?Asp?Tyr?Lys?Tyr?Thr?Asn?Asp?Val?Thr?Asn?Tyr?Ser?Lys?Asp
200 205 210
Met?Arg?Lys?Tyr?Gly?Arg?Gly?Asp?Ala?Asp?Ser?Val?Val?Pro?Val
215 220 225
Gly?Gly?Gly?Glu?Ala?Lys?Lys?Glu?His?Gln?Ile?Tyr?Asp?Leu?Asn
230 235 240
Phe?Gln?His?Gln?Lys?Phe?Gln?Leu?Gly?Cys?Asp?Tyr?Glu?Ala?Ser
245 250 255
Asn?Gly?Gly?Tyr?Ser?Tyr?Pro?Ala?Ser?Arg?Gly?His?Ser?Val?Ser
260 265 270
Met?Ser?Ser?Leu?Asp?Val?Gly?Val?Val?Pro?Glu?Ser?Ser?Ile?Ser
275 280 285
Ser?Ser?Arg?Ser?Ser?Lys?Gly?Thr?Thr?Asp?Leu?Phe?Ser?Gly?Thr
290 295 300
Ser?Ile?Gln?Met?Pro?Thr?Gln?Leu?Thr?Pro?Leu?Asp?Arg?Glu?Ala
305 310 315
Arg?Val Leu?Ser?Tyr?Arg?Glu?Lys?Lys?Lys?Thr?Arg?Lys?Phe?Glu
320 325 330
Lys?Thr?Ile?Arg?Tyr?Ala?Ser?Arg?Lys?Ala?Tyr?AlaGlu?Thr?Arg
335 340 345
Pro?Arg?Ile?Lys?Gly?Arg?Phe?Ser?Lys?Arg?Thr?Asn?Val?Asp?Val
350 355 360
Glu?Val?Asp?Gln?Met?Phe?Ser?Thr?Thr?Leu?Met?Thr?Glu?Gly?Gly
365 370 375
Tyr?Cys?Ile?Val?Pro?Ser?Phe
380
Embodiment 3: the structure of expression vector pROK3-CO
The segmental pMD18-T carrier of CmCO that contains that makes up from embodiment 1 with SmaI and XbaI downcuts this fragment, is connected with the Yeast expression carrier pROK3 of same restrictions endonuclease digestion.Connect product and transform Agrobacterium LBA4404 competent cell, on the YEP solid plate that contains that mould penicillin of card and Pyocianil, cultivate then, bacterium colony is carried out the restriction analysis of PCR screening, evaluation and plasmid DNA, obtain to have the expression vector pROK3-CO of CmCO.
Embodiment 4: the acquisition of transgenosis chrysanthemum plant
With reference to the method for Mc Cormick (1986) and Van Roekel (1993), and do suitably to improve.Transform the chrysanthemum blade with Ye Panfa with the in advance cultured Agrobacterium LBA4404 that is loaded with pROK3-CO, cut blade tip and cardinal extremity, be cut into 0.5 * 0.5cm, face down places on the pre-culture medium [MS+1.5mg/L6-BA+0.5mg/L NAA], 28 ℃ of dark 2d that cultivate.Afterwards pre-incubated explant is immersed in the agrobacterium tumefaciens suspension with the dilution of MS liquid nutrient medium, infect 20-30min, shake gently during this time, inhale the bacterium liquid that goes explant surperficial unnecessary with aseptic filter paper, rotate back into cultivate 2-3d altogether on the former substratum after, be transferred to screening culture medium [MS+1.5mg/L 6-BA+0.5mg/L NAA+15mg/L Km+400mg/L Carb] and go up screening differentiation bud.When 20-30d grows to 2.0-3.0cm when the resistant buds that transforms, root media [1/2MS+0.1mg/L NAA+10mg/L Km+400mg/L Carb] root induction is put in the bud cutting-out.About 35d, regenerated T1 directly moves into for plantlet and is equipped with in the nutrition pot of the peat composed of rotten mosses and vermiculite, and the experienced seedling 3-5d that preserves moisture that shelters from heat or light grows under the greenhouse natural lighting.Treat that 30d left and right sides robust plant is colonizated in the field.
Embodiment 5:
The transgenosis chrysanthemum that obtains is carried out the flowering time analysis revealed, and the overexpression of CmCO can impel chrysanthemum to bloom in advance in the transgenosis chrysanthemum.
