CN1259417C - Coding sequence of arisaema agglutinant protein and its application - Google Patents
Coding sequence of arisaema agglutinant protein and its application Download PDFInfo
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Abstract
The present invention provides a novel a-Lectin protein expressed in rivier giantarum rhizome, a coding sequence and an application method thereof on pest resistance of modified plants. The present invention relates to a fusion gene construction body containing the gene, a novel recombinant expression carrier with the construction body, a plant cell converted by the recombinant expression carrier, a transgenic plant of the gene produced by the converted cell and offspring thereof. The present invention comprises plant seeds and plant tissue. The gene of the present invention is expressed in plants, and the obtained transgenic plants have reinforced resistance on plant pest.
Description
Technical field
The present invention relates to fields such as molecular biology, zymetology, physiology and genetically engineered.Particularly, the present invention relates to a kind of ah-Lectin albumen of in Rhizoma Arisaematis, expressing (arisaema agglutinant protein, Arisaemaheterophyllum agglutinin, AHA) and nucleotide sequence.The invention still further relates to the preparation method and the purposes of this albumen and nucleotide sequence.
Background technology
The applied research development of lectin aspect biology rapidly over nearly 20 years, the characteristics of its maximum are that it can discern sugar and carbohydrate, each lectin has has single-minded bonded ability (Ann Biochem, 1981,50:207～231) to a certain special carbohydrate.Some agglutinin gene then has stronger anti-Homoptera class insect and comprises aphid and plant hopper function (Entomol Exp Appl, 1993,66:119～126; Transgenic Res, 1995,4:18～25; Entomol Exp Appl, 1995,75:51～59).
Among the present invention, we are cloned into the homologous gene ah-Lectin of snowdrop g-Lectin lectin (GNA) gene in Araeceae (Araceae) plant Rhizoma Arisaematis (Arisaema heterophyllum).
Before the present invention comes forth, any Rhizoma Arisaematis ah-Lectin protein sequence and nucleotide sequence of mentioning in the present patent application thereof that disclose or reported do not arranged as yet.
Summary of the invention
First purpose of the present invention just provides a kind of new Rhizoma Arisaematis gene ah-Lectin (aha), and this gene is a Rhizoma Arisaematis ah-Lectin protein gene.
Second purpose of the present invention provides a kind of new Rhizoma Arisaematis albumen ah-Lectin.
The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and produces the above-mentioned new Rhizoma Arisaematis ah-Lectin albumen and the method for nucleotide sequence.
The present invention also provides this Rhizoma Arisaematis ah-Lectin protein polypeptide and encoding sequence in the application that utilizes on the transgenic technology controlling plant disease and pest.
In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of Rhizoma Arisaematis ah-Lectin protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 85-861 position dna molecular among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 85-861 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.4.More preferably, described sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 85-861 position.
In another aspect of this invention, provide a kind of isolated Rhizoma Arisaematis ah-Lectin protein and peptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.
In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is BL21 and tobacco in example.
In another aspect of this invention, also provide a kind of generation to have the method for the polypeptide of Rhizoma Arisaematis ah-Lectin protein active, its step is as follows:
(1) nucleotide sequence that coding is had a purifying of Rhizoma Arisaematis ah-Lectin protein active polypeptide operationally is connected in expression regulation sequence, form Rhizoma Arisaematis ah-Lectin protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 85-861 position among described nucleotide sequence and the SEQ ID NO.3;
(2) change the expression vector in the step (1) over to prokaryotic host cell, form the proteic reconstitution cell of Rhizoma Arisaematis ah-Lectin;
(3) be fit to express under the condition of Rhizoma Arisaematis ah-Lectin protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate pure substantially polypeptide with Rhizoma Arisaematis ah-Lectin protein-active.
Preferably, the nucleotide sequence that uses in the method has the sequence of 85-861 position among the SEQ ID NO.3.
