CN101173001B - Gene for regulation of auxin sensibility and blade area, and uses thereof - Google Patents
Gene for regulation of auxin sensibility and blade area, and uses thereof Download PDFInfo
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Abstract
The invention discloses new auxin conjugated protein having the function for adjusting the sensitivity of the plant to the auxin or adjusting the plant leaf area. The invention also discloses the code gene of the protein, a carrier and a cell which contain the gene. The invention also discloses a method for using the gene to prepare the plant with high auxin sensitivity, and the method for using the gene to prepare the plant with a proper leave area. The protein of the invention has wide application value in the plant implantation field, and keeps the plant more sensitive to plant, thereby greatly reducing the auxin putting amount in field and effectively reducing the agriculture cost; or according to the actual product requirement, the leaf size is changed to improve the yield and the quality of plants.
Description
Technical field
The invention belongs to the genetically engineered field, relate to a kind of plant hormone auxin-binding protein AtABP2 gene and regulating plant the application in growth hormone susceptibility and the blade area.
Background technology
Growth hormone (Auxin) is a kind of plant hormone of finding the earliest, and it extensively is present in the organs such as root, stem, leaf, flower, fruit, seed and coleoptile of plant.Growth hormone also is to use the earliest and the widest broad-spectrum plant growth regulator, is widely used in agriculture production.Utilize synthetic growth hormone that the effect of following several broad aspect can be arranged on producing: (1) promotes that the branch of cuttage is taken root, (2) promote fruit development, (3) to prevent fruit drop.In addition, the growth hormone analogue of high density also can be used as weedicide, removes the dicotyledonous class weeds in field.
(Auxin Binding Protein ABP1) all has discovery to auxin-binding protein 1 in many species.Concerning plant, known auxin-binding protein 1 influences the growth of plant body early embryo, processes such as cell elongation (Chen etc., GENES ﹠amp; DEVELOPMENT 15:902-911).
Yet, in present plant growing is cultivated, grow well in order to make plant, still need to grant a large amount of growth hormone, improved planting cost greatly, so this area presses for and find some to reduce the approach of plants to the growth hormone demand, thereby reduce the agricultural planting cost.
Summary of the invention
The object of the present invention is to provide a kind of new auxin-binding protein 2 (ABP2) and uses thereof.
The present invention also aims to provide the method for utilizing described auxin-binding protein 2 or its encoding gene improvement plant.
In a first aspect of the present invention, a kind of isolated polypeptide is provided, this polypeptide is selected from down group:
(a) has the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2;
(b) aminoacid sequence shown in the SEQ ID NO:2 is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have (a) described polypeptide function by (a) polypeptides derived.
In another preference of the present invention, (a) function of described polypeptide is: regulate plant to growth hormone susceptibility function and/or adjusting plant blade area.
In another preference of the present invention, this polypeptide is the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and described polynucleotide are selected from down group:
(a) polynucleotide of coding said polypeptide;
(b) with polynucleotide (a) complementary polynucleotide.
In another preference of the present invention, described polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
In another preference of the present invention, described polynucleotide have the sequence shown in the SEQ ID NO:1.
In a third aspect of the present invention, a kind of carrier is provided, it contains described polynucleotide.
In a fourth aspect of the present invention, provide a kind of genetically engineered host cell, described host cell:
Contain described carrier; Or
Be integrated with described polynucleotide in the genome.
In a fifth aspect of the present invention, a kind of preparation method of described polypeptide is provided, this method comprises:
(a) under conditions suitable for the expression, cultivate described host cell, obtain culture;
(b) from culture, isolate described polypeptide.
In a sixth aspect of the present invention, the purposes of described polypeptide is provided, be used for:
Regulate susceptibility or the plant blade area of plant to growth hormone; Or
The plant that preparation is high to growth hormone susceptibility, or the plant that the preparation blade area increases or blade area reduces.
In another preference of the present invention, described polypeptide can be used for improving the susceptibility of plant to growth hormone, or increases the area of plant leaf.
In another preference of the present invention, described plant is selected from (but being not limited to) group down: Gramineae, the Rosaceae, Cruciferae.
In another preference of the present invention, described plant includes, but is not limited to: Arabidopis thaliana, paddy rice, tobacco, melon and fruit, vegetables, rape etc.
In a seventh aspect of the present invention, the method for a kind of preparation to the big plant of growth hormone susceptibility height or blade area is provided, described method comprises:
(1) described polynucleotide is changed over to vegetable cell, tissue or organ (as petal), obtain to be transformed into vegetable cell, tissue, organ or the seed of described polynucleotide; With
(2) vegetable cell that has changed described polynucleotide over to, tissue, organ or the seed regeneration plant that step (1) is obtained.
