CN101935342B - Moth orchid development B gene-PhAP3 coded sequence and application thereof - Google Patents
Moth orchid development B gene-PhAP3 coded sequence and application thereof Download PDFInfo
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Abstract
The invention provides a moth orchid development B gene-PhAP3 coded sequence and application thereof in the field of botany, and in particular relates to a B gene PhAP3 gene nucleotide coded sequence expressed in moth orchid and application thereof. The invention provides a moth orchid MASD box B AP3 protein, a coded sequence, a vector with the coded sequence, transformed cells and a method for preparing transgenic plants by using the coded sequence and also provides a method for detecting an entogenous expression mode and analyzing and identifying the expression mode of the endogenous gene of the moth orchid. The invention also can be used for molecular breeding, and polynucleotide transgenic plant perianth with the moth orchid development B gene-PhAP3 coded sequence has the advantages of healing integrity, multiple inflorescences, smaller calyx and higher plant height of individual plants, thereby increasing ornamental.
Description
Technical field
The invention belongs to the phytology field, specifically, the present invention relates to a kind of cell and genetically modified plant of the gene PhAP3 of B family gene, the expression vector that contains this gene, conversion or the transduction of in butterfly orchid, expressing.The invention also discloses the application of PhAP3 gene aspect plant molecular breeding and the improvement of ornamental gardening characteristic.
Background technology
Along with to the research of flower development associated problem going deep into progressively, A, B, C genoid in more and more different genera plants are cloned in succession.The overwhelming majority in these floral organ identity genes belongs to an ancient gene family MADS-box family.The MADS-box gene family is gained the name in the initial of identifying at first four members out: yeast
MCM1 gene, Arabidopis thaliana
AGAMOUS gene, Common Snapdragon
DEFICIENS gene and people's
SThe RF gene.The transcription factor of this gene family coding is in the different steps of plant, and the different tissues organ affects it and grows.The people such as Bonhomme are double influence (Bonhomme F from the morphogenesis of separating the SaMADS gene pairs inflorescence development obtain and floral organ the Sinapisalba, Sommer H, Bernier G, Jacqmard A., Characterization of SaMADS D from Sinapis alba suggestsa dual function of the gene:In inflorescence development and floral organogenesis, Plant Molecular Biology, 1997,34:573-582.).The people such as Richard separate the petunia gene PFG gene that obtains; it almost expresses (Immink RGH at all sites (except root, stamen and the seedling) or the developmental stage of plant; Hannapel DJ; Ferrario S; Busscher M; Franken J; CampagneMML; Angenent GC.; A petunia MADS box gene involved in the transition fromvegetative to reproductive development.Development; 1999,126:5117-5126).The people such as Purugganan from the angle of evolving think " development of plants the MADS-box gene from different sides or level produce different effects " this is pleiotropy (the Purugganan MD of the particularly noticeable gene of vital role of MADS-box, Rounsley SD, Schmidt RJ, Yanofsky MF (1995) .Molecularevolution of flower development:diversification of the plant MADS-box regulatorygene family.Genetics 140:345-56).
In Arabidopis thaliana, B family gene can divide two subclass AP3 and PI, their (McGonigle B. that has an effect that interosculates, Bouhidel K., Irish V.F. (1996) Nuclear localization of theArabidopsis APETALA3 and PISTILLATA homeotic gene products depends on theirsimultaneous expression.Genes Dev.10:1812-1821).To connecting the reporter gene GUS (PI::GUS of AP3 and PI promotor; AP3::GUS) two subclass PI in the expression analysis proof B family gene family and AP3 only are activated in floral organ.AP3 not only takes turns expression at second and third, and in some tissues of the first round, expression (Jack T is arranged also, Brockman LL, Meyerowitz EM. (1992), The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and isexpressed in petals and stamens.Cell 68:683-97; Weigel D, Meyerowitz, E.M. (1994) .The ABCs of floral homeotic genes.Cell 78:203-209; Goto K, MeyerowitzEM. (1994) .Function and regulation of the Arabidopsis floral homeotic genePISTILLATA.Genes Dev 8:1548-60), its conserved sequence corresponding in the K district is (H/Q) YExM (Kramer EM, Dorit RL, Irish VF. (1998) .Molecular evolution of genescontrolling petal and stamen development:duplication and divergence within theAPETALA3 and PISTILLATA MADS-box gene lineages.Genetics 149:765-83).
Although first MADS gene that separation obtains in the monocotyledons is from orchid (Lu ZX, Wu M, Loh CS, Yeong CY, Goh CJ. (1993) .Nucleotide sequence of a flower-specificMADS box cDNA clone from orchid.Plant Mol Biol 23:901-4), but most research all concentrates on paddy rice, the corn for over ten years, the molecular studies of relevant the orchid family flower development are comparison basis still, mainly concentrates on butterfly orchid and the dendrobium.The orchid family belongs to one of four especially big sections in the world, old origin, evolutionary rate is extremely asynchronous, the peak of spermatophyte phylogeny in monocotyledons, have typicalness and representativeness (Wu Zhengyi, pacify the people in the road, Tang Yancheng etc. and (2003) Chinese angiosperm section belongs to combines opinion. Science Press, 275).Butterfly orchid floral structure eggcase, the height variation (such as sepal and the lip of floweriness lobe) of its perianth and the appearance (Bowman of gynostemium, John L. (1997) .Developmental genetics offlower pattern formation, American Journal of Botany, 84:67), for research ABC functional gene provides new visual angle and platform, and may expand the extension of classical ABC functional gene concept.
