CN1778813A - Plant epidermic hair control gene - Google Patents
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- CN1778813A CN1778813A CN 200410084399 CN200410084399A CN1778813A CN 1778813 A CN1778813 A CN 1778813A CN 200410084399 CN200410084399 CN 200410084399 CN 200410084399 A CN200410084399 A CN 200410084399A CN 1778813 A CN1778813 A CN 1778813A
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Abstract
A plant crust hair regulating protein-GaMYB2 protein, polynucleotide for coding this protein and production by recombinant technology are disclosed. The GaMYB2 is a key gene for regulating cotton fibre growth.
Description
Technical field
The invention belongs to agricultural sciences and plant improvement genetically engineered field.Specifically, the present invention relates to new plant epidermic hair control gene GaMYB2 and proteins encoded thereof.The invention also discloses the purposes of GaMYB2 gene, especially the application in improvement cotton fibre genetically engineered.
Background technology
Plant epidermal hair be a modular system studying cytodifferentiation, polar growth and morphogenesis on the unicellular level (Hulskamp, 2004, Nat.Rev.Mol.Cell Biol.5,471-480).Plant epidermal hair is divided into glandular hairs and nonglandular hair, and main function is a protective plant, as resist insect pest, reduce evaporation, improve freeze proof power and antiultraviolet etc. (Marks etc., 1997, Annu.Rev.Plant Physiol.Plant Mol.Biol.48,137-163).Wherein, cotton fibre and glandular hairs also have important economic value (Wilkins etc., 2000, Crit.Rev.Plant Sci.19,511-550; Wagner etc., 2004, Ann.Bot. (Lond) 93,3-11).
Cotton fibre is the epidermal hair of cotton seeds, and it is the most important natural fiber raw material of textile industry.Cotton fibre be one unicellular, be the longest known vegetable cell, generally long 2.2～3.0cm; Cotton fiber cell does not contain xylogen, 95% (Kim and Triplett, 2001, Plant Physiol.127:1361-1366) of cellulose comprises dry cell weight.The cotton fiber development progress of research mainly is to have separated some cotton fibre advantages or specific expression gene (John﹠amp; Crow, 1992, Proc.Natl.Acad.Sci USA 89:5769-5773; Li etc., 2002, Biochim.Biophys.Acta 1487:106-111; Ji etc., 2003, Nucleic.AcidsRes.31:2534-2543; Ruan etc., 2003, Plant Cell 15:952-964).First subunit CesA1/CesA2 of plant cellulose synthetic enzyme at first clones in cotton (Pear etc., 1996, Proc.Natl.Acad.Sci.USA 93:12637-12642).
The MYB genoid is the focus of clone's cotton fiber development key gene, some isolated M YB expression of gene feature and evolutionary relationships in cotton fibre come into one's own (Cedroni etc., 2003, Plant Mol.Biol.51:313-325).Up to now, it is relevant with cotton fiber development to have only cotton sucrose synthase gene Sus (sucrose synthase gene) to confirm in vegetable lamb.The sense-rna analysis revealed, Sus participates in cotton fibre elongation (Ruan etc., 2003, Plant Cell 15:952-964).
In sum, current position is also known little about it to the gene of regulation and control plant epidermal hair, therefore grows in order to regulate and control plant epidermis, especially improves the quality trait of cotton fibre, and this area presses for the new plant epidermal hair regulatory gene of exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of new plant epidermal hair modulin GaMYB2 albumen with and active fragments, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated GaMYB2 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ IDNO:2 aminoacid sequence.
Preferably, this polypeptide is selected from down group:
(a) has the polypeptide of 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, and have the regulation and control trichome development function by (a) polypeptides derived;
(c) have the R2R3MYB structural domain shown in the 12-117 position and the activation domain shown in the 171-197 position among the SEQ ID NO:2, and have the regulation and control trichome development function by (a) polypeptides derived.
More preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise-nucleotide sequence, this nucleotide sequence be selected from down group-kind of a nucleotides sequence shows at least 70% (more preferably at least 80%, best at least 90%) homogeny: (a) the encode polynucleotide of above-mentioned cotton GaMYB2 polypeptide; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 74-745 position among the SEQ ID NO:1; (b) has the sequence of 1-818 position among the SEQ ID NO:1; Or (c) has a sequence of 1-594 position among the SEQ ID NO:3; (d) has the sequence of 1-597 position among the SEQ ID NO:3.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or host cell, the especially vegetable cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.The regenerated plant by plant transformed cell institute also is provided.
In a fourth aspect of the present invention, the method for preparing cotton GaMYB2 polypeptide is provided, this method comprises: (a) under conditions suitable for the expression, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate cotton GaMYB2 polypeptide.