In another preference of the present invention, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains described polynucleotide;
(s2) vegetable cell, tissue, organ or seed are contacted with Agrobacterium in the step (s1), thereby make described polynucleotide change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell, tissue, organ or the seed that has changed described polynucleotide over to; And
(s4) with the vegetable cell in the step (s3), tissue, organ or seed regeneration plant.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 is wild-type plant and gene overexpression strain system and reduces strain system to growth hormone susceptibility comparison diagram.Figure 1A is not for adding the wild-type of growth hormone and variant system to the susceptibility of growth hormone relatively; Figure 1B for the wild-type of adding corresponding growth hormone and variant system to the susceptibility of growth hormone relatively.Wherein, WT is the wild-type Arabidopis thaliana, and abp2 is the strain system of ABP2 down regulation of gene expression.The strain system that abp2-d raises for ABP2 genetic expression.Abp2 compares with wild-type, growth hormone has been lost part susceptibility, promptly poor to the susceptibility of growth hormone than wild-type, with the abp2 of gene downward modulation to the reaction of growth hormone just in time opposite be, the strain that gene raises be abp2-d when comparing with wild-type to the growth hormone hypersensitization.
Fig. 2 is that wild-type plant and gene overexpression strain are the blade area comparison diagram, and WT is the wild-type Arabidopis thaliana, and abp2-d is the strain system that ABP2 genetic expression is raised.The strain that gene raises is that abp2-d compares with WT, and the abp2-d blade area is obviously big than wild-type, illustrates that the ABP2 gene participates in the positive regulation process that blade increases.File the 1st row refer to the 1st on lotus throne leaf, and the 2nd on lotus throne leaf is shown in the 2nd tabulation, afterwards and the like.
Fig. 3 is that wild-type plant and gene overexpression strain are blade area statistics relatively.Wherein, " ☆ " expression has been compared significance with wild-type, p＜0.05, and " ☆ ☆ " expression has been compared significance with wild-type, p＜0.01.
Embodiment
The inventor is through long term studies, find a kind of new albumen---auxin-binding protein 2 (Auxin Binding Protein 2, ABP2), and it is associated with the growth hormone susceptibility of plant, the proteic expression excessively of discovery ABP2 can be improved the susceptibility of plant to growth hormone, and the low expression of ABP2 or loss of expression make plant insensitive to growth hormone.And the inventor finds that also the ABP2 gene has also participated in regulating the size of plant blade area, and the high expression level of ABP2 in leaf makes the blade area of plant increase, and low expression or loss of expression make the blade area of plant reduce.Finished the present invention based on this.
As used herein, described " plant " includes but not limited to: Cruciferae, Gramineae, the Rosaceae.Such as, described " plant " includes but not limited to: Arabidopis thaliana, paddy rice gramineous, corn, rosaceous cherry that the Cruciferae rape belongs to comprise tobacco, melon and fruit, vegetables, rape or the like in addition.
As used herein, described " plant high to growth hormone susceptibility " is meant after applying exogenous auxin, the verified response index to growth hormone of some of described plant is (as issuing the elongation of taking root in the plain concentration of certain growth, lateral root increase and blade increase etc.) be higher than kindred plant (plant or ABP2 as normal wild type express plant low or that do not express), the index of " height " is based on the value (such as P＜0.01) of significant difference in the data analysis herein.
As used herein, described " plant that blade area is big " or " blade area increase plant " is meant a kind of plant, and (as bolting time) was significantly bigger (as big more than 10% than the blade area of kindred plant (plant or ABP2 as normal wild type express plant low or that do not express) under the same conditions when it grew to certain phase under suitable growth conditions; As big 10-40%, preferred big 30-40%).Described " plant that blade area is little " or " plant that blade area reduces " are meant when growing to certain phase under suitable growth conditions that (as bolting time) is than the remarkable little plant of the blade area of kindred plant under the same conditions.
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 ABP2 albumen " or " isolating ABP2 polypeptide " is meant that ABP2 albumen 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 ABP2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably 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, insect and mammalian cell).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 ABP2, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of ABP2 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 fusion rotein).These fragments of definition, derivative and analogue according to this paper belong to the known scope of those skilled in the art.