Butterfly orchid gene B family gene all has expression (Tsai WC in one or two three-wheel floral organs, Kuoh CS, Chuang MH, Chen WH, Chen HH. (2004) .Four DEF-like MADS box genesdisplayed distinct floral morphogenetic roles in Phalaenopsis orchid.Plant CellPhysiol 45:831-44.).There is a need in the field to provide new separation butterfly orchid category-B gene, and its spatial and temporal expression pattern is determined, for the genetic mechanism of establishing the orchid flower development provides certain theoretical foundation, thereby the ABC model of classics is made further additional.On the other hand, this area also needs the molecular breeding method that provides new, improves the fancy horticulture characteristic of plant and then improves its economic worth.
Summary of the invention
One of technical problem to be solved by this invention is to disclose a kind of butterfly orchid MADS box B AP3 of family albumen (protein of B class MADS box gene AP3) and fragment thereof, analogue or derivatives thereof.
Two of technical problem to be solved by this invention provides the polynucleotide of these polypeptide of coding.
Three of technical problem to be solved by this invention provides the purposes of polynucleotide of these polypeptide of coding, especially at molecular breeding or improve purposes aspect the plants ' aesthetics gardening characteristic.
Of the present invention aspect first, a kind of butterfly orchid MADS box B AP3 of family albumen of novelty is provided, this peptide source comprises from butterfly orchid: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments, or derivatives thereof.Preferably, this polypeptide is selected from lower group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, have the polypeptide of being derived by (a) of the MADS box B AP3 of family protein function.
Second aspect of the present invention, provide the coding aforementioned polypeptides polynucleotide, these polynucleotide comprise a nucleotide sequence.Preferably this nucleotide sequence be selected from a kind of nucleotide sequence at least 70% homogeny of lower group: (a) polynucleotide of the above-mentioned butterfly orchid MADS box B AP3 of the family albumen of coding; (b) polynucleotide complementary with polynucleotide (a).Preferably, this polynucleotide encoding has the polypeptide of SEQ ID NO:2 aminoacid sequence.More preferably, this polynucleotide sequence is selected from lower group:
(a) has the sequence of SEQ ID NO:1; With,
(b) has among the SEQ ID NO:1 1～684 sequence.
A third aspect of the present invention provides the carrier that contains above-mentioned polynucleotide, and is transformed or the host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
A fourth aspect of the present invention provides the method for preparing the butterfly orchid MADS box B AP3 of family albumen, and the method comprises:
(i) under suitable condition, cultivate the above-mentioned host cell that is converted or transduces;
(ii) from culture, reclaim the butterfly orchid MADS box B AP3 of family albumen.
In a fifth aspect of the present invention, a kind of method for detection of butterfly orchid PhAP3 gene copy number in the sample is provided, comprise step:
(a) extracting sample gene group DNA obtains endonuclease bamhi with digestion with restriction enzyme;
(b) utilize aforementioned polynucleotide sequence SEQ ID NO:1, the about 250bp of special section 3 ' UTR as probe, the endonuclease bamhi in (a) is carried out Southern hybridization, the combination of testing goal fragment and probe.
In a sixth aspect of the present invention, a kind of method that detects the butterfly orchid B PhAP3 of family gene mRNA expression pattern is provided, its step is as follows:
(1) total RNA of extraction butterfly orchid floral organ;
(2) utilize the reverse transcription test kit that total RNA reverse transcription is become cDNA, according to SEQ ID NO:3 design primer, carry out PCR.
In another aspect of this invention, provide the method for a kind of PhAP3 of detection gene in the former plant tissue of butterfly orchid expression and distribution situation, its step is as follows:
(1) gets the fritter organization material, process and the gradient dehydration through Paraformaldehyde 96, be embedded in the paraffin organization;
Get section that rehydration (reconstitution process: adopt to add one by one ethanol that concentration raises gradually so that material recovers high saturation state) and Proteinase K processed and prior dna probe molecule bulk crossing in hybridization solution of preparation when (2) experiment detects, the rear observation that develops the color is taken pictures; And the dna probe molecule that hybridization is used is the dna sequence dna that is contained in certain special section 3 ' UTR of PhAP3 gene.
In another aspect of this invention, provide to change the method that plant forms builds up, comprise step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence, and the described butterfly orchid MADS box B AP3 of family albumen is selected from lower group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; With,
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, have the polypeptide of being derived by (a) of the MADS box B AP3 of family protein function;
(2) vegetable cell or tissue or organ are contacted with the Agrobacterium of step (1), thereby make the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or tissue or the organ that changes the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
Other aspects of the present invention will be readily apparent to persons skilled in the art via of the present invention open.
Description of drawings:
Fig. 1 PhAP3 gene and the comparison of other AP3 gene homology and phylogenetic analysis
Fig. 2 is that the Southern blot of PhAP3 gene in the butterfly orchid analyzes.Two kinds of commercially available enzyme EcoR I that E, D are respectively that digested genomic dna uses and Dra I.