In another aspect of this invention, also provide a kind of transgenic technology of utilizing that the nucleotide sequence that coding has Rhizoma Arisaematis ah-Lectin protein active polypeptide is transformed into plant to improve the method for plant to the disease and pest resistance, its step is as follows:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Rhizoma Arisaematis ah-Lectin protein-active operationally is connected in the expression of plants regulating and controlling sequence, formation contains the plant expression vector of Rhizoma Arisaematis ah-Lectin protein gene, shows at least 70% homology from the nucleotides sequence of Nucleotide 85-861 position among described nucleotide sequence and the SEQ ID NO.3;
(2) change the expression vector in the step (1) over to Agrobacterium, the Agrobacterium that will contain expression vector is cultivated altogether with eukaryotic host cell, under 22-28 ℃ of condition, the dark cultivation after 1-2 days, by screening as antibiotic-screening, acquisition contains transformant and the final regeneration of transgenic plant and the offspring thereof of Rhizoma Arisaematis ah-Lectin protein gene, comprises plant seed and plant tissue.The transfer-gen plant that contains Rhizoma Arisaematis ah-Lectin protein gene has the effect of enhanced resistance to plant pest.
Preferably, the nucleotide sequence that uses in the method has the sequence of 85-861 position among the SEQ ID NO.3.
The present invention also provides and ah-Lectin protein polypeptide specificity bonded antibody, and it comprises polyclonal antibody and monoclonal antibody.
In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, term " Rhizoma Arisaematis ah-Lectin albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with Rhizoma Arisaematis ah-Lectin protein-active is as 85-861 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 85-861 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 85-861 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 85-861 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 85-861 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.
This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ IDNO.3 with natural Rhizoma Arisaematis ah-Lectin identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " Rhizoma Arisaematis ah-Lectin albumen or polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of Rhizoma Arisaematis ah-Lectin protein-active.This term also comprises having and variant form natural Rhizoma Arisaematis ah-Lectin identical function, SEQ ID NO.4 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 at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of Rhizoma Arisaematis ah-Lectin and reactive derivative.
The variant form of Rhizoma Arisaematis ah-Lectin polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of Rhizoma Arisaematis ah-LectinDNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-Rhizoma Arisaematis ah-Lectin polypeptide to obtain.The present invention also provides other polypeptide, as comprises Rhizoma Arisaematis ah-Lectin polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of Rhizoma Arisaematis ah-Lectin polypeptide.Usually, this fragment have Rhizoma Arisaematis ah-Lectin peptide sequence at least about 10 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.
In the present invention, " Rhizoma Arisaematis ah-Lectin conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.4, have 10 at the most, preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Set |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Invention also comprises the analogue of Rhizoma Arisaematis ah-Lectin albumen or polypeptide.The difference of these analogues and natural Rhizoma Arisaematis ah-Lectin polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, Y-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing Rhizoma Arisaematis ah-Lectin polypeptide of the present invention, Rhizoma Arisaematis ah-Lectin encoding sequence operationally can be connected in expression regulation sequence, thereby form Rhizoma Arisaematis ah-Lectin protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " is protokaryon or eukaryotic cell.Prokaryotic host cell commonly used comprises BL21, and eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
Whether and quantity the expression of also available Northern blotting technical Analysis Rhizoma Arisaematis ah-Lectin gene product, the existence of rna transcription thing in cell of promptly analyzing Rhizoma Arisaematis ah-Lectin.
The Western engram analysis of the Northern engram analysis of Rhizoma Arisaematis ah-Lectin RNA and Rhizoma Arisaematis ah-Lectin specific antibody can be united use, to confirm the expression of Rhizoma Arisaematis ah-Lectin in biological specimen.
In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 continuous nucleotide of Rhizoma Arisaematis ah-Lectin nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding Rhizoma Arisaematis ah-Lectin.
The present invention also provides the method that whether has Rhizoma Arisaematis ah-Lectin nucleotide sequence in the test sample, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Rhizoma Arisaematis ah-Lectin nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In addition, according to Rhizoma Arisaematis ah-Lectin nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Rhizoma Arisaematis ah-Lectin homologous gene or homologous protein.
In order to obtain the dot matrix with the Rhizoma Arisaematis cDNAs of Rhizoma Arisaematis ah-Lectin gene-correlation, can screen Rhizoma Arisaematis cDNA library with dna probe, these probes are under low stringent condition, use
32P Rhizoma Arisaematis ah-Lectin all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Rhizoma Arisaematis.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, PaloAlto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with Rhizoma Arisaematis ah-Lectin.