The proteic bioactive fragment of any ABP2 can be applied among the present invention.Here, the implication of the proteic bioactive fragment of ABP2 is meant that as a peptide species it still can keep the proteic all or part of function of ABP2 of total length.Generally, described bioactive fragment keeps 50% the proteic activity of total length ABP2 at least.Under preferred condition, described active fragments can keep proteic 60%, 70%, 80%, 90%, 95%, 99% or 100% the activity of total length ABP2.
In the present invention, term " ABP2 albumen " refers to have the SEQ ID NO:2 polypeptide of sequence of ABP2 protein-active.This term also comprises having and variant form ABP2 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best, also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is 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 ABP2 and reactive derivative.
The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with coded albumen of the DNA of ABP2 protein D NA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-ABP2 to obtain.The present invention also provides other polypeptide, as comprises ABP2 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of ABP2.Usually, this fragment have the ABP2 protein sequence at least about 20 continuous amino acids, usually at least about 30 continuous amino acids, preferably 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 ABP2 albumen or polypeptide.These analogues and the proteic difference of natural A BP2 can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have 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.Modify and also comprise glycosylation.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, " ABP2 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 20 at the most, preferably at the most 10, 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
| Amino-acid 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 |
The present invention also provides the polynucleotide sequence of code book invention ABP2 albumen or its conservative property variation polypeptide.
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:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
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 coding said polypeptide, 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 50%, preferably at least 70%, the polynucleotide of at least 80% 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 ABP2.
Should understand, though ABP2 gene of the present invention is preferably available from Arabidopis thaliana, but available from other plant and other gene Arabidopis thaliana ABP2 gene height homology (as have more than 80%, as 85%, 90%, 95% even 98% sequence homogeny) also within the scope that the present invention considers.The Method and kit for of aligned sequences homogeny also is that this area is known, for example BLAST.
ABP2 pyrenoids thuja acid 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 ABP2 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 express or produce the ABP2 albumen of reorganization.In general following steps are arranged:
(1). with the proteic polynucleotide of coding ABP2 of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, ABP2 albumen 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, mammalian 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 ABP2 encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be 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.
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 kantlex 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, streptomyces, Agrobacterium; 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.
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, paddy rice rataria conversion method etc.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain plant that growth hormone susceptibility or blade area are changed.
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 ABP2 albumen or the polypeptide of reorganization are of use in many ways.For example be used to screen antibody, polypeptide or other part that promotes or resist the ABP2 protein function.Can be used for seeking the valuable peptide molecule that can suppress or stimulate the ABP2 protein function with the reorganization ABP2 protein screening peptide library of expressing.
Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of ABP2 albumen and also can detect the proteic transcription product of ABP2.
The invention provides the proteic purposes of described ABP2, be used to regulate plant the susceptibility of growth hormone or the area of adjusting plant leaf; Or be used to screen and regulate plant to growth hormone susceptibility or regulate the material (that is: described material is regulated susceptibility or the blade area of plant to growth hormone by regulating the proteic expression of ABP2) of plant blade area.As a kind of optimal way, described ABP2 albumen can be used for improving the susceptibility of plant to growth hormone, makes plant apply growth fast under the situation of growth hormone seldom, thereby reaches the purpose of the cost that reduces domestication of plants.Such as, for some ABP2 protein expression low plant or plant, can improve the transfer-gen plant of expressing by the ABP2 albumen of this kind of plant of preparation or plant, improve its susceptibility to growth hormone.
As another kind of optimal way, described ABP2 albumen can be used for regulating the blade area of plant, changes the blade size thereby reach according to needs of production, the purpose of improvement plant biomass and quality.
ABP2 albumen of the present invention can be regulated the susceptibility of plant for multiple growth hormone, described growth hormone include but not limited to indolylacetic acid (IAA), naphthylacetic acid (NAA) or 2,4 dichlorphenoxyacetic acids (2,4-D).
Described ABP2 albumen also can be used for preparing the composition of adjusting plant to the blade area of growth hormone susceptibility or adjusting.
The invention still further relates to the agonist of ABP2 or antagonist and uses thereof.Because the activity of the agonist of ABP2 or the expression that antagonist can be regulated ABP2 and/or adjusting ABP2 etc., therefore, the agonist of described ABP2 or antagonist also can be by regulating plant to the susceptibility of growth hormone or regulate plant blade area to the influence of ABP2, thereby reach the purpose of the cost that reduces domestication of plants.
