CN102321163B - Sea island cotton lipid transfer protein and application in fiber improvement thereof - Google Patents
Sea island cotton lipid transfer protein and application in fiber improvement thereof Download PDFInfo
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- CN102321163B CN102321163B CN201110305355.8A CN201110305355A CN102321163B CN 102321163 B CN102321163 B CN 102321163B CN 201110305355 A CN201110305355 A CN 201110305355A CN 102321163 B CN102321163 B CN 102321163B
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Abstract
The invention provides a new lipid transfer protein gene lpt and coded LTP protein polypeptides thereof which can increase the quality of cotton fibers, and a method for producing the LTP protein by recombination technology. The invention also discloses a function and an application of the lpt gene in the improvement of cotton fiber length and strength.
Description
Technical field
The present invention relates to a kind of albumen with lipid transfer function, in cotton fiber, the encode gene of this albumen of specifically expressing can improve the staple length of cotton variety.
Background technology
The fibrous quality that utilizes genetic engineering means to improve cotton comprises that the staple length of cotton is the important directions of current Molecular breeding in upland cotton:.
Studies of Lipid-transfer Protein (LTP) is small molecule basic protein, accounts for 4% left and right of plant total soluble protein.LTP is a kind of multi-functional protein family, the member of LTP protein family has not only participated in the transportation of phosphatide between microbial film, but also has brought into play important effect in the bioprocesss such as formation, the reproductive development of plant and the transduction of signal of microbial film, cutin and lipid.Recent research shows, LTP albumen has signal peptide, can and be positioned cell walls from cell internal secretion to extracellular.The cell walls positioning function of LTP albumen has vital role for the disease resistance response of plant, degeneration-resistant border reaction (high temperature, high salt, drought stress) and the formation of microbial film and lipid.
In the Fibre Development process of cotton, important effect has been brought into play in lipid metabolism in cotton fibre rapid elongation.In cotton embryo culture base, add the Fiber elongation that the lipoid substance of carbon chain lengths more than 20 can promote fiber.In tobacco, the research of lipid transport protein gene shows, lipid transport protein has the function of loose cell walls.But up to the present, which kind of lipoid substance more than albumen participation transportation C20 is also unclear, and the staple length of utilizing genetic engineering means to improve cotton is not also reported.
Summary of the invention
The object of this invention is to provide a kind of new lipid transfer protein gene with and fragment, analogue and derivative.
Another object of the present invention is to provide the polynucleotide of these polypeptide of coding.
Another object of the present invention is to provide produces the method for these polypeptide and the purposes of this polypeptide and encoding sequence, thereby especially promotes the purposes aspect cotton fiber extension improving the expression level of cotton lipid transfer protein in protoblast.
The inventor is through extensive and deep research, adopt first molecular cloning method to separate from the cDNA of sea island cotton embryo specifically expressing and obtained new ltp gene, and confirm that by experiment it has the elongate fiber of promotion function, can cause cotton fiber extension after ltp gene transferred plant.Complete on this basis the present invention.
The present invention includes following content:
1, provide a kind of new isolated lipid transfer protein (being designated hereinafter simply as " LTP " protein), comprised:
There is polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is selected from lower group: the polypeptide (a) with 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 (preferably 1-20) amino-acid residue, and have promote cotton fiber extension function LTP protein active by (a) derivative polypeptide.
The polynucleotide that 2, coding aforementioned polypeptides is provided, are selected from:
(a) the encode polynucleotide of above-mentioned LTP protein and peptide; (b) polynucleotide complementary with polynucleotide (a).Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2.More preferably, the sequence of these polynucleotide is to be selected from the one of lower group: the sequence (a) with 1-363 position in SEQ ID NO:1; (b) there is the sequence of 1-400 position in SEQ ID NO:1.
3, provide the carrier that contains above-mentioned polynucleotide, and transformed by this carrier or the host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide.
4, provide the method for preparing above-mentioned LTP protein and peptide, the method comprises: (a) under conditions suitable for the expression, cultivate the above-mentioned host cell that is converted or transduces; (b) from culture, isolate the activated polypeptide of tool.