Rhizoma Arisaematis ah-Lectin Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book invention Rhizoma Arisaematis ah-Lectin.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., SanFrancisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
Utilize Rhizoma Arisaematis ah-Lectin albumen of the present invention,, can filter out with Rhizoma Arisaematis ah-Lectin interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
Table 2 is that the homology of arisaema agglutinant of the present invention (AHA) albumen and the proteic nucleotide sequence of aroid pinellia agglutinin (PTA) compares (GAP) table.
Table 3 is that the homology of Rhizoma Arisaematis ah-Lectin albumen of the present invention and the proteic aminoacid sequence of aroid pinellia agglutinin (PTA) compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".
Table 4 is that the homology of Rhizoma Arisaematis ah-Lectin albumen of the present invention and the proteic aminoacid sequence of amrallid snowdrop g-Lectin lectin (GNA) (GenPept Accession No.AAA33349.1) compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".
Description of drawings
Fig. 1 is the prokaryotic expression figure of arisaema agglutinant (AHA).
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of Rhizoma Arisaematis ah-Lectin gene
1. separate tissue (isolation)
The Rhizoma Arisaematis bulb derives from Shanghai Univ. of Traditional Chinese Medicine, take arisaema tuber inflorescence material after, place the freezing preservation of liquid nitrogen immediately.。
2.RNA separation (RNA isolation)
Get portion of tissue, grind, add the 50mL pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to 50ml after the homogenate and newly manage, and extracted total RNA (TRIzol Reagents, Gibco, NY, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis.
3. the full-length clone of gene (Cloning of Full-length cDNA)
According to snowdrop and other Araeceae lectin amino acid conserved sequence, the design degenerated primer utilizes homologous genes clone principle, adopts RACE method (GibcoBRL test kit)
Carry out the cDNA full-length clone, divide three phases to carry out:
(1)3′-RACE
PCR (AP+TNX001) obtains 2001TNX3 ' (570bp), reclaim, be connected on the T-Easy carrier, with SP6 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of agglutinin gene of knowing its nucleotide sequence and proteins encoded and known amrallid such as snowdrop (Galanthusnivalis) and aroid such as taro (Colocasia esculenta), boundary Southern Star (Arum maculatum) is very high, so think that tentatively it is an agglutinin gene.
(2).5′-RACE
First round PCR (AAP+TNX02)
Second takes turns PCR (AUAP+TNXR03) obtains 2001TNX 5 ' (560bp) (process is with (1))
(3) .PCR amplification 2001TNX coding region (TNX01+R1) obtains 2001TNX coding region (770bp) (process is with (1)).
The gene that result's proof of BLAST newly obtains from Rhizoma Arisaematis really is a phytohemagglutinin gene.Because known homology lectin such as GNA (GNA) gene have stronger anti-Homoptera class insect and comprise aphid and plant hopper function (Hilder etc., 1994; Powell etc., 1993,1995), and the lectin that is purified into from arisaema tuber also is proved to be in to the test of the feeding of aphid (cotten aphid) cotten aphid is had very strong resistance (Mao Xue etc., JShanxi Agric Univ, 1999,19:122～124), the gene (aha) of the arisaema agglutinant of therefore encoding also has identical functions.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of Rhizoma Arisaematis ah-Lectin.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further R1:5 '-CCAGCATCAHAGGGAAGAAG-3 ' (SEQ ID NO.1) is a forward primer to the design primer, oligonucleotide R2:5 '-TATGCAACTCAGGGTAGCCA-3 ' (SEQ ID NO.2) is a reverse primer, with the total RNA of Rhizoma Arisaematis is template, carry out the RT-PCR amplification, the PCR condition of RI/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 1 minute with 94 ℃ 45 seconds, 58 ℃ 45 seconds and 72 ℃ thereupon, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1169bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.3.
The sequence information and the homology analysis of Rhizoma Arisaematis ah-Lectin gene
The length of the Rhizoma Arisaematis ah-Lectin full-length cDNA that the present invention is new is 1169bp, and detailed sequence is seen SEQ IDNO.3, and wherein open reading frame is positioned at 85-861 position Nucleotide.Derive the aminoacid sequence of Rhizoma Arisaematis ah-Lectin according to full-length cDNA, totally 258 amino-acid residues, molecular weight is 28464 dalton, pI is 7.71.Detailed sequence is seen SEQ ID NO.4.