The material of transcribing and translating of the proteic activity of any ABP2 of adjusting, the proteic stability of adjusting ABP2, promotion or the proteic expression of inhibition ABP2, prolongation or minimizing ABP2 albumen effective acting time or promotion or reduction ABP2 all can be used for the present invention, as can be used for regulating the active substance of plant to growth hormone susceptibility or adjusting plant blade area.
The invention still further relates to a kind of method that improves plant, this method comprises regulates ABP2 gene or its homogenic expression in the described plant.
On the one hand, the invention provides and a kind ofly make plant to growth hormone susceptibility height or the big method of blade area, described method comprises: make described plant overexpression ABP2 albumen.
On the other hand, the method that the present invention also provides a kind of blade area that makes plant to reduce, described method comprises: reduce the proteic expression of ABP2 in the described plant (comprise ABP2 albumen is not expressed or low the expression).
After getting the proteic purposes of the described ABP2 of cicada, can adopt several different methods well known in the art to regulate the proteic expression of described ABP2.Be delivered on the target spot such as can carrying ABP2 expression of gene unit (such as expression vector or virus etc.), and make it the ABP2 albumen of expression activity by certain approach.In addition, also can adopt several different methods well known in the art to reduce the proteic expression of ABP2 or make it loss of expression, be delivered on the target spot such as carrying antisense ABP2 expression of gene unit (such as expression vector or virus etc.), make cell or plant tissue not express or reduce and express ABP2 albumen.
As one embodiment of the present invention, the proteic gene of coding ABP2 is cloned in the appropriate carriers by the method for routine, the described recombinant vectors that has foreign gene imported to express in the proteic vegetable cell of described ABP2, make described vegetable cell express ABP2 albumen.Can obtain the proteic plant of overexpression ABP2 by with described vegetable cell regeneration plant.
Preferably, described preparation comprises the method for the big plant of growth hormone susceptibility height or blade area:
(1) changes the proteic encoding gene of the ABP2 of external source over to vegetable cell, tissue or organ (as petal), obtain to be transformed into vegetable cell, tissue, organ or the seed of the proteic encoding gene of ABP2; With
(2) vegetable cell that has changed the proteic encoding gene of external source ABP2 over to, tissue, organ or the seed regeneration plant plant that step (1) is obtained.
As a kind of preferred examples, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains the proteic encoding gene of ABP2;
(s2) vegetable cell, tissue, organ are contacted with Agrobacterium in the step (s1), thereby make the proteic encoding gene of ABP2 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell, tissue, organ or the seed that changes the proteic encoding gene of ABP2 over to; And
(s4) with the vegetable cell in the step (s3), tissue, organ or seed regeneration plant.
Can adopt any suitable conventional means, comprise that reagent, temperature, pressure condition wait the described method of implementing.
Other method that increases ABP2 gene or the expression of its homologous gene is that this area is known.For example, thus can be by drive to strengthen ABP2 gene or its homogenic expression with strong promoter.Perhaps strengthen this ABP2 expression of gene by enhanser (as paddy rice waxy gene first intron, Actin gene first intron etc.).The strong promoter that is applicable to the inventive method includes but not limited to: the Ubi promotor of 35s promotor, paddy rice, corn etc.
In addition, the invention still further relates to and utilize plant the susceptibility of growth hormone or plant blade area proterties tracking mark as a kind of gene transformation plant offspring.In addition, also can utilize the blade area of this gene or growth hormone susceptibility characteristic cue mark as true hybrid in the hybrid seeding process.
In addition, the present invention also provides screening to regulate the method for plant to the potential material of growth hormone susceptibility or adjusting plant blade area.After getting the proteic purposes of the described ABP2 of cicada, can adopt several different methods well known in the art to screen and regulate the potential material of plant growth hormone susceptibility or adjusting plant blade area.
In a kind of optimal way of the present invention, a kind of method of plant to the potential material of growth hormone susceptibility or adjusting plant blade area of regulating of screening is provided, described method comprises: candidate substances is contacted with expressing the proteic system of ABP2, detect candidate substances to the proteic influence of ABP2; If described candidate substances can improve or reduce the proteic expression of ABP2, or promote or the proteic activity of inhibition ABP2, show that then it is to can be used for regulating susceptibility or the adjusting plant blade area of plant to growth hormone.
The material that these preliminary screening go out can constitute a screening storehouse so that people finally can therefrom filter out can be for regulating plant to growth hormone susceptibility or regulate the useful material of plant blade area.
Therefore, the present invention also comprises the material that obtains by described screening method, and described material can be used for regulating the susceptibility or the adjusting plant blade area of plant to growth hormone.Have more using value ground, described material is to promote plant to growth hormone susceptibility, or increases the blade area of plant.