Fig. 3 is that RT-PCR identifies the expression pattern of PhAP3 in each organ of butterfly orchid.Actin is contrast, ped1: three joint position bennets, ped2: four joint position bennets, ped3: five joint position bennets, bud: bud, root: root, leaf: blade, Sepal: calyx, petal: petal, lip: lip, column: stamen post, ped+ov: bennet and ovary.
The in situ hybridization result of Fig. 4 PhAP3 gene.
First flower primordium of A begins the inflorescence meristem (* 100) of eruption; The inflorescence meristem (* 200) that the former base of B floral organ germinates; The bud (* 160) that the former base of C floral organ all possesses; The unfertilized ovary of D (* 40); The ovary (* 40) of E fertilization; The rip cutting of F ovary (* 40).Ac, pollen wall; B, bract; C, column; Fm, floral meristem; Fp, flower primordium; Im, inflorescence meristem; Lp, the former base of lip; Lpj: lip nidus; Pp, the former base of petal; Pl, pollinium; Sp, the former base of lip; S, column cap.
The PCR of the hygromycin gene of Fig. 5 transfer-gen plant (Hyg) and goal gene (AP3) identifies.Ap3-1 is for turning PhAP3 gene masculine tobacco strain numbering, M be DL2000Marker from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.
The phenotype of Fig. 6 transgene tobacco.Ap3-1 is for turning PhAP3 gene masculine tobacco strain numbering, and A and C are perianth; EV is the contrast of empty carrier transgenic line, and B and D are inflorescence.A compares the perianth healing with C more complete; It is more that B and D compare inflorescence.
Embodiment
The inventor is through extensive and deep research, screening has obtained the new butterfly orchid MADS box B AP3 of a family protein gene (PhAP3) from butterfly orchid, and its function is studied, show the growth of the syngenesis organ of this effect gene transfer-gen plant, improve the growth of plant petal and stamen etc., therefore at molecular breeding, improve the fancy horticulture characteristic of plant and then improve its economic worth and have application prospect.
In the present invention, term " the butterfly orchid MADS box B AP3 of family albumen ", " the butterfly orchid MADS box B AP3 of family polypeptide " etc. are used interchangeably, and all refer to have protein or the polypeptide of the butterfly orchid MADS box B AP3 of family Argine Monohydrochloride sequence (SEQ ID NO:2).
When not particularly pointing out, term " the butterfly orchid MADS box B AP3 of family albumen " comprises wild-type and mutant sequence.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide.Recombinant polypeptide preferably.Polypeptide of the present invention can be the product of natural purifying or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host, for example produces in bacterium, yeast, higher plant cell, insect cell or the mammalian cell.The host who uses according to recombination method is different, and polypeptide of the present invention can be glycosylated or nonglycosylated.Polypeptide of the present invention can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analogue of the butterfly orchid MADS box B AP3 of family albumen.Used such as this specification sheets, term " fragment ", " derivative " refer to basically keep the natural butterfly orchid MADS box B identical biological function of the AP3 of family albumen of the present invention or active polypeptide with " analogue ".Polypeptide fragment/derivative of the present invention or similar position include but not limited to: (1) has one or more conservative or substituted polypeptide of nonconservative amino-acid residue (preferred conservative amino-acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code; (2) in one or more amino-acid residues, has the polypeptide of substituted radical; (3) mature polypeptide and another compound, for example polyoxyethylene glycol merges the polypeptide that forms; (4) additional aminoacid sequence is fused to the polypeptide that this sequence forms, such as leader sequence or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying and be fused to the polypeptide that this peptide forms.According to the instruction of this specification sheets, these fragments, derivative or analogue belong to the known scope of those of ordinary skill in the art.
Hit at this, term " the butterfly orchid MADS box B AP3 of family albumen " comprises the polypeptide with SEQ ID NO:2 sequence.This term also comprises having and the butterfly orchid MADS box B AP3 of family albumen variant form identical function, SEQ ID NO:2 sequence.These variant forms include but not limited to one or more aminoacid deletion, insertion and/or its replacement and add one or more amino acid at C-terminal and/or N-terminal.Amino acid whose disappearance, insertion and/or its replace normally 1-50, preferably 1-30, more preferably be 1-20, most preferably be 1-10.When C-terminal and/or N-terminal add one or more amino acid, generally be 20 with interior, preferably 10 with interior, more preferably be in 5.For example, in this area, when replacing with the close or similar amino acid of performance, usually can not change the function of protein.Again such as, add the function that one or more amino acid also can not change protein usually at C-terminal and/or N-terminal.Term " the butterfly orchid MADS box B AP3 of family albumen " also comprises its active fragments and reactive derivative.
The variant form of the butterfly orchid MADS box B AP3 of family albumen of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, albumen that can be coded with the DNA of " the butterfly orchid MADS box B AP3 of family albumen " DNA hybridization under high or low stringent condition.
The present invention also provides other polypeptide, for example comprises the fusion rotein of " the butterfly orchid MADS box B AP3 of family albumen " or its fragment.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of " the butterfly orchid MADS box B AP3 of family albumen ".Usually, this fragment has at least 10 continuous amino acids of " the butterfly orchid MADS box B AP3 of family albumen " sequence, preferably 30 continuous amino acid, more preferably at least 50 continuous amino acids, at least 100 continuous amino acids most preferably.