Full length cDNA sequence and the coded protein thereof of ah-Lectin are carried out Nucleotide and protein homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and aroid pinellia agglutinin (PTA) (Yao etc., 40:461-464) there is certain homology in Fudan Journal 2001 with amrallid GNA (GNA) gene.On nucleotide level, it and pinellia agglutinin gene (pta) have 89% homogeny (subordinate list 2); On amino acid levels, it and the proteic 1-258 amino acids residue of pinellia agglutinin (PTA) have 78% homogeny and 86% similarity, also there is higher homology (subordinate list 3) with the 19-126 amino acids residue of snowdrop g-Lectin albumen (GNA, GenPept Accession No.AAA33349.1).This shows that all there are higher homology in ah-Lectin gene and pinellia agglutinin (PTA) gene and snowdrop g-Lectin (GNA) gene on nucleic acid still is protein level, can think that both also have very high similarity on function.
The proteic 26S Proteasome Structure and Function research of Rhizoma Arisaematis ah-Lectin
1. the proteic aminoacid sequence domain analyses of Rhizoma Arisaematis ah-Lectin.With the proteic aminoacid sequence of Rhizoma Arisaematis ah-Lectin ncbi database (network address is: index structure territory http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) obtains following result:
(1) in aminoacid sequence, there is following structural domain district:
Add the frame district (27-131,148-252): Lectin protein function module (Lectin).
(2) functional analysis
In this aminopeptidase gene acid sequence, there is Lectin protein function module, thereby can predicts it and have corresponding function really.
2. the signal peptide analysis of arisaema agglutinant protein.By to the comparative analysis of arisaema agglutinant protein with the nucleotide sequence and the aminoacid sequence of other phytohemagglutinin, find arisaema agglutinant protein genes encoding one precursor protein, and contain signal peptide sequence, press the rule of prediction such as van Heijine (1986) agglutinant protein signal peptides and to arisaema agglutinant protein with other phytohemagglutinin such as the proteic comparison of GNA, the signal peptide shearing site that identifies arisaema agglutinant protein is between the 24th amino acid (A) and the 25th amino acid (V), this position also meets single-minded bonded lectin of known most of seminoses such as GNA (GNA at present, Van Damme etc., 1991), the signal peptide shearing site of the agglutinant protein of kafir lily lectin (Van Damme etc., 1994) etc.
3. Rhizoma Arisaematis ah-Lectin is proteic single-minded in conjunction with the glycosylation analysis.By to Rhizoma Arisaematis ah-Lectin albumen single-minded with other phytohemagglutinin albumen such as GNA (GNA), kafir lily lectin, narcissus lectin etc. in conjunction with the glycosylation analysis, find Rhizoma Arisaematis ah-Lectin albumen with the single-minded bonded lectin of most of seminoses as GNA (GNA), kafir lily lectin, narcissus lectin etc., have 3 single-minded binding site boxes of typical seminose (QDNY or QDFY), See Figure (seminose single-minded binding site box QDNY and QDFY live with the square frame frame among the figure).Therefore prove that the also same GNA of Rhizoma Arisaematis ah-Lectin albumen (GNA), kafir lily lectin etc. are the same, be the single-minded bonded agglutinant protein of seminose.
The preparation and the purification of Rhizoma Arisaematis ah-Lectin polypeptide
In this embodiment, the Rhizoma Arisaematis ah-Lectin encoding sequence of total length or fragment are built among the prokaryotic expression carrier, to express and purification of recombinant proteins.
Construction of prokaryotic expression vector and conversion prokaryotic expression host bacterium competence cell
Aminoacid sequence according to Rhizoma Arisaematis ah-Lectin, design amplifies the primer of the protein-coding region behind the excision signal peptide, and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the prokaryotic expression plasmid pET-32a that selects for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, Rhizoma Arisaematis ah-Lectin gene is being guaranteed to be cloned into prokaryotic expression plasmid pET-32a (Novagen) under the correct prerequisite of reading frame.Transformed into escherichia coli DH5 α bacterial strain, order-checking is confirmed after PCR identifies, then at the extracting plasmid, is transformed in the competent cell of prokaryotic expression host bacterium BL21, is coated with the flat board of LB+Amp (100mg/L), cultivates 1d for 37 ℃.