In a fifth aspect of the present invention, provide and above-mentioned cotton GaMYB2 polypeptid specificity bonded antibody.
In a sixth aspect of the present invention, a kind of method that changes plant epidermal hair is provided, it is characterized in that it comprises step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the dna encoding sequence of GaMYB2 polypeptide, group under being selected from of described GaMYB2 polypeptide:
(a) has the polypeptide of 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, and have the regulation and control trichome development function by (a) polypeptides derived;
(c) have the R2R3MYB structural domain shown in the 12-117 position and the activation domain shown in the 171-197 position among the SEQ ID NO:2, and have the regulation and control trichome development function by (a) polypeptides derived;
(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make the dna encoding sequence of GaMYB2 polypeptide change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or the tissue or the organ of the dna encoding sequence that changes the GaMYB2 polypeptide over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
In another preference, described plant is Arabidopis thaliana or cotton.
In a seventh aspect of the present invention, a kind of purposes of GaMYB2 polypeptide is provided, it is characterized in that it is used to change the epidermal hair of plant.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the sequence of GaMYB2 gene.The sequence of underscore is the coding region, and the sequence in the square frame is an intron.
Fig. 2 has shown the protein sequence of GaMYB2 genes encoding.The sequence of underscore is the R2R3MYB structural domain, and the sequence in the square frame is an activation domain.
Fig. 3 has shown the expression characteristic of GaMYB2 gene in cotton.Fate (dayspost-anthesis) after the DPA representative is bloomed.His represents the histone3 gene.
Fig. 4 has shown the regulation and control of GaMYB2 gene pairs Arabidopis thaliana trichome development.
Embodiment
The inventor is separated to a plant epidermal hair regulatory gene, called after GaMYB2 first through extensive and deep research from Asiatic cotton (Gossypium arboreum) (being also referred to as Gossypium orboreum).It is the gene of a cotton fibre high expression level that in situ hybridization and RT-PCR analyze analysis revealed GaMYB2.Yeast one-hybrid and arabidopsis thaliana analysis experiment show GaMYB2 regulating cotton specific expression promoter GaRDL1.GaMYB2 can control the growth of Arabidopis thaliana epidermal hair, and the not only complete complementary Arabidopis thaliana gl1 mutant of GL1 ∷ GaMYB2, and 35S ∷ GaMYB2 can impel the Arabidopis thaliana seed to produce epidermal hair.Therefore, GaMYB2 is the key gene of a control cotton fiber development, the regulation and control trichome development.
In the present invention, term " GaMYB2 albumen ", " GaMYB2 polypeptide " or " plant epidermal hair modulin GaMYB2 " are used interchangeably, and all refer to have the albumen or the polypeptide of plant epidermal hair modulin GaMYB2 aminoacid sequence (SEQ IDNO:2).They comprise the plant epidermal hair modulin GaMYB2 that contains or do not contain initial methionine.
As used herein, term " GaMYB2 " is the abbreviation of Gossypium arboreum MYB 2.
As used herein, term " intron " is meant some dna sequence dnas on the gene, and they can be transcribed, but is that processed shearing has been removed before translation.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating GaMYB2 albumen or polypeptide " is meant that the GaMYB2 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying GaMYB2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of GaMYB2 polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of cotton GaMYB2, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural cotton GaMYB2 albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation 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, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " cotton GaMYB2 polypeptide " refers to have the SEQ IDNO:2 polypeptide of sequence of cotton GaMYB2 protein-active.This term also comprises having and variant form cotton GaMYB2 albumen identical function (promptly regulating and control the trichome development function), SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of cotton GaMYB2 and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of cotton GaMYB2DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-cotton GaMYB2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises cotton GaMYB2 polypeptide or its segmental fusion rotein (as the fusion rotein that forms with GST).Except the polypeptide of total length almost, the present invention has also comprised the fragment of cotton GaMYB2 polypeptide.Usually, this fragment have cotton GaMYB2 peptide sequence at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of cotton GaMYB2 albumen or polypeptide.The difference of these analogues and natural cotton GaMYB2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " cotton GaMYB2 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | 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.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
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 can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 70%, preferably at least 80%, the polynucleotide of at least 90% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, 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%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding GaMYB2.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
Cotton GaMYB2 Nucleotide full length sequence 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 storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or GaMYB2 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the GaMYB2 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention cotton GaMYB2 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, cotton GaMYB2 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains cotton GaMYB2 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria; Fungal cell such as yeast; Vegetable cell etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain the plant that epidermal hair changes.