Among the present invention, " the butterfly orchid MADS box B AP3 of family albumen " conservative property variation polypeptide refers to compare with the aminoacid sequence of SEQ IDNO:2,10 at the most, 5, the ground more preferably polypeptide that formed by similar performance or close amino acid substitution of 3 amino acid at the most at the most preferably.These conservative property variation polypeptide preferably carry out amino acid substitution according to table one and produce.
Table one
| Initial residue | The preferred replacement | Preferred replacement |
| Ala | Val，Leu，Ile | Val |
| Arg | Lys，Gln，Asn | Lys |
| Asn | Gln，His，Lys，Arg | Gln |
| Asp | Glu | Glu |
| Cys | Ser | Ser |
| Gln | Asn | Asn |
| Glu | Asp | Asp |
| Gly | Pro，Ala | Ala |
| His | Asn，Gln，Lys，Arg | Arg |
| Ile | Leu，Ala，Phe | Leu |
| Leu | Ile，Val，Met，Ala，Phe | Ile |
| Lys | Arg，Gln，Asn | Arg |
| Met | Leu，Ile，Phe | Leu |
| Phe | Leu，Val，Ile，Ala，Tyr | Leu |
| Pro | Ala | Ala |
| Ser | Thr | Thr |
| Thr | Ser | Ser |
| Trp | Tyr，Phe | Tyr |
| Tyr | Trp，Phe，Thr，Ser | Phe |
| Val | Ile，Leu，Met，Phe，Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.The encoding sequence of the polypeptide of encoding mature can with SEQ ID NO; The varient of the identical or degeneracy of the encoding sequence shown in 1.In this manual, term " varient of degeneracy " refer to encode protein with SEQ ID NO:2 but with the nucleotide sequence of the encoding sequence shown in the SEQ ID NO:1 by difference.For example according to host's difference, select corresponding host to like the codon coding SEQ ID NO of usefulness; The corresponding amino-acid residue of 2 sequence.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of the encoding sequence of an encoding mature polypeptide, the encoding sequence of mature polypeptide and various additional code sequence, mature polypeptide.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide is the allelic variant of natural generation or the varient that non-natural occurs.These polynucleotide varients comprise and replace varient, deletion mutation body and insert varient.As known to those skilled in the art, allelic variant is the replacement form of a Nucleotide, for example replacement of one or more Nucleotide, disappearance or insertion, but can not change the function of coded polypeptide.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have the polynucleotide of at least 50%, preferably at least 70%, more preferably at least 80% homogeny.The present invention particularly including under stringent condition with the polynucleotide of multi-nucleotide hybrid of the present invention.In the present invention, " stringent condition " and " stringent condition " is used interchangeably, and all refers to: hybridize and wash-out under than low ionic strength and comparatively high temps (1), such as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time has denaturing agent, such as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, better is just to hybridize when above 95%, and the polypeptide of interfertile polynucleotide encoding has identical biological function or activity with the mature polypeptide shown in the SEQID NO:2.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.At least 15 Nucleotide of the length of " nucleic acid fragment ", preferably at least 25 Nucleotide, at least 50 Nucleotide more preferably are most preferably more than at least 100 Nucleotide.This nucleic acid fragment can be used for nucleic acid amplification technologies, such as PCR, RT-PCR etc. to determine and/or to separate the polynucleotide of the coding butterfly orchid MADS box B AP3 of family albumen.
The Nucleotide full length sequence of the butterfly orchid MADS box B AP3 of family albumen of the present invention or its fragment obtain with the method for pcr amplification method, recombination method or synthetic usually.When adopting the pcr amplification method, can be disclosed according to the present invention about Nucleotide, especially the opening code-reading frame sequence designs primer, and commercially available cDNA storehouse or according to the prepared cDNA storehouse of those skilled in the art's ordinary method known as template, amplification and obtain relevant sequence.In a preferred embodiment of the invention, adopt SEQ ID NO:3 and SEQID NO:4 respectively as 3 '-primer and 5 '-primer, amplification obtains PhAP3 full length gene sequence.
In case obtained relevant sequence, just can obtain in large quantity relevant sequence according to the common recombination method in this area.Normally it is cloned into carrier, is changing host cell over to, then by conventional this engineered host cell of method propagation, from the host cell after the propagation, separate relevant sequence.
In addition, can also synthesize relevant sequence, especially fragment more in short-term with the method for synthetic.Usually synthesize first a plurality of little nucleotide fragments, and then connect the very long fragment of acquisition sequence.
At present, can be fully by chemosynthesis obtain the encoding dna sequence dna of the butterfly orchid MADS box B AP3 of family albumen of the present invention or its fragment or derivatives thereof, then this dna sequence dna can be introduced in dna molecular known in the art (for example carrier) and the cell.Can will suddenly change by chemosynthesis in addition and introduce in the protein sequence of the present invention.
The invention still further relates to the carrier that comprises polynucleotide of the present invention.
The invention still further relates to the method for the host cell that produces with the polynucleotide of carrier of the present invention or the coding butterfly orchid MADS box B AP3 of family albumen of the present invention.
By the recombinant DNA technology of routine, can utilize polynucleotide sequence of the present invention to express or produce the described butterfly orchid MADS box B AP3 of family albumen.Usually, comprise the steps:
(1) with polynucleotide or its varient of the coding butterfly orchid MADS box B AP3 of family albumen of the present invention, or transforms or transduction appropriate host cell with the recombinant expression vector that contains these polynucleotide;
(2) in suitable medium, cultivate host cell;
(3) separation, this protein of purifying from substratum or in the cell.