5, the purposes of above-mentioned polynucleotide and LTP protein and peptide is provided, that is, and the application aspect the cotton variety of cultivation raising cotton fiber length.
6, provide a kind of change Studies of Lipid-transfer Protein, to improve the method for cotton fiber quality, it comprises step:
(1) provide the Agrobacterium of carrying expression vector, the polynucleotide that described expression vector contains sequence shown in SEQ ID NO:1, described LTP is selected from lower group:
(a) there is 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 promote cotton fiber extension by (a) derivative polypeptide;
(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thereby make lpt gene coded sequence proceed to vegetable cell, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or tissue or the organ of the DNA encoding sequence that proceeds to lpt gene;
(4) vegetable cell in step (3) or tissue or neomorph are become to plant.
In the present invention, term " LTP protein ", " LTP polypeptide " or " lipid transfer protein " are used interchangeably, all refer to have LTP aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They comprise the LTP protein that contains or do not contain initial methionine.
As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.
As used herein, " LTP albumen or the polypeptide of separation " refers to that LTP polypeptide does not basically contain natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be purified LTP protein with the purified technology of protein of standard.
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 for example, to produce from protokaryon or eucaryon host (, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to recombinant production scheme, polypeptide of the present invention can be glycosylated, can be maybe nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analogue of LTP protein.As used herein, term " fragment ", " derivative " refer to and substantially keep biological function or the active polypeptide that natural LTP protein of the present invention is identical with " analogue ".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 (preferably conservative amino acid residue), and the amino-acid residue of such replacement can not be also to be encoded by genetic code, or (ii) in one or more amino-acid residues, there is the polypeptide of substituted radical, or (iii) mature polypeptide and another compound merge the polypeptide forming, 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 of this polypeptide of purifying).According to described herein, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " LTP polypeptide " refers to the polypeptide of the SEQ ID NO.2 sequence with lipid transfer activity.This term also comprises having and variant form LTP protein 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) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally in 20 at C-terminal and/or N-terminal, being preferably in 10, is more preferably in 5) amino acid.For example, in the art, while replacement with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, the function of adding or several amino acid and conventionally also can not change protein at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of LTP albumen.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, albumen that can be coded with the DNA of ltp DNA hybridization under high or low rigorous degree condition.The present invention also provides other polypeptide, as the fusion rotein that comprises LTP polypeptide or its fragment.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of LTP polypeptide.Conventionally, this fragment have LTP peptide sequence at least about 10 continuous amino acids, conventionally 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 LTP protein or polypeptide.The difference of these analogues and natural LTP polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.Induce variation body can obtain by various technology.
In the present invention, " LTP protein conservative property variation polypeptide " refers to, compared with the aminoacid sequence with SEQ ID NO:2, have 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid are replaced by the similar or close amino acid of character and form polypeptide at the most best.These conservative property variation polypeptide preferably carry out amino acid substitution according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | 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 SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " refers to that coding has the protein of SEQ ID NO:2 in the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in 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 (with optional additional code sequence) and the non-coding sequence of mature polypeptide.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and can be also 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 the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.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 in fact the function of polypeptide of its coding.
The invention still further relates between above-mentioned sequence hybridization and two sequences and have at least 50%, preferably at least 70%, the more preferably polynucleotide of at least 80% homogeny.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 " refers to: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization 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 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 SEQ ID NO:2.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better 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 to separate the polynucleotide of coding LTP protein.
Polypeptide in the present invention preferably provides with the form separating with polynucleotide, is more preferably purified to homogeneous.
LTP Nucleotide full length sequence of the present invention or its fragment can use the method for synthetic, pcr amplification method or recombination method to obtain conventionally.For example first carrying out complete sequence according to the sequence of SEQ ID NO:1 synthesizes.
For pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and by the LTP Nucleotide full length sequence of synthetic or its fragment as template, amplification and must be about sequence.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.