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, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The cotton GaMYB2 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: be used to screen antibody, polypeptide or other part that promotes or resist the GaMYB2 protein function.
On the other hand, the present invention also comprises cotton GaMYB2DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into cotton GaMYB2 gene product or fragment.Preferably, refer to that those can combine with cotton GaMYB2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of cotton GaMYB2, comprise that also those do not influence the antibody of cotton GaMYB2 protein function.The present invention also comprise those can with modify or without the cotton GaMYB2 gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the cotton GaMYB2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing cotton GaMYB2 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block cotton GaMYB2 protein function and the antibody that does not influence cotton GaMYB2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of cotton GaMYB2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of cotton GaMYB2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-cotton GaMYB2 can be used for whether existing in the test sample cotton GaMYB2 albumen.Whether having the proteic method of GaMYB2 in a kind of detection test sample is to utilize the proteic specific antibody of GaMYB2 to detect, and it comprises: sample is contacted with the GaMYB2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GaMYB2 albumen.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from Asiatic cotton.Its genome sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 818 bases, comprises 2 sections CDS (being 74-209 and 288-745 position); Corresponding cDNA sequence is listed in SEQ ID NO:3, and wherein open reading frame is positioned at 1-594 position (wherein), and the coding total length is 198 amino acid whose cotton GaMYB2 albumen (SEQ ID NO:2).GaMYB2 provides new approach for the epidermal hair (the especially cotton fibre of cotton) of improvement plant, thereby has huge potential application foreground.
Below in conjunction with specific embodiment, further set forth 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, as molecular cloning laboratory manual (Molecular cloning:A laboratorymanual, 3rd ed., Sambrook etc., Cold Spring Harbor Laboratory, 2001) and molecular biology of plants laboratory manual (Plant Molecular Biology-A Laboratory Mannual, Clark etc., Springer-Verlag, 1997) condition described in, or the condition of advising according to manufacturer.
The separation of embodiment 1 GaMYB2 gene
Cotton DNA extracts the cold phenol method that adopts.2g material (cotton fibre of Asiatic cotton (Gossypium arboreum)), in liquid nitrogen, clay into power, transfer in the 50ml centrifuge tube, add 8ml and extract damping fluid (1M TrisHCl, 50mM EDTA, 1%SDS, pH9.0) and isopyknic water-saturated phenol: chloroform: primary isoamyl alcohol (25: 24: 1), the concussion mixing was placed 1 hour, every 10 minutes mixings once on ice.4 ℃, centrifugal 20 minutes of 13000g.Repeat phenol: chloroform: primary isoamyl alcohol extracting 2～4 times, use chloroform at last: primary isoamyl alcohol (24: 1) extracting once.Get supernatant, add the high level salt solution (0.8M Trisodium Citrate, 1.2M NaCl) and the 1/2 volume Virahol of 1/2 volume, mixing was placed 1 hour for-70 ℃.4 ℃, centrifugal 20 minutes of 13000g.Followingly take corresponding operation according to extracting DNA and RNA.Remove supernatant liquor, use 1ml 70% ethanol washing and precipitating 2 times, room temperature was blown 20 minutes, and precipitation is dissolved in the 1ml sterilized water.4 ℃, centrifugal 10 minutes of 13000g.Get supernatant liquor, add 5～10 μ l RNase (10mg/ml), 37 ℃, 30 minutes.
With following primer GaMYB2-F 5 '-CCTTCCCT TGTTTCTCACTAATC-3 ' (SEQ ID NO:4) and GaMYB2-R 5 '-CACACCAATAATTGTTGTTTTTTT CTTC-3 ' (SEQ ID NO:5).PCR reaction conditions: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of 30 seconds of pre-sex change then, in 60 ℃ of 30 seconds of renaturation, 72 ℃ are extended 30 seconds, totally 35 circulations; Last 72 ℃ were extended 10 minutes.Check order behind the subclone, confirm whether sequence is correct.
Sequencing result such as SEQ ID NO:1 and shown in Figure 1, this is a genome sequence, the polynucleotide sequence total length that it comprises is 818 bases, comprises 2 sections CDS (being 74-209 and 288-745 position); Corresponding cDNA sequence is listed in SEQ ID NO:3, and wherein open reading frame is positioned at 1-594 position (wherein), and the coding total length is 198 amino acid whose cotton GaMYB2 albumen (SEQ ID NO:2 and Fig. 2).
With the DNA of the various tissues of above-mentioned same procedure extracting cotton, carry out conventional RT-PCR with same primers as.Also use the probe based on SEQ ID NO:1 to carry out the in situ hybridization test, the result shows that GaMYB2 is the gene (Fig. 3) of a cotton fibre high expression level.