In the present invention, the butterfly orchid MADS box B AP3 of family albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to well known to a person skilled in the art bacterial plasmid, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carrier.As long as can copy in the host cell body and stablize, any plasmid or carrier can adopt.A key character of expression vector is usually to contain replication orgin, promotor, marker gene and translation controlling elements.
The known method of those skilled in the art can be used for make up and contain the butterfly orchid MADS box B AP3 of family encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods include but not limited to extracorporeal recombinant DNA technology, DNA synthetic technology, extracorporeal recombination etc.Described dna sequence dna can be effectively connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
Expression vector preferably comprises one or more selected markers, phenotypic character with the host cell that is provided for selecting transforming, for example eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness, or is used for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable dna sequence dna and the suitable carrier of promotor or control sequence, can be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cell; Or higher eucaryotic cells, such as vegetable cell.Representational example has: intestinal bacteria, streptomyces, Agrobacterium, yeast, vegetable cell.In a preferred embodiment of the invention, adopt Agrobacterium to make host cell.
Skilled in the art will recognize that the suitable carrier of How to choose, promotor and host cell.
Technology with recombinant DNA conversion or transduction host cell is being routine techniques to one skilled in the art.When host cell was prokaryote such as intestinal bacteria, the competent cell that can absorb DNA can in exponential growth after date results, be used CaCl
2Method is processed, and used step is known in this area.Another kind method is to adopt MgCl
2If necessary, conversion also can be adopted electroporation.When host cell is eukaryotic cell, can select following DNA transfection method: calcium phosphate precipitation, microinjection, electroporation, liposome packing etc.
Conversion of plant can use the methods such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, for example leaf dish method.The vegetable cell, tissue or the organ that transform can be used the known ordinary method regeneration of those skilled in the art plant, thereby obtain the plant that sight is improved.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, cultivate the substratum that used substratum can be selected from various routines.Under the condition of host cell growth, cultivate.According to used host cell, albumen of the present invention be constitutive expression or inducible expression.When inducible expression, after host cell grows into suitable density, abduction delivering and then cultivation for some time.The method of abduction delivering is selected corresponding induction method according to the promotor of selecting, such as temperature-induced or chemical induction.
In the above methods, the extracellular be expressed or be secreted into to recombinant polypeptide can in cell or at cytolemma.If necessary, can utilize its physics, chemistry or other characteristic by the albumen of the separation of various separation methods and purification of Recombinant.These methods are that the technician is known in this area.The example of these methods includes but not limited to: the combination of conventional refolding method, salt analysis method, centrifugal, the broken bacterium of infiltration, ultrafiltration, sieve chromatography, ion exchange chromatography or additive method.
The butterfly orchid MADS box B AP3 of the family albumen of restructuring is of use in many ways.For example screening promotes or antibody, polypeptide or other parts of the antagonism butterfly orchid MADS box B AP3 of family protein function.The butterfly orchid MADS box B AP3 of the family albumen of restructuring can be used in seeks the valuable peptide molecule that can stimulate the butterfly orchid MADS box B AP3 of family protein function.
Part or all of polynucleotide of the present invention can be used as probe and is fixed on microarray (Microarry) or the DNA chip, is used for analyzing the differential expression of tissue gene.Carry out the transcription product that the RT-PCR amplification in vitro can detect the butterfly orchid MADS box B AP3 of family albumen with the butterfly orchid MADS box B AP3 of family albumen special primer.
Has the obvious growth that improves plant petal and stamen etc. at the butterfly orchid MADS box B AP3 of family albumen of the present invention.At vegetative growth phase, plant is higher relatively; Inflorescence number is more compared with the control, and the perianth healing is more complete, and is solid normal.Show that PhAP3 gene of the present invention has extensive use in plant molecular breeding, can improve the growth of plant syngenesis organ, thereby improve its sight.
Below in conjunction with the real further explaination of implementation the present invention.Should be understood that these examples only limit the scope of the invention to be not used in for explanation the present invention.The experimental technique of unreceipted actual conditions in the following example, all can carry out according to normal condition, such as Sambrook etc., molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) or molecular biology of plants-laboratory manual (PlantMolecular Biology-A Laboratory Manual, Melody S.Clark compiles, Springer-verlagBerlin Heidelberg, 1997) condition described in, or according to the condition of making production firm and advising.
Butterfly orchid PhAP3 gene cloning
1 butterfly orchid (Phalaenopsis hybrida) kind Formosa rosa is from vegetable gardening institute of Shanghai Academy of Agricultural Sciences; Growth conditions is photoperiod 16h/8h (L/D), 25 ℃.
2RNA extracts.Get 100 milligrams of materials that the left and right sides is fresh, liquid nitrogen fully grinds.Add 1ml TriBlue reagent, firmly shake 15s, room temperature is placed 5min.Add the 0.2ml chloroform, Deproteinization, supernatant is transferred to new centrifuge tube behind the centrifugal 10min of 12k rpm, adds the equal-volume Virahol, abundant mixing, room temperature is placed 10min, the centrifugal 10min of 12krpm, abandon supernatant, 75% ethanol 1ml washing precipitation with the preparation of DEPC treated water repeats once.Drying at room temperature 5～10min is dissolved in the 20 μ l DEPC water, surveys the OD value, electrophoresis detection.