In an example of the present invention, a kind of ABP2 gene available from Arabidopis thaliana is provided, this full length gene 663bp, the polypeptide of coding 220AA, described gene is shown in SEQ ID NO:1.Its encoded protein is shown in SEQ ID NO:2.
The inventor passes through this ABP2 gene of clone, the dna fragmentation structure protokaryon protein expression vector with this ABP2 full-length gene, utilize then [
14C] IAA proved the growth hormone binding ability of ABP2.
In order to prove the purposes of this ABP2, the inventor inserts the library screening from Salk T-DNA and has obtained ABP2 and cross the Arabidopis thaliana strain system of expression and the Arabidopis thaliana strain system that the ABP2 downward modulation is expressed, and wild-type ABP2 is expressed and the strain of downward modulation expression compares research between being with crossing.Studies show that, compare the wild-type Arabidopis thaliana, it is responsive more for the growth effect that growth hormone causes that the mistake that T-DNA inserts sudden change is expressed the Arabidopis thaliana of ABP2 gene, the elongation of ABP2 gene at the root of growth hormone control is described, blade area increases (because of playing a part positive regulating factor in the processes such as ABP2 control cell fission, thereby ABP2 gene overexpression strain system is very responsive for the growth hormone that external source applies, based on this characteristic, the ABP2 gene can be used to regulate the susceptibility of plant for growth hormone, be applied to produce, can reduce the amount of application of field growing element greatly, reduce agricultural cost.And ABP2 also can the render transgenic roots of plants change the blade size according to needs of production for the adjusting of blade area, thus improvement plant biomass and quality.
Utilize the susceptibility of ABP2 generegulation plant for growth hormone (as indolylacetic acid, 2,4 dichlorphenoxyacetic acids), the growth hormone that can the render transgenic plant applies for external source is very responsive.This transfer-gen plant is applied to produce, and can reduce the amount of application of field growing element greatly, thereby reduces agricultural cost.Utilize the function of ABP2 generegulation blade area, can the render transgenic roots of plants change the blade size according to needs of production, thus improvement plant biomass and quality.This discovery not only has theory significance, has also shown the application prospect of ABP2 gene in modern agriculture simultaneously.
Major advantage of the present invention is:
To separate a kind of new albumen first---auxin-binding protein 2 (ABP2), and it is associated with the growth hormone susceptibility of plant, find that ABP2 can be used in to regulate the susceptibility of plant to growth hormone.And this albumen can also be regulated the blade area of plant.Described albumen is with a wide range of applications for the domestication of plants field, and it can make plant more responsive to growth hormone, thereby can reduce the plain amount of application of field growing greatly, can reduce agricultural cost very effectively; Perhaps can change the blade size according to needs of production, thus improvement plant biomass and quality.
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 people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the method for announcing in the following document: Carl W.Dieffenbach and Gabriela S.Devksler eds.PCRPrimer:A Laboratory Manual.Cold Spring Harbor Laboratory Press, 1995.Or the condition of advising according to manufacturer.
Embodiment 1The acquisition of ABP2 gene
The contriver according to one it is predicted may encode the ABP gene unknown cDNA fragment (be the fragment of GenBank accession number AC068602, wherein the record this segmental any function), design following primer:
Forward primer: 5 '-ATGGCGTTAGAACTATGGC-3 (SEQ ID NO:3);
Reverse primer: 5 '-CTACTCCTCCTTCTTCAAC-3 (SEQ ID NO:4).
Phage PCR sieve storehouse method by routine is screened the library of Arabidopis thaliana hypocotyl cDNA (available from Arabidopis thaliana biological study center, ABRC), found the new gene of a coding ABP, described full length gene 663bp, the polypeptide of coding 220AA, the nucleotide sequence of described gene is shown in SEQ ID NO:1, and its encoded protein is shown in SEQ ID NO:2.
Then, the inventor is BLAST with said gene and analyzes in http://www.ncbi.nlm.nih.gov/ website, found the position (No. 1 karyomit(e)) of this gene on arabidopsis thaliana chromosome.The inventor is ABP2 (Auxin Binding Protein 2) with this unnamed gene.Up to the present, do not see any bibliographical information as yet for the functional study and the application thereof of ABP2 gene.