Embodiment 2 vector constructions and Agrobacterium-mediated Transformation
Complete encoding sequence according to the GaMYB2 gene, design amplifies complete coding and reads the primer of frame (corresponding to the most preceding 20bp among the SEQ ID NO:3 and last 20bp), behind pcr amplification, with the genomic dna cloning of GaMYB2 to intermediate carrier pBluescript (available from Stratagene company), further be cloned into binary expression vector pBI121 (available from Clontech company), under the prerequisite that guarantees the reading frame, identify good expression vector, in it being changed over to Agrobacterium (Agrobacterium tumefaciens), obtain positive colony, be used for plants such as converting cotton.
Freeze-thaw method is adopted in the conversion of Agrobacterium.With single bacterium colony LBA4404 or GV3101 (Invitrogen), 3mlLB substratum (25 μ g/ml rifomycin Rif and 50 μ g/ml kalamycin Kan or gentamicin Gen), 28 ℃, 220rpm, incubated overnight.2ml bacterium liquid, 50ml LB substratum (25 μ g/ml Rif and 50 μ g/mlGen), 28 ℃, 220rpm cultivates OD
600=0.5 (about 6 hours).Placed 30 minutes on ice, 4 ℃, centrifugal 5 minutes of 5000g.Be resuspended in 10ml 0.15M NaCl.4 ℃, centrifugal 5 minutes of 5000g.Be resuspended in 1ml 20mM CaCl
2, 50 μ l/ manage packing, and liquid nitrogen flash freezer is preserved competent cell for-70 ℃.Mix and contain goal gene binary vector and 50 μ l/ pipe competent cell, placed 30 minutes liquid nitrogen flash freezer 1 minute on ice.Bacterium liquid was melted in 5 minutes in 37 ℃ of water-baths, add 1ml LB substratum, 28 ℃, 220rpm cultivated 2～4 hours.Get 50～100 μ l and be coated with LB culture medium flat plate (25 μ g/ml Rif, 50 μ g/ml Gen and 50 μ g/ml kalamycin Kan or Totomycin Hyg).
The screening of embodiment 3 Plant Transformation and transgenic progeny
In the present embodiment, be example with the transgenosis of cotton and Arabidopis thaliana, the conversion of other plant can be as reference.
A. the transgenosis of cotton
Adopt the agrobacterium mediation method converting cotton.
Getting the aseptic seedling hypocotyl top segment (0.5cm) of 5 ages in days cultivated 36 hours in advance.Substratum: SH+2,4-D 0.1mg/L+KT 0.1mg/L.
Hypocotyl segment soaked 20 minutes in Agrobacterium bacterium liquid (SH+2,4-D 0.1mg/L+KT 0.1mg/L+ Syringylethanone 100 μ mol/L).
Cultivated altogether 3 days.Substratum: SH+2,4-D 0.1mg/L+KT 0.1mg/L+ Syringylethanone 100 μ mol/L.
Callus is initial induced 25～30 days.Substratum: SH+2,4-D 0.1mg/L+KT 0.1mg/L+Km50mg/L+ cephamycin (cef) 300mg/L or MSB+2,4-D 0.1mg/L+IAA0.1mg/L+ZT0.1mg/L+Km 50mg/L+cef 300mg/L.
Embryonic callus induction and subculture screening, per 15 days propagation once.Select soft, light green or gray callus and be used for inducing of embryo callus subculture; Select fresh, yellow, fine and close, granular embryo callus shoot proliferation.Substratum: SH or MSB (NH
4NO
3Be 0.3mg/L)+IAA 0.5mg/L+KT 0.5mg/L+Km75mg/L+Cef 300mg/L.
Embryoid forms and is ripe.Substratum: MSB (KNO
3Doubling does not have hormone)+Km 75mg/L+Cef 300mg/L.
Embryoid (sprouting) elongation, growth.Substratum: MSB (no hormone)+gac 250mg/L+Km75mg/L+Cef 300mg/L.
The growth of embryoid true leaf.Substratum: MSB+IBA0.4mg/L+KT0.1mg/L+Km75mg/L+Cef300mg/L.Part becomes normal plantlet; Part is taked grafting.
Results suggest, GaMYB2 regulating cotton specific expression promoter GaRDL1 (being designated hereinafter simply as GL1) (SEQID NO:6), the trichome development of regulation and control cotton.