3 gene clonings.Take butterfly orchid bud cDNA first chain of reverse transcription as template, utilize forward primer AP3F (SEQ ID ID NO:3 and reverse primer B
25(SEQ ID NO:4) carries out PCR, obtain full length gene, for having the dna molecular of particular sequence, total length 838bp, its nucleotides sequence is classified as shown in the SEQ IDNO:1, carry out sequence analysis with other member in the MADS-box gene family, see Table two, the result shows to have very high homology.
The phylogenetic analysis of PhAP3 gene and other AP3 gene is to utilize PAUP software, according to the nucleotide sequence of these gene conserved regions, makes up maximum brief tree, Fig. 1.PhAP3 belongs to B family gene, the DEF class MADS-box gene PeMADS2 of PhAP3 and pink butterfly orchid, PeMADS3, PeMADS4 is in the same place with the PeMADS5 cluster, and and the monocotyledonous AP3 subclass such as corn consist of a subfamily.According to this cluster analysis tree: orchid category-B MADS-box gene may originate from germanus twice with this genoids of monocotyledons corn, paddy rice etc. and copy.The tapping point value (1000 times copy) of bootstrapping provides at tapping point, and PhAP3 indicates with " ◆ ".
Embodiment 2
The copy number analysis of butterfly orchid PhAP3 gene
Extract the butterfly orchid genome with the CTAB method, get 10 μ gDNA and use respectively Eco R I (E) and two kinds of commercially available enzymic digestions of Dra I (D), 37 ℃ of enzymes are cut and are spent the night.With 0.8% sepharose enzyme is cut product and carry out electrophoretic separation, transfer DNA also is fixed on the nylon membrane probe hybridization.Because the high conservative of MADS-box gene family MADS box, so we select the special section 3 ' UTR of butterfly orchid PhAP3 gene, and about 250bp determines that it is single copy as probe, sees Fig. 2.
Other member in table two PhAP3 and the MADS-box gene family carries out sequence analysis
Embodiment 3
Butterfly orchid PhAP3 Gene Expression Profile Analysis
Extract respectively total RNA of root, blade, bennet, bud and the floral organ (calyx, ala, lip and stamen post) of butterfly orchid plant.Measure OD
260Rear quantitatively taking-up 1 μ g RNA, reverse transcription.(referring to SEQ ID NO:3) carries out PCR with special primer.Reaction product is separated with 1.0% agarose gel electrophoresis, and electrophoresis result as shown in Figure 3.From electrophorogram, can find out, AP3 class butterfly orchid MADS-box gene, PhAP3 has expression nourishing and growing of butterfly orchid with reproductive growth period, and all expresses in all floral organs of butterfly orchid flower.
Embodiment 4
In situ hybridization
1) obtains less organization material, fix 12 hours with 4% Paraformaldehyde 96.With Gradient elution using ethanol, be embedded in the middle of the paraffin mass again.
2) slide glass and cover glass are dipped in the chromic acid lotion 2 days, then distilled water rinsing.Soak or smear again 7 ℃ or 42 ℃ of dry for standby with poly-lysine (PLL, 1mg/ml).
3) the specific probe molecule is the one section special dna sequence dna that is about 250bp in gene 3 ' terminal non-transcribed zone.
4) take off after the paraffin sample section cured, rehydration and with the protease digestion tissue protein.Through behind the prehybridization with the probe molecule of prior denaturing treatment in hybridization solution 50 ℃ bathe altogether 16h.
5) after the hybridization, with serum sealing probe, utilize the probe molecule of specific antibody in hybridization to be combined, and dyeed.
6) water-soluble mountant mounting, microscopic examination is also taken a picture.
The result shows that the signal of PhAP3 is enriched in inflorescence meristem at first, in the flower primordium, but does not express in bract.Along with the appearance of the former base of floral organ, PhAP3 obviously is better than the former base of other floral organ (Fig. 4 A, B) at lip and stamen post place.On the rip cutting figure of developmental stamen post, PhAP3 has strong expression signal (Fig. 4 C) at the anther cap place, and in fertilization and unfertilized ovary, PhAP3 has expression (Fig. 4 D, E, F) in ovule.
Embodiment 5
Butterfly orchid PhAP3 gene carries out eukaryotic expression and transfer-gen plant in tobacco cell phenotypic evaluation 1 contains the structure of goal gene (butterfly orchid PhAP3 gene) expression vector
According to butterfly orchid PhAP3 full length gene encoding sequence, design and amplify the primer of complete coding reading frame, and on forward and reverse primer, introduce respectively restriction enzyme enzyme recognition site (deciding on the carrier of selecting), so that construction of expression vector.The amplified production that obtains in the embodiment 1 is as template, holds at 5 ' end and 3 ' to add respectively Sac I and Xba I restriction enzyme site.Through Xba I be connected with Sac I double digestion the PCR product with is connected through the pHB plant expression vector of same double digestion, transform, check order, guarantee that open reading frame is correct.It is changed in the Agrobacterium.Utilize leaf disc law technology transformation mode plant tobacco.