Sequence table
＜110〉Shandong Agricultural University
＜120〉clone of chrysanthemum flowers development gene CmCO and application thereof
<160>2
<210>1
<211>1289
<212>DNA
＜213〉chrysanthemum (Chrysanthemum morflorium)
<400>1
1?atgttgaaac?aagagagtaa?cattaacgct?agtggagcga?acagttgggc?acgactctgt
61?gacacatgcc?ggtcagctcc?ctgcaccgtg?tattgccgag?cagattctgc?ctacttgtgc
121?gccggctgtg?atgcccatgt?tcacgctgcc?aatcgtgttg?cttcccgcca?taaacgcgtc
181?agggtctgcg?aggcgtgtga?gcgtgctcca?gctgcttttt?tatgtaaggc?agatgcagcc
241?tctctttgca?ccgcctgtga?tgcagatatt?cactctgcaa?accctcttgc?acgacgccac
301?caacgtgtcc?cggttattcc?tatttcaggg?tctacgtatg?aatctcaagg?caggtttttt
361?ccccaagggt?cagacggaac?tgttaataaa?gaagaagaag?acgaagcagc?gtcgtggctg
421?ctattcgata?ctcctgctaa?aaacaaccaa?aaccaagaat?atacgaacga?gtttttgttt
481?aatggagagg?ggggtgtaga?tgagtatttg?gatcttgtgg?actacaattc?ttgtcaagat
541?actcagttta?gtgatgatca?taaatgcaat?aatttgcaat?ttaatgatga?ctataagtat
601?acgaatgatg?ttactaatta?tagtaaggat?atgcggaagt?atggcagagg?tgatgcagat
661?agcgtagtgc?cagttggagg?tggagaggct?aagaaggagc?atcaaattta?tgatcttaac
721?ttccaacatc?aaaaatttca?attgggatgt?gattatgagg?cttcaaatgg?tggctacagc
781?taccctgctt?cacgtggtca?tagtgtttct?atgtcatcac?tggatgttgg?agtagtacca
841?gaatcttcaa?tctcaagttc?aagatcttca?aaagggacaa?ctgacttatt?ctcaggtact
901?tcaattcaga?tgccaacgca?acttactccg?ttagacagag?aggcaagagt?cttgagttac
961?agggaaaaga?agaaaacaag?aaaatttgag?aaaacgatta?ggtacgcatc?tagaaaagcg
1021?tatgcagaaa?caaggcctag?gatcaaaggc?cgtttctcaa?agcgaacaaa?tgttgatgtc
1081?gaggtggatc?aaatgttttc?gacaacatta?atgacagaag?gcggatactg?tattgtccct
1141?tcgttttaat?gaatggttag?gagcaataga?tgccaatatt?gtaactggtt?tttttcattc
1201?aagtcatgag?gttattatag?taaatctcgt?aactgtatgt?tcaacgcttc?tcatttcctt
1261?aaatgcaagc?aaactttcca?gttttggtt
<210>2
<211>382
<212>PRT
＜213〉chrysanthemum (Chrysanthemum morflorium)
<400>2
Met?Leu?Lys?Gln?Glu?Ser?Asn?Ile?Asn?Ala?Ser?Gly?Ala?Asn?Ser
1 5 10 15
Trp?Ala?Arg?Leu?Cys?Asp?Thr?Cys?Arg?Ser?Ala?Pro?Cys?Thr?Val
20 25 30
Tyr?Cys?Arg?Ala?Asp?Ser?Ala?Tyr?Leu?Cys?Ala?Gly?Cys?Asp?Ala
35 40 45
His?Val?His?Ala?Ala?Asn?Arg?Val?Ala?Ser?Arg?His?Lys?Arg?Val
50 55 60
Arg?Val?Cys?Glu?Ala?Cys?Glu?Arg?Ala?Pro?Ala?Ala?Phe?Leu?Cys