B. the transgenosis of Arabidopis thaliana
The conversion of arabidopsis thaliana adopt bud infusion method (floral dip) (method is pressed Clough and Bent, 1998, Plant J.16,735-743 carries out).Single bacterium colony GV3101 (Invitrogen) that will contain binary vector, 3ml LB substratum (25 μ g/ml Rif, 50 μ g/ml Gen and 50 μ g/ml Kan or Hyg), 28 ℃, 220rpm, 12 hours.2ml bacterium liquid, 50ml LB substratum (25 μ g/ml Rif, 50 μ g/ml Gen and 50 μ g/ml Kan or Hyg), 28 ℃, 220rpm, 12 hours.50ml bacterium liquid, 250ml LB substratum (50 μ g/ml Gen and 50 μ g/ml Kan or Hyg), 28 ℃, 220rpm, 12 hours.4200rpm (2900g), 15 minutes.Thalline is resuspended in 5% sucrose solution that 500ml contains 0.02%Silwet L-77.Above-ground plant parts soaks 5sec in bacterium liquid, lie against in the plastic tub, preserves moisture lucifuge, 16～24 hours.T0 at 4 ℃ of vernalization 2～4d, handled 15 minutes sterile water wash 3～4 times for seed with 20% drift ice.Be suspended from 0.5% agarose (55 ℃), be layered on the LB substratum (50 μ g/ml Kan or Hyg) of 0.6% agar, 22 ℃, continuous illumination, after the about week, green resistance transplantation of seedlings is to nutrition soil (peat: vermiculite: growth perlite 1: 1: 1).
The results are shown in Figure 4.Fig. 4 shows, change Arabidopis thaliana over to after, GL1 ∷ GaMYB2 reduces the wild-type epidermal hair.In addition, the complete complementary Arabidopis thaliana gl1 mutant of GL1 ∷ GaMYB2, the chaeta that makes gl1 not have the chalaza variant recovers.In addition, 35S ∷ GaMYB2 can impel the Arabidopis thaliana seed to produce epidermal hair.These results show that GaMYB2 can control the growth of Arabidopis thaliana epidermal hair.
The molecular biology identification of embodiment 4 transgenic plant
In the present embodiment, the Arabidopis thaliana T3 that chooses embodiment 3 acquisitions adopts methods such as antibiotic-screening, PCR, GUS dyeing and Southern hybridization analysis that transfer-gen plant is verified for homozygous lines.
A.PCR analyzes
DNA extraction method and PCR reaction conditions are seen embodiment 1.Primer is SEQ ID NO:4 and 5.
The b.GUS staining analysis
The GUS staining fluid soaks vegetable material, 37 ℃, 12～24 hours.70% ethanol decolorization, sample is preserved in 70% ethanol.GUS staining fluid (100mM pH7.0 phosphoric acid buffer, 50mM K
3[Fe (CN)
6], 50mMK
4[Fe (CN)
6], 10mM EDTA, 1mM X-gluc, 0.1%Triton X-100).
The c.Southern hybridization analysis
10 μ g DNA select 3～5 kinds of restriction enzymes, complete degestion (spending the night).After finishing, 0.7% agarose gel electrophoresis, electrophoresis cut the nonuseable part of gel.Soaked 45 minutes in sex change liquid (1.5M NaCl, 0.5M NaOH), gentleness is shaken.Rinsed with deionized water, (pH7.4) middle immersion is 45 minutes for 1.5M NaCl, 1M TrisHCl, and gentleness is shaken at neutralizer.
Adopt the capillary transfer method that DNA is transferred to nylon membrane Hybond-XL (Amersham-PharmaciaBiotech), transfering buffering liquid is 20XSSC (3M NaCl, a 0.3M Trisodium Citrate), and be 12-18 hour transfer time.With 6XSSC rinsing nylon membrane, be clipped in the middle of the filter paper, be pressed between the sheet glass, 80 ℃ were dried by the fire 2 hours.
25ng adopts Primer-a-Gene Labeling System (Promega) label probe through the PCR product of rubber tapping alcoholization.α-
32P-dCTP, 50 μ l reaction systems, 37 ℃, 1 hour.Adopt QIAquick NucleotideRemoval Kit (QIAGEN) purifying probe.In the boiling water bath 10 minutes, place on ice.
Nylon membrane is put into hybrid pipe, add 10ml prehybridization solution (50% methane amide, 5XSSC, 5XDenhardt, 1%SDS, 100 μ g/ml sex change milt DNA), 42 ℃, 2～4 hours.Add the probe of sex change, hybridized 16～24 hours.