2 utilize leaf dish method transformation of tobacco
● the positive colony with on the sterilizing toothpick picking YEB selection substratum is inoculated in 2ml YEB liquid (Rifampin 40mg/L, kantlex 50mg/L, gentamicin 100mg/L), 28 ℃ of 200rpm shaking culture 24-36h;
● the centrifugal 10min of 4000g under the room temperature;
● 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;
● 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;
● blade is put into the bacterium liquid for preparing, soak 2～5min, blot bacterium liquid at aseptic filter paper;
● the blade that will infect is put on the MS substratum, 28 ℃ of dark 48h that cultivate;
● blade is forwarded on the callus substratum (MS+6-BA 1.0mg/L+NAA 0.1mg/L+ Totomycin 50mg/L+cef250mg/L), cultivate under 25～28 ℃ of illumination, saw that callus formed in 7～15 days;
● about 20 days afterwards visible Bud Differentiation grow, after bud is grown up, downcut, place (1/2MS+NAA 0.5mg/L+ Totomycin 50mg/L+cef250mg/L) on the root media;
● behind well developed root system, 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 3 transfer-gen plants
Genomic dna with SDS method a small amount of extracting part transfer-gen plant according to hygromycin gene on the carrier and goal gene primers (referring to SEQ ID NO:3), carries out PCR and detects, and detected result as shown in Figure 5.PCR result shows that the expression plasmid that carries external source butterfly orchid PhAP3 gene has been inserted in the tobacco gene group.
4 transfer-gen plants carry out phenotypic evaluation
Altogether obtain 3 strain transfer-gen plants, the part phenotype of transgene tobacco as shown in Figure 6.Contrast with turning zero load, at vegetative growth phase, plant is higher relatively; Inflorescence number is compared many with zero load, the perianth healing is more complete, and is solid normal.In the host tobacco, realize overexpression according to RT-PCR results presumption external source PhAP3 gene, affected the growth of transfer-gen plant petal and stamen etc.Test-results shows that the butterfly orchid PhAP3 gene that obtains among the embodiment 1 is the gene that function is arranged, and can affect the growth of plant syngenesis organ.
Implement the six butterfly orchid MADS box B AP3 of family albumen Preparation and identifications
Cultivate embodiment five and transform the Agrobacterium that obtains, separation, the purifying butterfly orchid MADS box B AP3 of family albumen from culture.Use the SDS-PAGE determining molecular weight, be about 26.5kDa; The point point such as recording with capillary gel electrophoresis is 9.3.
Sequence table
＜110〉Fudan University
＜120〉moth orchid development B gene-PhAP 3 coded sequence and application thereof
<130>memo
<160>4
<170>PatentIn version 3.4
<210>1
<211>838
<212>DNA
＜213〉butterfly orchid (Phalaenopsis sp.)
<220>
<221>CDS
<222>(1)..(684)
<400>1
atg ggg aga gga aag ata gag ata aag aag ata gag aat ccg acg aac 48
Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn
1 5 10 15
agg caa gtt aca tat tct aag agg aga gtt ggg ata ctg aag aag gcc 96
Arg Gln Val Thr Tyr Ser Lys Arg Arg Val Gly Ile Leu Lys Lys Ala
20 25 30
aag gag ctc act gtt ctc tgt gat gct cag gtc tct ctc atc atg ttc 144
Lys Glu Leu Thr Val Leu Cys Asp Ala Gln Val Ser Leu Ile Met Phe
35 40 45
tca agc aca gga aaa ttg gct gat tac tgc agc ccc tct act gat att 192
Ser Ser Thr Gly Lys Leu Ala Asp Tyr Cys Ser Pro Ser Thr Asp Ile
50 55 60
aag ggg ata tat gag agg tac cag gtt gtg act ggt atg gat cta tgg 240
Lys Gly Ile Tyr Glu Arg Tyr Gln Val Val Thr Gly Met Asp Leu Trp
65 70 75 80
aat gct cag tat gag agg atg cag aat acg ctg aag cat ctg aat gag 288
Asn Ala Gln Tyr Glu Arg Met Gln Asn Thr Leu Lys His Leu Asn Glu
85 90 95
att aac caa aac ctg agg aag gag att agg agg agg aag ggg gag gaa 336
Ile Asn Gln Asn Leu Arg Lys Glu Ile Arg Arg Arg Lys Gly Glu Glu
100 105 110
ttg gag ggc atg gac ata aag caa ctg cgc ggt ctt gag caa act ttg 384
Leu Glu Gly Met Asp Ile Lys Gln Leu Arg Gly Leu Glu Gln Thr Leu
115 120 125
gaa gag tct ctt aga att gtt agg cat aga aag tat cat gtg atc gcc 432
Glu Glu Ser Leu Arg Ile Val Arg His Arg Lys Tyr His Val Ile Ala
130 135 140
aca caa act gac act tac aag aaa aag ctt aaa agc aca agg gaa act 480
Thr Gln Thr Asp Thr Tyr Lys Lys Lys Leu Lys Ser Thr Arg Glu Thr
145 150 155 160
tac cgc gct cta ata cat gaa ctg gac atg aaa gag gag aat ccg aac 528
Tyr Arg Ala Leu Ile His Glu Leu Asp Met Lys Glu Glu Asn Pro Asn
165 170 175
tac ggt ttt aat gta gaa aac cag agt aga att tat gaa aat tcg att 576
Tyr Gly Phe Asn Val Glu Asn Gln Ser Arg Ile Tyr Glu Asn Ser Ile
180 185 190
cca atg gtg aac gag tgt cct cag atg ttt tcc ttt agg gtt gtt cat 624
Pro Met Val Asn Glu Cys Pro Gln Met Phe Ser Phe Arg Val Val His
195 200 205
ccg aat cag ccc aat ctg ctt ggt tta ggt tat gaa tca cat gat ctt 672
Pro Asn Gln Pro Asn Leu Leu Gly Leu Gly Tyr Glu Ser His Asp Leu
210 215 220
agc ctt gca taa ttagcagtga tattatgatt ttattgtatt tttattgttt 724
Ser Leu Ala
225
gtttgaaact ttagaattat gagacggggg atctattcag agagaactgt gctttaattt 784
gatttttcag tttgtttcct cttcatgttc agtgaaaaaa aaaaaaaaaa aaaa 838
<210>2
<211>227
<212>PRT
＜213〉butterfly orchid (Phalaenopsis sp.)