Embodiment 2The clone of ABP2 gene
With reference to " molecular cloning " (J. Sa nurse Brooker, D.W. Russell (U.S.) writes, Huang Peitang etc. translate, Science Press, Beijing, 2002), with the Arabidopsis leaf is raw material, adopt RNA to extract test kit (TrizolIV, Shanghai is along magnificent bio-engineering corporation), extract Arabidopis thaliana RNA according to the product description operation.
Adopting RNA reverse transcription test kit (FSK101, TaKaRa (Dalian) company), is cDNA according to the product description operation with the RNA reverse transcription that obtains.
The reverse transcription reaction condition is:
30℃，10min；42℃，30min；99℃，5min；4℃，5min。
The cDNA that obtains with reverse transcription is a template, adopts following primer to carrying out pcr amplification:
ABP2-1：5’-ATGGCGTTAGAACTATGGC-3’(SEQ?ID?NO：3)；
ABP2-2：5’-CTACTCCTCCTTCTTCAAC-3’(SEQ?ID?NO：4)。
With the described cDNA of pfu polymeric enzymatic amplification, the PCR reaction conditions is:
94 ℃, 3min, 1 circulation; 94 ℃, 49sec, 58 ℃, 40sec, 72 ℃, 50sec, 28 circulations; 72 ℃, 10min, 1 circulation.
Amplified production is through order-checking, and its sequence is shown in SEQ ID NO:1, and 663bp is long altogether not comprise 3 ' end polyA, shows that clone's product is the ABP2 gene.
Embodiment 3The structure of ABP2 protein expression vector and proteic expression
2.1 it is right to design following primer:
ABP2-3 (wherein, underscore is represented Nco I restriction enzyme site):
5’-CATG
CCATGTTTCCATGCAAATCAGCCG-3’(SEQ?ID?NO：5)；
ABP2-4 (wherein, underscore is represented Xho I restriction enzyme site):
5’-CCG
CTCGAGAGTTGTATCATCAGTCCTAAA-3’(SEQ?ID?NO：6)。
With the positive colony that obtains among the embodiment 1 is template, and with the pfu polymeric enzymatic amplification cDNA of (full-length proteins causes the prokaryotic expression host to cause death), the PCR reaction conditions is:
30℃，10min；42℃，90min；95℃，5min；4℃，60min。
The SEQ ID NO:2 sequence that is obtained according to embodiment 1, delete 20 amino acid (full-length proteins causes the prokaryotic expression host to cause death) of N end, thereby obtain the dna fragmentation that encoding sequence length is 600bp, with this fragment cloning to pET32C carrier (Novagen, Inc.Madison, Wisconsin, the U.S.) in, and add their confirmation, thereby construct the protokaryon protein expression vector of ABP2 gene through order-checking.
2.2 CaCl with routine
2Facture is (referring to " molecular biology experiment technology ", Hao Fuying etc. write, the BJ University Press, Beijing, 1998) preparation e. coli bl21 (DE3) (Novagen, Inc.Madison, Wisconsin, the U.S.) competence is by the electric shock conversion method prokaryotic expression carrier transformed into escherichia coli that embodiment 2.1 is constructed.
The picking positive colony, put into LB nutrient solution (the Tryptones 10g/L that contains kantlex (Km), yeast extract 5g/L, NaCl 10g/L, kantlex 50mg/L transfers pH to 7.5 with NaOH) in, in 37 degrees centigrade of incubators, utilize IPTG (final concentration 1mM) to induce the proteic expression of ABP2, separating and purifying protein, and then be used to carry out the growth hormone binding analysis.
Embodiment 4ABP2 crosses the evaluation of expressing strain system and downward modulation strain system
The T-DNA that the present invention has searched for ABP2 inserts mutant (available from Salk T-DNA insertion mutant library, available from SALK institute, or referring to website www.signal.salk.edu), the plant of finding accession number Salk_022981 is that ABP2 crosses expression strain system (being called abp2-d), and the plant of accession number Salk 657-F8 is the downward modulation strain system (being called abp2) of ABP2.
At first, show that with separating T-DNA inserts the insertion site of mutant and have only one in mutant gene by resistance screening than analyzing the result; Secondly, the information in the insertion site of the corresponding mutant that provides according to the SALK website, inserting two ends, site design primer ABP2-P1 and ABP2-P2, the detection primer LBa1 that provides with the SALK website detects by PCR and can determine that the insertion site of T-DNA is in ABP2 gene inside respectively.By above-mentioned 2 proofs, the mutable site of mutant only limits to the ABP2 gene.