Remove hybridization solution, wash film 2 times, each 5 minutes with the 2XSSC that contains 0.1%SDS in room temperature.Wash film 2 times, each 5 minutes in room temperature with the 0.2XSSC that contains 0.1%SDS.Wash film 2 times at 42 ℃ with the 0.2XSSC that contains 0.1%SDS, each 15 minutes.Wash film 2 times in room temperature with 2XSSC.Take out nylon membrane, the drip-dry hybridization solution, with the preservative film parcel, adhesive tape is fixed, and presses intensifying screen and X-ray film, and-70 ℃, 2d.Film develops with D-72 liquid.
Test-results shows, has changed the GaMYB2 gene of cotton in the genetically modified Arabidopis thaliana plant really over to, and the Arabidopis thaliana plant of contrast does not then contain the GaMYB2 gene.
Embodiment 5
The proteic expression of GaMYB2
(1) expression vector establishment
According to the complete encoding sequence (SEQ ID NO:3) of GaMYB2 gene, design amplifies the primer of complete coding frame.The full-length gene of GaMYB2 gene is behind pcr amplification, be cloned into expression vector pRS426-CUP (available from Clontech company), guaranteeing to identify expression vector under the correct prerequisite of reading frame, change it over to model animals yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
(2) electrotransformation transformed saccharomyces cerevisiae
A. use that yeast saccharomyces cerevisiae list bacterium colony is in 2mL liquid YEPD substratum in the aseptic toothpick picking YEPD flat board, 28 ℃, the 200rpm shaking culture is spent the night.
B. change in the 200mLYEPD substratum 28 ℃ next day over to, the 200rpm shaking culture is between OD600=1.0～1.3.
C. with cell transfer 4000rpm to the 50mL centrifuge tube, 4 ℃ of centrifugal 10min abandon or adopt supernatant.
D. isopyknic deionized water is added in the centrifuge tube re-suspended cell, 4000rpm, 4 ℃ of centrifugal 10min.Abandon or adopt supernatant.
E. the deionized water with half volume adds in the centrifuge tube re-suspended cell, 4000rpm, 4 ℃ of centrifugal 10min.Abandon or adopt supernatant.
F. the 1M sorbyl alcohol with half volume adds in the centrifuge tube re-suspended cell, 4000rpm, 4 ℃ of centrifugal 10min.Abandon or adopt supernatant.
G. the 1M sorbyl alcohol with 500 μ L adds centrifuge tube.
H. with 100 μ L competence yeast cell and 100ng plasmid DNA mixing, be added to ice bath 20min in the 0.1cm electric shock cup, be positioned in the BioRed electroporation apparatus by 25 μ F the 1500V electric shock.
I. add 1mL 1M sorbyl alcohol then rapidly, behind the mixing part cell is coated dull and stereotyped (the no uridylic substratum) 30 ℃ of going up of selection and cultivated 3～6 days, thereby obtain to change over to the proteic yeast strain of GaMYB2.
The cell lysate that changes the proteic yeast strain of GaMYB2 over to is having a tangible GaMYB2 protein band corresponding to about 20KDa place.
The generation of anti-GaMYB2 protein antibodies
The reorganization cotton GaMYB2 albumen that obtains among the embodiment 5 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation cotton GaMYB2 protein gene translation product with it.Found that antibody can combine with GaMYB2 albumen specifically.
Embodiment 7
The mutant of GaMYB2
Site-directed mutagenesis method with routine, the nucleotide sequence of SEQ ID NO:3 is replaced or adds, thereby 195 the Leu that makes SEQ ID NO:2 becomes Ile (mutant 1), and 195 the Leu of SEQ ID NO:2 becomes ala (mutant 2) and add an Ala (mutant 3) after 198 of SEQ ID NO:2.Press method arabidopsis thaliana transformation identical among the embodiment 3b then.