<400>2
Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn
1 5 10 15
Arg Gln Val Thr Tyr Ser Lys Arg Arg Val Gly Ile Leu Lys Lys Ala
20 25 30
Lys Glu Leu Thr Val Leu Cys Asp Ala Gln Val Ser Leu Ile Met Phe
35 40 45
Ser Ser Thr Gly Lys Leu Ala Asp Tyr Cys Ser Pro Ser Thr Asp Ile
50 55 60
Lys Gly Ile Tyr Glu Arg Tyr Gln Val Val Thr Gly Met Asp Leu Trp
65 70 75 80
Asn Ala Gln Tyr Glu Arg Met Gln Asn Thr Leu Lys His Leu Asn Glu
85 90 95
Ile Asn Gln Asn Leu Arg Lys Glu Ile Arg Arg Arg Lys Gly Glu Glu
100 105 110
Leu Glu Gly Met Asp Ile Lys Gln Leu Arg Gly Leu Glu Gln Thr Leu
115 120 125
Glu Glu Ser Leu Arg Ile Val Arg His Arg Lys Tyr His Val Ile Ala
130 135 140
Thr Gln Thr Asp Thr Tyr Lys Lys Lys Leu Lys Ser Thr Arg Glu Thr
145 150 155 160
Tyr Arg Ala Leu Ile His Glu Leu Asp Met Lys Glu Glu Asn Pro Asn
165 170 175
Tyr Gly Phe Asn Val Glu Asn Gln Ser Arg Ile Tyr Glu Asn Ser Ile
180 185 190
Pro Met Val Asn Glu Cys Pro Gln Met Phe Ser Phe Arg Val Val His
195 200 205
Pro Asn Gln Pro Asn Leu Leu Gly Leu Gly Tyr Glu Ser His Asp Leu
210 215 220
Ser Leu Ala
225
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
atgaaaagag gaaagataga gataa 25
<210>4
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
gactcgagtc gacatcg 17
Claims (1)
1. change the method for tobacco plant morphogenesis, comprise step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence, and the described butterfly orchid MADS box B AP3 of family albumen is the polypeptide shown in the SEQ ID NO:2 aminoacid sequence;
(2) vegetable cell or tissue or organ are contacted with the Agrobacterium of step (1), thereby make the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or tissue or the organ that changes the butterfly orchid MADS box B AP3 of family protein D NA encoding sequence over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
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| CN108949773B (en) * | 2017-05-18 | 2023-12-26 | 萧郁芸 | Method for producing transgenic plants |
| CN112626076B (en) * | 2020-11-25 | 2022-11-11 | 上海师范大学 | Application of gene PeNAC67 in breeding of Phalaenopsis miniata |
| CN112522281B (en) * | 2020-12-07 | 2022-11-11 | 上海师范大学 | Gene PeGRAS sequence for regulating and controlling petal development of phalaenopsis miniata and application thereof |
| CN114410646B (en) * | 2021-12-13 | 2023-09-29 | 上海师范大学 | Gene PeARF18 for regulating organ development of butterfly orchid and application thereof |
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Non-Patent Citations (6)
| Title |
|---|
| Bin Guo,et al.Cloning and Characterization of a PI-like MADS-Box Gene in Phalaenopsis Orchid.《Journal of Biochemistry and Molecular Biology》.2007,第40卷(第6期), |
| Cloning and Characterization of a PI-like MADS-Box Gene in Phalaenopsis Orchid;Bin Guo,et al;《Journal of Biochemistry and Molecular Biology》;20071130;第40卷(第6期);845-852 * |
| GenBank.MADS box AP3-like protein 17[Phalaenopsis hybrid cultivar].《GenBank:AAV28492.1》.2007, |
| GenBank.Phalaenopsis hyrid cultivar MADS box AP3-like protein 17 mRNA,complete cds.《GenBank:AY771993.1》.2007, |
| MADS box AP3-like protein 17[Phalaenopsis hybrid cultivar];GenBank;《GenBank:AAV28492.1》;20071130;1 * |
| Phalaenopsis hyrid cultivar MADS box AP3-like protein 17 mRNA,complete cds;GenBank;《GenBank:AY771993.1》;20071130;1 * |
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