65 70 75
Lys?Ala?Asp?Ala?Ala?Ser?Leu?Cys?Thr?Ala?Cys?Asp?Ala?Asp?Ile
80 85 90
His?Ser?Ala?Asn?Pro?Leu?Ala?Arg?Arg?His?Gln?Arg?Val?Pro?Val
95 100 105
Ile?Pro?Ile?Ser?Gly?Ser?Thr?Tyr?Glu?Ser?Gln?Gly?Arg?Phe?Phe
110 115 120
Pro?Gln?Gly?Ser?Asp?Gly?Thr?Val?Asn?Lys?Glu?Glu?Glu?Asp?Glu
125 130 135
Ala?Ala?Ser?Trp?Leu?Leu?Phe?Asp?Thr?Pro?Ala?Lys?Asn?Asn?Gln
140 145 150
Asn?Gln?Glu?Tyr?Thr?Asn?Glu?Phe?Leu?Phe?Asn?Gly?Glu?Gly?Gly
155 160 165
Val?Asp?Glu?Tyr?Leu?Asp?Leu?Val?Asp?Tyr?Asn?Ser?Cys?Gln?Asp
170 175 180
Thr?Gln?Phe?Ser?Asp?Asp?His?Lys?Cys?Asn?Asn?Leu?Gln?Phe?Asn
185 190 195
Asp?Asp?Tyr?Lys?Tyr?Thr?Asn?Asp?Val?Thr?Asn?Tyr?Ser?Lys?Asp
200 205 210
Met?Arg?Lys?Tyr?Gly?Arg?Gly?Asp?Ala?Asp?Ser?Val?Val?Pro?Val
215 220 225
Gly?Gly?Gly?Glu?Ala?Lys?Lys?Glu?His?Gln?Ile?Tyr?Asp?Leu?Asn
230 235 240
Phe?Gln?His?Gln?Lys?Phe?Gln?Leu?Gly?Cys?Asp?Tyr?Glu?Ala?Ser
245 250 255
Asn?Gly?Gly?Tyr?Ser?Tyr?Pro?Ala?Ser?Arg?Gly?His?Ser?Val?Ser
260 265 270
Met?Ser?Ser?Leu?Asp?Val?Gly?Val?Val?Pro?Glu?Ser?Ser?Ile?Ser
275 280 285
Ser?Ser?Arg?Ser?Ser?Lys?Gly?Thr?Thr?Asp?Leu?Phe?Ser?Gly?Thr
290 295 300
Ser?Ile?Gln?Met?Pro?Thr?Gln?Leu?Thr?Pro?Leu?Asp?Arg?Glu?Ala
305 310 315
Arg?Val?Leu?Ser?Tyr?Arg?Glu?Lys?Lys?Lys?Thr?Arg?Lys?Phe?Glu
320 325 330
Lys?Thr?Ile?Arg?Tyr?Ala?Ser?Arg?Lys?Ala?Tyr?Ala?Glu?Thr?Arg
335 340 345
Pro?Arg?Ile?Lys?Gly?Arg?Phe?Ser?Lys?Arg?Thr?Asn?Val?Asp?Val
350 355 360
Glu?Val?Asp?Gln?Met?Phe?Ser?Thr?Thr?Leu?Met?Thr?Glu?Gly?Gly
365 370 375
Tyr?Cys?Ile?Val?Pro?Ser?Phe
380
<210>3
<211>11
<212>DNA
＜213〉upstream primer
<400>3
atgttgaaac?aagagagtaa?c 11
<210>4
<211>382
<212>DNA
＜213〉downstream primer
<400>4
aaccaaaact?ggaaagtttg?c 11
Claims (3)
1. chrysanthemum flowers development gene
CmCO, it is characterized in that: its nucleotide sequence is shown in SEQ IN NO.1; Its nucleotide coding aminoacid sequence is shown in SEQ IN NO.2.
2. chrysanthemum flowers development gene
CmCOApplication, it is characterized in that: the albumen of this coded by said gene comprises B-box1, B-box2, CCT structural domain and COOH zone can regulation florescence, cultivate insensitive new variety of plant of photoperiod.