The result shows, these the three kinds of mutant under promotor GL1 control equally can complementary Arabidopis thaliana gl1 mutant, and the chaeta that makes gl1 not have the chalaza variant recovers.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉plant epidermic hair control gene
<130>044446
<160>6
<170>PatentIn version 3.1
<210>1
<211>818
<212>DNA
＜213〉cotton (Gossypium arboreum)
<220>
<221>CDS
<222>(74)..(209)
<223>
<220>
<221>CDS
<222>(288)..(745)
<223>
<400>1
ccttcccttg tttctcacta atcatctctg tcttcccttc tcactctttg cctcttctca 60
ctgtcggcta ata atg gct cca aag aag gat gga gtg agc aaa agg gtt 109
Met Ala Pro Lys Lys Asp Gly Val Ser Lys Arg Val
1 5 10
ttt aac aaa ggt tct tgg aca gct gag gaa gat aga aga ttg gct aaa 157
Phe Asn Lys Gly Ser Trp Thr Ala Glu Glu Asp Arg Arg Leu Ala Lys
15 20 25
tat att gag att cat ggc gca aag aga tgg aaa aca atc gcc att aaa 205
Tyr Ile Glu Ile His Gly Ala Lys Arg Trp Lys Thr Ile Ala Ile Lys
30 35 40
tca g gtaatttact ttctgttgaa gagaaactcc attttttgga gcattggaat 259
Ser
45
taacgggtta ttttgttttg atgtatag gt ttg aat cga tgc ggc aag agt 310
Gly Leu Asn Arg Cys Gly Lys Ser
50
tgc agg ttg aga tgg ttg aac tac ttg aga cct aac att aag aga ggc 358
Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn Ile Lys Arg Gly
55 60 65
aac ata tca gat gaa gaa gag gac tta att att agg ctt cat aaa ctg 406
Asn Ile Ser Asp Glu Glu Glu Asp Leu Ile Ile Arg Leu His Lys Leu
70 75 80 85
ctg ggg aac agg tgg tct ttg att gct ggg aga ctt cca ggg cga aca 454
Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro Gly Arg Thr
90 95 100
gac aat gaa att aag aac tac tgg aat tcc cat ttg agc aag aaa ata 502
Asp Asn Glu Ile Lys Asn Tyr Trp Asn Ser His Leu Ser Lys Lys Ile
105 110 115
ata aac cat gat gtc aga aca gaa caa act tcc tcc tcg gaa caa att 550
Ile Asn His Asp Val Arg Thr Glu Gln Thr Ser Ser Ser Glu Gln Ile
120 125 130
gtg cct cac aaa gca tgg gaa act gtc cat atg gaa gaa gaa gag gta 598
Val Pro His Lys Ala Trp Glu Thr Val His Met Glu Glu Glu Glu Val
135 140 145
gta aaa gga agt gat gaa att gaa aac tct gaa ttc agc att gat gtg 646
Val Lys Gly Ser Asp Glu Ile Glu Asn Ser Glu Phe Ser Ile Asp Val
150 155 160 165
gac gaa ttc ttt gac ttc aca acg gaa ggt tgc ttt act ttg gat tgg 694
Asp Glu Phe Phe Asp Phe Thr Thr Glu Gly Cys Phe Thr Leu Asp Trp
170 175 180
gtg aat aag ttc ctt gaa ctt gat gat caa cag gat cca tta gca atg 742
Val Asn Lys Phe Leu Glu Leu Asp Asp Gln Gln Asp Pro Leu Ala Met
185 190 195
gta taataagttt gtaattaaca tgtttccttg gataaataaa taaagagatg 795
Val
ttctgagtct aaaaatggaa gat 818
<210>2
<211>198
<212>PRT
＜213〉cotton (Gossypium arboreum)
<400>2
Met Ala Pro Lys Lys Asp Gly Val Ser Lys Arg Val Phe Asn Lys Gly
1 5 10 15
Ser Trp Thr Ala Glu Glu Asp Arg Arg Leu Ala Lys Tyr Ile Glu Ile
20 25 30
His Gly Ala Lys Arg Trp Lys Thr Ile Ala Ile Lys Ser Gly Leu Asn
35 40 45
Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro
50 55 60
Asn Ile Lys Arg Gly Asn Ile Ser Asp Glu Glu Glu Asp Leu Ile Ile
65 70 75 80
Arg Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg
85 90 95
Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Ser His
100 105 110
Leu Ser Lys Lys Ile Ile Asn His Asp Val Arg Thr Glu Gln Thr Ser
115 120 125
Ser Ser Glu Gln Ile Val Pro His Lys Ala Trp Glu Thr Val His Met
130 135 140
Glu Glu Glu Glu Val Val Lys Gly Ser Asp Glu Ile Glu Asn Ser Glu
145 150 155 160
Phe Ser Ile Asp Val Asp Glu Phe Phe Asp Phe Thr Thr Glu Gly Cys
165 170 175
Phe Thr Leu Asp Trp Val Ash Lys Phe Leu Glu Leu Asp Asp Gln Gln
180 185 190
Asp Pro Leu Ala Met Val
195
<210>3
<211>597
<212>DNA
＜213〉cotton (Gossypium arboreum)
<400>3
atggctccaa agaaggatgg agtgagcaaa agggttttta acaaaggttc ttggacagct 60
gaggaagata gaagattggc taaatatatt gagattcatg gcgcaaagag atggaaaaca 120
atcgccatta aatcaggttt gaatcgatgc ggcaagagtt gcaggttgag atggttgaac 180
tacttgagac ctaacattaa gagaggcaac atatcagatg aagaagagga cttaattatt 240
aggcttcata aactgctggg gaacaggtgg tctttgattg ctgggagact tccagggcga 300
acagacaatg aaattaagaa ctactggaat tcccatttga gcaagaaaat aataaaccat 360
gatgtcagaa cagaacaaac ttcctcctcg gaacaaattg tgcctcacaa agcatgggaa 420
actgtccata tggaagaaga agaggtagta aaaggaagtg atgaaattga aaactctgaa 480
ttcagcattg atgtggacga attctttgac ttcacaacgg aaggttgctt tactttggat 540
tgggtgaata agttccttga acttgatgat caacaggatc cattagcaat ggtataa 597