Particularly, above-mentioned strain to the ABP2 down regulation of gene expression is abp2, and the strain system that ABP2 genetic expression is raised verifies that experimental technique and step are as follows:
1, the extraction of genomic dna
Extract arabidopsis thaliana genomic dna with conventional CTAB (cetyl trimethylammonium bromide) method.
2, reverse transcription
The reverse transcription reaction condition is:
30℃，10min；42℃，30min；99℃，5min；4℃，5min。
The cDNA that obtains with reverse transcription is a template, adopts following primer to carrying out pcr amplification.
3, pcr amplification and detection
Primer on the T-DNA (LBa1):
5’-TGGTTCACGTAGTGGGCCATCG-3’(SEQ?ID?NO：7)；
ABP2-P1(SEQ?ID?NO：8)：
5’-AATTATCTCAGCTTTCTCCAAC-3’；
ABP2-P2(SEQ?ID?NO：9)：
5’-TGAATACCATACGTTGACAATG-3’。
Use the RT-PCR method, qualification result abp2 is the strain system of ABP2 down regulation of gene expression; The strain system that abp2-d raises for ABP2 genetic expression.And the mutable site of mutant only limits to the ABP2 gene.
Arabidopis thaliana in the controlled environment chamber, every day was in 16 hours dark culturing of 22 ℃ of following illumination cultivation 8 hours.
Embodiment 5Susceptibility experiment to several growth hormone of different concns
Get the seed of WT, abp2 (ABP2 down regulation of gene expression), three strain systems of abp2-d (ABP2 genetic expression rise), after 16-18 minute, use rinsed with sterile water three times through 20% drift ice+1%Tween 20 concussion rinsings.Seed after disinfecting places on the MS media surface, 4 degrees centigrade of vernalization 48 hours, after vertically cultivating 3 days in the controlled environment chamber, choose the good sprigging of growth conditions to containing 0.01nM indolylacetic acid (IAA), 2,4 dichlorphenoxyacetic acids (2,4-D), naphthylacetic acid (NAA), or 1mM IAA, 2,4-D, NAA, and do not contain on the MS substratum of any interpolation growth hormone and vertically cultivated again three days, observe phenotype.Elongation has restraining effect to the growth hormone of high density to the main root of plant, and the growth hormone of lower concentration has promoter action to the main root elongation of plant, so the root length of plant is the important indicator of mensuration plant to growth hormone susceptibility itself.
The inventor has chosen the mutant plant that the growth hormone of two concentration (1um and 0.01nm) is handled wild-type plant and ABP2 respectively, observes plant whether the susceptibility of growth hormone can be changed owing to the expression amount of ABP2.In the experimental result, the plant phenotype the results are shown in Table 1 as shown in Figure 1.Wherein, root length is the per-cent of the length (being made as 100%) of main root under the IAA disposition with respect to the plant main root length that is untreated relatively.
Table 1 ABP2 influences the susceptibility of plant to growth hormone
Because of last table as seen, compare with wild-type, abp2 the shortening than wild-type of root under high density IAA condition to few, the short trend of the root of abp-d is then more violent, illustrate that ABP2 reduces expression and makes plant lose part susceptibility to IAA, promptly poor to the susceptibility of IAA than wild-type.The plant of overexpression ABP2 then has higher IAA susceptibility.
In like manner, the minimizing of ABP2 makes plant to 2, and the susceptibility of 4-D also reduces, and the increase of its expression also can cause 2, the increase of 4-D susceptibility.
Embodiment 6The variation of ABP2 generegulation blade area
Choose the seed of two strain systems of WT, abp2-d (ABP2 genetic expression rise), after 16-18 minute, use rinsed with sterile water three times through 20% drift ice+1%Tween20 concussion rinsing.Seed after disinfecting places on the MS media surface, and 4 degrees centigrade of vernalization 48 hours is vertically cultivated in the controlled environment chamber after 5 days and transplanted to soil, and the seedling when getting 20 days carries out phenotype to be observed and the blade electron-microscope scanning.Phenotype as shown in Figure 2, the experiment statistics result is as shown in Figure 3.
As seen from the figure, the lotus throne leaf of abp2-d (ABP2 genetic expression rise) strain system all enlarges markedly than the area of wild-type since the 2nd, illustrates that the ABP2 gene plays the effect of positive regulatory factor in blade increase process.