In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic multiple small segments, and then connect and can obtain the fragment that sequence is very long.
At present, can obtain the DNA sequence dna of code book invention albumen (or its fragment and derivative) completely by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (or as carrier) and cell.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or ltp gene coded sequence, and produce the method for polypeptide of the present invention through recombinant technology.
By conventional recombinant DNA technology (Science, 1984; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to the LTP1 polypeptide of expression or Restruction.In general there are following steps:
(1). with the polynucleotide (or varient) of coding of the present invention LTP polypeptide, or with the recombinant expression vector conversion that contains these polynucleotide or the suitable host cell of transduceing;
(2). the host cell of cultivating in suitable substratum;
(3). separation, protein purification from substratum or cell.
In the present invention, LTP protein polynucleotide sequence can be inserted in 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 copy in host and stablize, any plasmid and carrier can use.A key character of expression vector is conventionally to contain replication orgin, promotor, marker gene and translation controlling elements.
Method well-known to those having ordinary skill in the art can be used for building containing ltp genes encoding 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 be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.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 the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell; Insect cell etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe insert enhancer sequence in carrier time.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene conventionally.
Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl
2method processing, step used is well-known in this area.Another kind method is to use MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packing etc.Conversion of plant also can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, for example leaf dish method.Can use ordinary method regeneration plant for the vegetable cell, tissue or the organ that transform, thereby obtain the plant that lipid transfer protein resistance improves.
The transformant obtaining can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.
Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cytolemma.If needed, 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 processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
On the other hand, the present invention also comprises that DNA or the polypeptide of its fragment coding to ltp gene have specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Preferably, refer to that those can be combined with LTP protein gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.In the present invention antibody comprise those can in conjunction with and suppress the molecule of LTP protein, also comprise that those do not affect the antibody of LTP protein function.The present invention also comprise those can with modify or the antibody of being combined without the LTP protein gene product of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the LTP protein gene product of purifying or its have antigenic fragment, can be applied to the generation of animal with induction polyclonal antibody.Similarly, expression LTP protein or its cell with antigenic fragment can be used to immune animal and produce antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize fragment or the functional zone of LTP protein gene product, obtains by routine immunization technology.These fragments or functional zone can utilize recombination method preparation or utilize Peptide synthesizer synthetic.The antibody of being combined with the unmodified form of LTP protein gene product can for example, carry out immune animal and produce by the gene product of producing in prokaryotic cell prokaryocyte (E.Coli); The antibody (as the albumen of glycosylation or phosphorylation or polypeptide) of being combined with posttranslational modification form, can for example, carry out immune animal and obtain by the gene product producing in eukaryotic cell (yeast or insect cell).The antibody of anti-LTP protein can be used for detecting the LTP protein in sample.
The available LTP protein of production or the polypeptide immune animal of polyclonal antibody, as rabbit, mouse, rat etc.Multiple adjuvant can be used for strengthening immune response, includes but not limited to freund's adjuvant etc.
The invention still further relates to the testing method of quantitative and detection and localization LTP protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.
In a kind of detection sample, whether having LTP method of protein is to utilize the specific antibody of LTP protein to detect, and it comprises: sample is contacted with LTP protein-specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in sample LTP protein.
Part or all of polynucleotide of the present invention can be used as probe and is fixed on microarray (microarray) or DNA chip (being called again " gene chip "), for the expression analysis of gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect the transcription product of LTP protein with the special primer of LTP protein.
Research of the present invention is found, the lipid transfer protein LTP of cotton embryo specifically expressing is at cotton fiber rapid elongation predominant expression, and in the epidermal hair cell of embryonic epidermis and plant specifically expressing, LTP participates in lipoid substance more than transportation C20, strengthens expression ltp gene and can improve cotton fiber length in cotton embryo.
In an example of the present invention, a kind of polynucleotide of separation are provided, its coding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2.Polynucleotide of the present invention are from adopting hybridization in situ technique to separate from the sea island cotton cDNA library storehouse building.Its sequence is as shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 400 bases, and its open reading frame is positioned at 1-363 position, and coding total length is 120 amino acid whose LTP protein (SEQ ID NO:2).