<210>4
<211>23
<212>DNA
＜213〉cotton (Gossypium arboreum)
<400>4
ccttcccttg tttctcacta atc 23
<210>5
<211>28
<212>DNA
＜213〉cotton (Gossypium arboreum)
<400>5
cacaccaata attgttgttt ttttcttc 28
<210>6
<211>302
<212>DNA
＜213〉cotton (Gossypium arboreum)
<400>6
aattagttat gtttggtaaa tgaatttgaa tacatgcacc accactctca atgctcaccc 60
acctaagccc atgacataaa ctgcatttaa gtgaaccagt ggcagttgga gccattatgt 120
tggttgaaaa atattgttgg cagtgttatt cagcagtact tgttctaagg ctcctactac 180
atttctcatt ataaatgtgg agaaatcttg ttctactttc cattccatgc aacactaact 240
tttagattcc ctttccattg cactgctctt tctttctata gaattagtca ctcctgttct 300
ag 302
Claims (10)
1. isolating GaMYB2 polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of 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, and have the regulation and control trichome development function by (a) polypeptides derived;
(c) have the R2R3MYB structural domain shown in the 12-117 position and the activation domain shown in the 171-197 position among the SEQ ID NO:2, and have the regulation and control trichome development function by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 74-745 position among the SEQ ID NO:1;
(b) has the sequence of 1-818 position among the SEQ ID NO:1;
(c) has the sequence of 1-594 position among the SEQ ID NO:3;
(d) has the sequence of 1-597 position among the SEQ ID NO:3.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. the preparation method of a peptide species is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;
(b) from culture, isolate the GaMYB2 polypeptide.
9. energy and the described GaMYB2 polypeptid specificity of claim 1 bonded antibody.
10. method that changes plant epidermal hair is characterized in that it comprises step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the dna encoding sequence of GaMYB2 polypeptide, group under being selected from of described GaMYB2 polypeptide:
(a) has the polypeptide of 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, and have the regulation and control trichome development function by (a) polypeptides derived;
(c) have the R2R3MYB structural domain shown in the 12-117 position and the activation domain shown in the 171-197 position among the SEQ ID NO:2, and have the regulation and control trichome development function by (a) polypeptides derived;
(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make the dna encoding sequence of GaMYB2 polypeptide change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or the tissue or the organ of the dna encoding sequence that changes the GaMYB2 polypeptide over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
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|---|---|---|---|
| CN 200410084399 CN1778813A (en) | 2004-11-22 | 2004-11-22 | Plant epidermic hair control gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410084399 CN1778813A (en) | 2004-11-22 | 2004-11-22 | Plant epidermic hair control gene |
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| Publication Number | Publication Date |
|---|---|
| CN1778813A true CN1778813A (en) | 2006-05-31 |
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ID=36769277
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576284A (en) * | 2018-12-21 | 2019-04-05 | 中国农业科学院北京畜牧兽医研究所 | One multi-functional myb transcription factor gene and application thereof |
| CN109593768A (en) * | 2019-01-07 | 2019-04-09 | 华中农业大学 | Application of the Platanus acerifolia PaGL1 gene in regulation plant epidermal hair |
-
2004
- 2004-11-22 CN CN 200410084399 patent/CN1778813A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576284A (en) * | 2018-12-21 | 2019-04-05 | 中国农业科学院北京畜牧兽医研究所 | One multi-functional myb transcription factor gene and application thereof |
| CN109576284B (en) * | 2018-12-21 | 2021-09-17 | 中国农业科学院北京畜牧兽医研究所 | Multifunctional MYB transcription factor gene and application thereof |
| CN109593768A (en) * | 2019-01-07 | 2019-04-09 | 华中农业大学 | Application of the Platanus acerifolia PaGL1 gene in regulation plant epidermal hair |
| CN109593768B (en) * | 2019-01-07 | 2020-12-15 | 华中农业大学 | Application of Pagl1 gene in regulating and controlling plant epidermal hair |
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