3. chrysanthemum flowers development gene
CmCOApplication, it is characterized in that: the overexpression of this gene can impel chrysanthemum to bloom in advance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101501009A CN102229936A (en) | 2011-06-07 | 2011-06-07 | Cloning and applications of chrysanthemum morflorium floral development gene CmCO |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101501009A CN102229936A (en) | 2011-06-07 | 2011-06-07 | Cloning and applications of chrysanthemum morflorium floral development gene CmCO |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102229936A true CN102229936A (en) | 2011-11-02 |
Family
ID=44842541
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101501009A Pending CN102229936A (en) | 2011-06-07 | 2011-06-07 | Cloning and applications of chrysanthemum morflorium floral development gene CmCO |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102229936A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312780A (en) * | 2017-07-13 | 2017-11-03 | 中国农业大学 | Chrysanthemum nuclear factor CmNF YB8 and its application in florescence control and childhood leaf regulating and controlling of quantities |
| CN107502604A (en) * | 2017-09-07 | 2017-12-22 | 南京农业大学 | Chrysanthemum witchweed lactone synthase gene CmCCD8 and its application for changing chrysanthemum florescence |
| CN112390867A (en) * | 2020-11-17 | 2021-02-23 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
-
2011
- 2011-06-07 CN CN2011101501009A patent/CN102229936A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312780A (en) * | 2017-07-13 | 2017-11-03 | 中国农业大学 | Chrysanthemum nuclear factor CmNF YB8 and its application in florescence control and childhood leaf regulating and controlling of quantities |
| CN107312780B (en) * | 2017-07-13 | 2021-08-03 | 中国农业大学 | Chrysanthemum nuclear factor CmNF-YB8 and application thereof in flowering phase regulation and juvenile leaf number regulation |
| CN107502604A (en) * | 2017-09-07 | 2017-12-22 | 南京农业大学 | Chrysanthemum witchweed lactone synthase gene CmCCD8 and its application for changing chrysanthemum florescence |
| CN112390867A (en) * | 2020-11-17 | 2021-02-23 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
| CN112390867B (en) * | 2020-11-17 | 2022-02-01 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2363980T3 (en) | USE OF A NUCLEIC ACID SEQUENCE FOR THE GENERATION OF TRANSGENIC PLANTS THAT HAVE TOLERANCE TO IMPROVED DROUGHT. | |
| CN109825510A (en) | A kind of Ming River lily LrWRKY2 gene and application | |
| CN107641627B (en) | H gene and H protein for regulating formation of tomato type I glandular hairs and application of H gene and H protein | |
| CN101265293A (en) | Flowering time correlated albumen from arabidopsis, and coding gene and application thereof | |
| CN102229936A (en) | Cloning and applications of chrysanthemum morflorium floral development gene CmCO | |
| CA3002575C (en) | Synthetic promotor induced by abiotic stress, genetic construct containing same and plant cells transformed therewith | |
| CN117286150A (en) | Notoginseng disease course related protein 1 gene PnPR1-3 and its application | |
| CN108976293B (en) | Negative regulation gene for biosynthesis of litchi anthocyanin and application of negative regulation gene | |
| CN108570469A (en) | Sedum lineare resistant gene of salt SLTATS and its application | |
| CN108570471A (en) | Sedum lineare resistant gene of salt SLEIPP and its application | |
| CN110607307A (en) | Sedum lineare salt-tolerant gene SlNAC and application thereof | |
| CN109913466A (en) | The sedum lineare gene of resistance to Cd SlSERK3 and its application | |
| CN115992171A (en) | Method for enhancing resistance of plants to pseudomonas syringae | |
| CN109402135A (en) | Sedum lineare resistant gene of salt SLBHLH and its application | |
| CN108017696B (en) | Chrysanthemum CmTFL1c gene and application thereof | |
| CN103570813A (en) | Plant-stress-resistance related protein Gh01399, and coding gene and application thereof | |
| CN103374061B (en) | Protein coming from leymus chinensis and relevant to salt resistance, coding genes and applications | |
| CN112725355A (en) | Dragon fruit HuNIP6 and 1 gene for promoting early flowering of plants and application thereof | |
| CN107176983B (en) | Application of protein PpLEA3-3 in regulation and control of plant stress resistance | |
| CN110551733A (en) | sedum lineare salt-tolerant gene SlR2R3-MYB and application thereof | |
| CN106244595B (en) | China fir phytosulfokine-α CLPSK1 gene and its application | |
| CN114875043B (en) | Betula alba BpPIF4 gene participating in adventitious root development and application thereof | |
| CN109486833A (en) | A kind of Ming River lily LrWRKY25 gene and application | |
| CN108752442A (en) | Color potato salt tolerance correlation StDof2 albumen and its encoding gene and application | |
| CN110699362B (en) | AFP5 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111102 |