LTP protein of the present invention has following characteristic: A) LTP albumen specifically expressing in cotton elongated fibers, can transport the lipid acid (>=C20) of long carbochain; B) lipid transfer protein fibers for improved definite functions, in cotton fiber, this gene of overexpression can significantly promote elongate fiber.
Confirm through experiment, turn the cotton plants of ltp gene compared with the control, the cotton fiber quality of transfer-gen plant has obtained obvious improvement, and staple length increases 2.7-3.5mm; Fibre strength increases 0.3-3.3CN/tex compared with the control.
LTP protein provides new approach for promoting vegetable fibre to extend, thereby has huge application prospect.For example, by ltp gene is imported to variety of crops (cotton), change the staple length of existing cotton variety, can obtain the cotton variety of crops of lipid transfer protein, solve the practical problems existing in agriculture production.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of lpt gene genetic conversion carrier.In figure, LB, RB are respectively the left and right border sequence of T-DNA.CaMV35S is tobacco mosaic virus (TMV) 35S promoter.NOS is terminator.NPTII is kalamycin resistance gene.Fbp is petunia embryo specific expression promoter (seeing embodiment 3).
Fig. 2 turns lpt gene cotton seedling (T0) blade PCR to detect figure.M is DL2000 molecular weight marker.The molecular weight of mark is respectively 2000,1000,750,500,250 and 100bp; The positive contrast of P (lpt plasmid); 1-7 is the transgene cotton seedling (seeing embodiment 4) through transforming lpt gene.
Fig. 3 is the Southern hybridization analysis situation that turns the cotton plants of lpt gene.(P:lpt gene plasmid DNA; 1-6: transfer-gen plant (seeing embodiment 5).
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.Other side of the present invention, because technology herein discloses, is apparent to those skilled in the art.
The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The CDNA fragment clone of embodiment 1 lipid transfer protein
(1) sea island cotton embryo organizing specific cDNA's is synthetic
The synthetic method enforcement that adopts the cDNA library of Clontech company to build test kit of sea island cotton embryo organizing specific expression cDNA, the concrete steps of the method are as follows.
Choose embryo tissue that sea island cotton kind Pima-90 the blooms latter 2 days total RNA for extracting cotton.Take 0.5g sea island cotton embryo, become fine powder by liquid nitrogen grinding, be sub-packed in the eppendorf pipe of two 1.5ml.Every pipe adds 1ml TRIZOL firmly to shake and make to mix, and under room temperature, places 5min.Then at 4 ℃, centrifugal 10min under the condition of 12,000g, sucks supernatant liquor in the eppendorf pipe of clean 1.5ml.Every pipe adds 0.2ml chloroform, uses forced oscillation 15s, and room temperature is placed 2～3min.Then at 4 ℃, 12, centrifugal 15min under the condition of 000g.Supernatant liquor is transferred in the eppendorf pipe of clean 1.5ml, added equal-volume Virahol, put upside down and mix, room temperature is placed 10min.At 4 ℃, centrifugal 10min under the condition of 12,000g.Abandon supernatant, add 1ml 75% washing with alcohol, at 4 ℃, the centrifugal 5min of 7,500g.Abandon supernatant, after drying at room temperature 15-20min, be dissolved in (55～60 ℃ of water-bath 10min fully dissolve RNA) in appropriate RNA-free water.The RNA obtaining is for cDNA building-up reactions.
Be mRNA according to the method for the mRNA purification kit of QIAGEN company by total RNA purifying, then carry out strand cDNA according to the method providing in ClonTECHTM test kit at once and synthesize with double-stranded cDNA synthetic.