Embodiment 7The structure of ABP2 up-regulated expression plant and downward modulation plant
The SEQ ID NO:2 sequence that is obtained according to embodiment 1, the positive dirction subclone to the p35S1301 carrier (referring to Liu W, Xu ZH, Luo D, Xue HW. (2003) .Roles of OsCKI1, a rice caseinkinase I, in root development and plant hormone sensitivity.Plant is J.36, in multiple clone site 189-202), construct the sense expression vector of ABP2; With this sequence in the other direction subclone to the multiple clone site of p1300 carrier, construct the antisense expression vector of ABP2, utilize conventional Agrobacterium-mediated Transformation method to change in the Arabidopis thaliana plant more respectively, make up up-regulated expression gene plant and the downward modulation expressing gene plant of ABP2.
In ABP2 up-regulated expression plant, lotus throne leaf phenotype is consistent with the abp2 phenotype, and the trend of increase is all arranged.
Embodiment 8The mutant gene of ABP2 gene and function thereof
The mutant gene Mut.1 of ABP2 gene to preparation, method according to embodiment 7 makes up proteic transfer-gen plant of overexpression ABP2 and low transfer-gen plant of expressing, measure the susceptibility of described plant with the method for embodiment 5 to several growth hormone of different concns, and compare with wild-type, the result shows with the transfer-gen plant of described mutant gene preparation the susceptibility of growth hormone is significantly higher than wild-type, and blade area is significantly greater than wild-type.
The inventor finds that in Arabidopis thaliana ABP2 gene functional research process gene downward modulation and rise plant phenotype are obvious, and sex character can heredity.And the regulating effect of ABP2 gene also can be used for other economic plants for example quality-improving and the production of paddy rice, tobacco, melon and fruit, vegetables, rape etc.
Discuss:
Arabidopis thaliana has the advantages that as a kind of typical model plant growth cycle is short, individual morphology is little and genome is little, has become very important instrument in molecular biology of plants research.Arabidopis thaliana is a kind of experimental model of preciousness, and understand the total primary process of higher organism for the functions peculiar of understanding photosynthesis and other plant and on molecule and cell levels significant.
Though below only be experiment material, be protein expression vector, as the transgenic line of gene overexpression and downward modulation technical scheme of the present invention set forth with T-DNA insertion mutant with the BL21 plasmid with the Arabidopis thaliana, but according to of the present invention open, adopt other expression vectors and transgenic method can construct ABP2 overexpression and downward modulation (reaching the purpose that gene raises or reduces) transgenic line equally easily, this is conspicuous for a person skilled in the art.Therefore, adopt other expression vectors and transgenic method to construct overexpression and the downward modulation transgenic line of the ABP2 of other kind plants, should belong to scope of the present invention equally.
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
Claims (4)
1. the purposes of a peptide species, this polypeptide is selected from down group:
(a) polypeptide of the aminoacid sequence shown in the SEQ ID NO:2;
(b) aminoacid sequence shown in the SEQ ID NO:2 is formed through replacement, disappearance or the interpolation of 1-5 amino-acid residue, and have (a) described polypeptide function by (a) polypeptides derived;
Be used for:
Regulate susceptibility or the cress blade area of cress to growth hormone; Or
The cress that preparation is high to growth hormone susceptibility, or the cress that the preparation blade area increases or blade area reduces.
2. purposes as claimed in claim 1 is characterized in that described cress is: Arabidopis thaliana.
3. method for preparing the big cress of growth hormone susceptibility height or blade area is characterized in that described method comprises:
(1) is that the polynucleotide of the polypeptide of SEQ ID NO:2 change cress cell, tissue or organ over to encoding amino acid sequence, obtains to be transformed into vegetable cell, tissue or the organ of described polynucleotide; With
(2) vegetable cell that has changed described polynucleotide over to, tissue or the neomorph that step (1) is obtained becomes plant.
4. method as claimed in claim 3 is characterized in that, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains the polynucleotide that encoding amino acid sequence is the polypeptide of SEQ ID NO:2;
(s2) cress cell, tissue or organ are contacted with Agrobacterium in the step (s1), thereby make described polynucleotide change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell, tissue or the organ that has changed described polynucleotide over to; And
(s4) vegetable cell, tissue or neomorph in the step (s3) are become plant.
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| Title |
|---|
| Clay Carter et al.Arabidopsis thaliana contains a large family of germin-like proteins: characterization of cDNA and genomic sequences encoding 12 unique family members.《Plant Molecular Biology》.Springer Netherlands,1998,第38卷(第6期),全文. * |
| Ecker J.R.et al.AAF79304.《Genbank》.2000,全文. * |
| Ecker J.R.et al.AC068602.《Genbank》.2000,全文. * |
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