(2) adopt PCR method to separate lipid transfer protein gene
2 of design pcr amplification primers, are respectively P1 and P2, and utilizing the cDNA being obtained take step (1) at 2 primers is template amplification ltp full-length gene.The reaction system of PCR is: 10 × PCR buffer, 3 μ L; DNTPs (dATP, dTTP, dGTP) 2 μ L; MgCl
2, 2 μ L; The each 2 μ L of primer P1 and P2; Taq enzyme, 2 units; The cDNA of cotton fiber tissue, 100ng, adds sterilized water to supply 30 μ L systems, mixes rapidly rear centrifugal.
Pcr amplification condition completes according to following program:
94℃，3min，1cycle；
94℃，1min；50℃，1min，72℃，1min，36cycles；
72℃，3min，1cycle。
After reaction finishes, PCR product carries out electrophoretic separation on 1% sepharose.Reclaim reagent and reclaim PCR fragment according to the PCR product of Invitrogen company.
(3) sequence verification of the cDNA fragment of lipid transfer protein gene
PCR fragment is connected with the PGEM-18T of Takara company carrier.Connect product and transform bacillus coli DH 5 alpha.Identify positive colony according to the PCR method of step (2).The escherichia coli plasmid of the extracting positive also carries out sequencing.
The synthetic of the LTP protein gene of embodiment 2 lipid transfer proteins
According to the completed nucleotide sequence containing 400bp coding region, first divide 8 sections respectively according to normal chain and secondary chain-ordering, synthesize respectively the about 150-200bp of length, there is the single stranded oligonucleotide fragment of sticky end.Normal chain and each 8 the complementary single stranded oligonucleotide fragments one to one of secondary chain are annealed respectively, form 8 double chain oligonucleotide fragments with sticky end.Mix double chain oligonucleotide fragment, through T
4dNA ligase catalysis is assembled into a complete ltp gene.The nucleotide sequence that this synthetic DNA fragmentation contains 1-363 position in SEQ ID NO:1, and the two ends of synthetic gene are containing XbaI and SacI site.
Be XbaI and SacI site ltp gene by 5 ' and 3 ' end restriction enzyme site of above-mentioned synthetic, for the structure of the LTP protein gene plant expression vector of lipid transfer protein below.
The structure of the LTP protein gene plant expression vector of embodiment 3 lipid transfer proteins
The concrete grammar of the LTP protein gene plant expression vector construction of lipid transfer protein is as follows:
HindIII and EcoRI double digestion for A.pBI121 and pCAMBIA2301, be connected into pCAMBIA2301 by pBI121 with the fragment of p35S-GUS-Nos-ter, forms intermediate carrier p35S-2301-GUS;
B. with XbaI and the two ltp genes of cutting p35S-2301-GUS and above-mentioned synthetic of SacI, replace the GUS of the corresponding restriction enzyme site of p35S-2301-GUS with ltp, thereby obtain the ltp gene plant expression vector (Fig. 1) of lipid transfer protein.Being proceeded to Agrobacterium is in LBA4404 again, for converting cotton.
The Agrobacterium that contains goal gene is rule on the LB of suitable resistance substratum, cultivate 2 days at 28 ℃, picking list colony inoculation is in the LB liquid nutrient medium of 50ml (concentration is 50mg/L kantlex).Substratum, at 200rpm, is cultivated 42-48 hour on shaking table under 28 degree conditions.In the time that OD600 reaches between 0.3-0.8, the centrifugal collection thalline of 3000g.Utilize 1/2MS liquid nutrient medium by collect mycelium dilution between OD600=0.2-0.4.
(1) the common cultivation of explant and Agrobacterium: the hypocotyl of getting above-mentioned 5-6 days seedling age, be cut into and be about the long segment of 0.5-0.8cm, in the Agrobacterium bacterium liquid of 1/2MS liquid nutrient medium dilution, soak 15 minutes, take out, blot with filter paper after the bacterium liquid on hypocotyl surface, proceed to and do not add in any antibiotic solid CB2.1 substratum, every ware 30-40 piece hypocotyl is cultivated altogether 50-60 hour at 25 ℃.
(2) induction of callus (callus): the hypocotyl of cultivating altogether with the aseptic washing that contains cef 250mg/l 3-4 time, hypocotyl is blotted with aseptic filter paper after the water on hypocotyl surface, hypocotyl is proceeded on CB3.1 substratum to 28 ℃-30 ℃ (16h light/8h secretly circulate illumination cultivation) and cultivate 3-4 week.Little callus greatly can be in sight in the time of 3 weeks, and then, succeeding transfer culture every other month, until callus reaches suitable quantity.
(3) induction of embryo callus and somatic embryo: the callus of some amount is transferred in CB4 substratum, 28 ℃-30 ℃ (16h light/8h secretly circulate illumination cultivation) cultivates, succeeding transfer culture every other month, until somatic embryo occurs.
(4) sprouting of somatic embryo: somatic embryo is forwarded in CB5 substratum, allow them sprout and grow up to seedling.
(5) acquisition of transfer-gen plant: (2-4cm is long to shift seedling, the leafed spray of tool) forward in CB6 substratum (needing larger container) to, 28 ℃-30 ℃ (16h light/8h secretly circulate illumination cultivation) growths 4 months.The transfer-gen plant with better root system is directly forwarded in the little basin that contains wet soil, and in culturing room, 28 ℃-30 ℃ (16h light/8h is dark, circulation illumination cultivation) cultivated 2 weeks, then, shifts these little basins in greenhouse.Water every day to transfer-gen plant, and it is applied fertilizer etc. until cotton boll maturation.Then, collect seed, preserve seed for 4 ℃.
(7) genetically modified PCR identifies:
Obtain plant genomic DNA by the method for a small amount of extracting plant genomic DNA, carry out pcr amplification take the total DNA of 1.5 μ l as template with primer (P1/P2), 36 sterile cotton flower seedlings are detected altogether, wherein have 17 positive plants (T0) that are detected specific band, part plant PCR product electrophoresis result as shown in Figure 2.
Utilize Southern blot evaluating objects gene integration in sea island cotton genome,
(1) enzyme of contrast and transfer-gen plant gene DNA group is cut and electrophoresis
Contrast and transfer-gen plant gene DNA group are cut after 24-48h in 37 ℃, BamHI enzyme according to the enzyme of table 2 system of cutting, get 5 μ L product electrophoresis, under UV lamp, detect whether enzyme cuts entirely, it is even dispersion shape band completely that enzyme is cut on swimming lane, then enzyme is cut to product and on lyophilize whizzer, be concentrated into 50 μ L, add after 6 μ L Loading buffer, at 1% agarose gel electrophoresis.First use 120V electrophoresis 20min, when DNA all runs out of behind point sample hole, voltage is adjusted to 2V/cm, electrophoresis 5h.
Table 2 cotton DNA restriction enzyme enzyme system.
| Composition | Volume |
| Cotton genomic dna | 60μg |
| 10 × restriction enzyme Buffer | 20μL |
| Restriction enzyme (15U/ μ L) | 5μL |
| ddH 2O | Cumulative volume to 200 μ L |
(2) transferring film
Electrophoresis finishes rear taking-up gel, cuts unnecessary blob of viscose, measures length and wide, cuts the upper right corner and serves as a mark, and then puts into depurination liquid 10-15min and carries out depurination treatment, the now its colour changed into yellow of tetrabromophenol sulfonphthalein.
Blob of viscose is transferred in a new pallet, by rinsed with deionized water 2 times, then sample is transferred in sex change liquid at least 50min and done DNA denaturing treatment.
After sex change finishes, blob of viscose is transferred in another new pallet, uses rinsed with deionized water 2 times, then sample is transferred to 30min in neutralizer.
Glass plate is placed on ceramic square plate, and spreads two Whatman 3MM filter paper as siphon bridge, adds enough transfering buffering liquid 10 × SSC in dish, drives the bubble in siphon bridge and sheet glass out of with glass stick.By a long and wide and gel nitrocellulose filter of the same size, cut one jiao, at 10 × SSC solution impregnation 3-5min.
By gel face down, be positioned over siphon bridge central authorities, drive the bubble between filter paper and blob of viscose out of with glass stick.Nitrocellulose membrane is covered on gel, and makes corner cut alignment.With four limits of Parafilm envelope gel, in case short circuit.On film, put three layers of 3MM filter paper, and drive bubble out of with glass stick.Multi-layer absorbent paper is covered on filter paper, and the sheet glass of a suitable size is put at top, and above sheet glass, places 500g weight, and transferring film 3-5d should constantly change thieving paper during this time.After transferring film completes, take off colloid, with the EB 10min that dyes, and carry out ultraviolet detection, check transferring film effect.Clamp nylon membrane with 3MM filter paper, be placed in room temperature 30min and dry, be then placed in after 80 ℃ of fixing 2-3h, nylon membrane is wrapped in tin pool paper, save backup in 4 ℃.
(3) probe preparation
According to promoting the relatively non-conservative zone design special primer of elongate fiber gene 3 ' end, the probe that the fragment (2800-3200) of amplification 400bp length detects as Southern blot.
Probe mark step is as follows: 20 μ L Cross-linker, 80 μ L water (providing in test kit) are provided and are diluted to working concentration.Working fluid can be preserved a week at 2-8 ℃.10ng/ μ L will be diluted to for the DNA of mark with the water providing in test kit.Salt concn in nucleic acid must be low as far as possible, is no more than 50mmol/L.Get DNA sample that 10 μ L diluted in micro-Eppendorf centrifuge tube, sex change 5min in boiling water bath.Immediately sample is placed in to cooled on ice 5min, centrifugal gently on Eppendorf centrifuge, mixture is collected to the pipe end.In cooling DNA sample, add 10 μ L Reaction buffer, mix lightly, be placed on ice.
Add 2 μ L Labeling reagent, mix lightly.Add 10 μ L Cross-linker working fluids, thoroughly mix, centrifugal gently on Eppendorf centrifuge, mixture is collected to the pipe end.Reaction mixture is in 37 ℃ of incubation 30min.Probe after mark can use immediately, or is placed in and preserves 2h on ice.Long-time preservation need to add 50% glycerine (v/v).
(4) hybridization:
Get volume required hybridization buffer, be preheated to 55 ℃.Buffer consumption is generally 0.25mL/cm2 Hybond membrane.For large Hybond membrane, Buffer consumption can reduce to 0.125mL/cm2; Film is placed in to Hybridization buffer, at least 15min of prehybridization in hybrid heater; In prehybridization Buffer, add the probe that mark is good, in general every milliliter of Buffer, add 5-10ng probe; In 55 ℃ of hybrid heaters, hybridize and spend the night.Can be by changing hybridization temperature (50-75 ℃) regulation and control rigor.
(5) wash film:
Primary wash buffer is preheated to 55 ℃, and its consumption is 2-5mL/cm2; Carefully tunica fibrosa is transferred in Primary wash buffer to 55 ℃ of rinsing 10min; With Primary wash buffer at 55 ℃ of rinsing 10min; Tunica fibrosa is transferred to a clean container, adds excessive Secondary wash buffer, at room temperature rinsing 5min; With Secondary wash buffer rinsing 5min again.
(6) detect:
Remove unnecessary Secondary wash buffer on film, and film is placed in above the smooth SanranWrap of one deck, sample is faced up; Detection reagent is dripped on film (30-40 μ L/cm2), place 2-5min, remove unnecessary detection reagent;
At the clean filter paper of X-ray folder middle berth one deck, screen before intensifying screen in filter paper underneath; Film is wrapped with preservative film, faced up and be placed on filter paper, fix with adhesive tape; In darkroom, getting an X-ray is placed on Hybond membrane.After putting intensifying screen, shield, the mating plate that closes folder, seals up adhesive tape again, exposure 2h left and right; In darkroom, developing solution is poured in large square plate, taken out X-ray, put in developing solution, rock gently liquid, development 3-5min manifests to black exposure band, immediately X-ray is transferred in stop bath, and photographic fixing 20min left and right, takes out X-ray and put in flowing water and rinse and spend the night;
The Southern hybridization analysis situation that turns lpt gene cotton plants is shown in Fig. 3.In Fig. 3,1-6 is for turning lpt gene plant, and the positive plasmid of P contrasts.The experimental result explanation lpt gene of Fig. 3 has been incorporated in the genome of cotton, and copy number is 1-2, and transfer-gen plant can be used for the functional analysis of gene to be identified.
In order further to understand ltp gene in the concrete application improving in cotton fiber quality, we carry out field test to 6 obtained different transgene cotton strains, relatively fibrous quality difference between transgenic cotton plant and adjoining tree (non-transgenic plant Ke-312).
6 different transgene cotton strains of obtain are seeded in Shanghai Communications University testing ground by May 25th, 2010, and it is 10m × 2m that completely randomized design, community area are taked in test, and distance between rows and hills is 90cm × 20cm, repeats for 4 times.Field management is all undertaken by high-yield culturing requirement.Since September 15,5 of the normotrophic middle part of results cotton bolls carried out the Fibre Quality of cotton.
Pull tape measurement according to GB/T 19617-2004 cotton length test method hand) measure cotton fiber length identify.
Carry out cotton fiber strength evaluation according to (mensuration/flat bundle method of GB/T13783-1992 cotton fibre strength).The mensuration of fibre strength is that fiber sample is mixed and makes sliver with the cotton fiber device of extending afterwards, measure the 3.2mm specific tenacity of spacing with domestic Y162A type bundle strength machine, survey and repeat mean values for 6 times as sample typical value, and with the standarded cotton sample correction of Chinese examination of fibers office.
6 the different transgene cotton strains that obtain are measured with the staple length contrasting, fibre strength, the results are shown in Table 3, and in table, strain 1～strain 6 is to turn ltp gene cotton, and from above-described embodiment 4, contrast strain is Ke-312.
The contrast of table 3 transfer-gen plant fibrous quality and adjoining tree
From table 3, can see, turn the cotton plants of ltp gene compared with the control, the cotton fiber quality of transfer-gen plant has obtained obvious improvement, and staple length increases 2.7-3.5mm; Fibre strength also increases to some extent, and fibre strength increases 0.3-3.3CN/tex compared with the control.The above results explanation is expressed ltp gene and is had the function that improves cotton fiber length and partly increase fibre strength.
With reference to the stage division national standard " GB 1103-2007 cotton medium staple cotton " of cotton fiber quality, the fibrous quality of genetically modified cotton has reached national high quality cotton standard.
Claims (2)
1. a lipid transfer protein for separation, its aminoacid sequence is as shown in SEQ ID NO:2.
2. the application of lipid transfer protein claimed in claim 1 aspect raising cotton fiber length and intensity.
3. polynucleotide for separation, its sequence is as shown in SEQ ID NO:1.
4. the application of polynucleotide claimed in claim 3 aspect raising cotton fiber length and intensity.
5. a carrier, is characterized in that containing polynucleotide claimed in claim 3.
6. a genetically engineered host cell, is characterized in that containing carrier claimed in claim 5.
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Non-Patent Citations (4)
| Title |
|---|
| Analysis of promoter activity of cotton lipid transfer protein gene LTP6 in transgenic tobacco plants.;Chuan-Yu Hsu et al.;《Plant Science》;19990507;第143卷(第1期);第63-70页 * |
| Chuan-Yu Hsu et al..Analysis of promoter activity of cotton lipid transfer protein gene LTP6 in transgenic tobacco plants..《Plant Science》.1999,第143卷(第1期),第63-70页. |
| RAPD标记在棉属种间杂种后代检测中的应用;聂以春等;《中国农业科学》;20001031;第33卷(第5期);第25-29页 * |
| 聂以春等.RAPD标记在棉属种间杂种后代检测中的应用.《中国农业科学》.2000,第33卷(第5期),第25-29页. |
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