The proteic expression of Rhizoma Arisaematis ah-Lectin
Single bacterium colony of growing of picking at random, 37 ℃ of overnight incubation of the liquid nutrient medium of LB+Amp (100mg/L) are drawn 50 μ l and are boiled 5min then.The centrifugal 1min of 12 000rpm/min, getting supernatant is template.Use the primer of Rhizoma Arisaematis ah-Lectin to carry out pcr amplification reaction.Compare with unconverted host bacterium simultaneously.Observe the gene fragment that can amplify Rhizoma Arisaematis ah-Lectin in the recon.PCR male reorganization bacterium is inoculated in LB+Amp (100mg/L) substratum of 5ml 37 ℃ of shaking culture (250rpm/min) 3h to OD
600=0.6, get 1ml bacterium liquid as contrast (induce before), adding IPTG in remaining 4ml bacterium liquid, to make final concentration be 1mM, continues 37 ℃ of shaking culture (250rpm/min) 3h, absorption 1ml bacterium liquid, the centrifugal 5min of bacterium liquid 12000rpm/min of twice absorption.Precipitation suspends with 100 μ l, 1 * PBS substratum, gets 10 μ l and carries out SDS-PAGE detection expression product (see figure 1).
Fig. 1. the prokaryotic expression figure of arisaema agglutinant (AHA).1: albumen size criteria (being followed successively by 97kD, 66kD, 43kD, 31kD, 20kD, 14kD from top to bottom); 2:pET-32a is transformed among the BL21 before the IPTG abduction delivering; 3:pET-32a is transformed among the BL21 behind the IPTG abduction delivering; 4:pET-32a-AHA is transformed among the BL21 before the IPTG abduction delivering; 5:pET-32a-AHA is transformed among the BL21 behind the IPTG abduction delivering; 6: the AHA fusion rotein of purifying.
The extraction purifying of Rhizoma Arisaematis ah-Lectin fusion rotein
Great expression Rhizoma Arisaematis ah-Lectin albumen as stated above adds bacterium liquid to the centrifuge tube back of weighing, and the centrifugal 10min of 10000g abandons supernatant, weighs once more, calculates the weight in wet base of bacterium.Ratio in the 5ml/g bacterium adds BugBusterreagent (Novagen), adds 1 μ lBenzonase (Novagen), resuspended bacterial precipitation in every milliliter of BugBuster reagent.Room temperature is slowly shaken incubation 20min, and the centrifugal 20min of 4 degree 16000g moves into supernatant liquor (containing soluble albumen) in the clean pipe.With the resuspended precipitation of isopyknic BugBuster Reagent, adding lysozyme is 200mg/L to final concentration, mixing, and room temperature is placed 5min, and the centrifugal 10min of 10000g abandons supernatant.Add 6 times of volumes dilution 10 times BugBuste reagent, mixing, 4 degree 5000g centrifugal 15min collect inclusion body, abandon supernatant.With the dilution of 1/2 former culture volume 10 times the resuspended inclusion body of BugBuste reagent, mixing, the centrifugal 15min of 4 degree 5000g.Again with the dilution of 1/2 former culture volume 10 times BugBuste reagent washing once, a same step.Washing once more, the centrifugal 15min of 4 degree 16000g abandons supernatant, the weight in wet base of weighing inclusion body.With the resuspended inclusion body of 1*IB Solubilization Buffer/0.3%N-lauroylsarcosine (Novagen), make final concentration reach 10-20mg/ml, mixing is blown and beaten repeatedly with broken inclusion body.Room temperature, incubation 15min.Room temperature, the centrifugal 10min of 10000g changes supernatant (containing soluble inclusion body protein) in the new pipe over to, does not carefully suck precipitation.With dialysis buffer (Novagen) dialysis that is 50 times of sample volumes at least, the middle dialysisbuffer that changes dialyses twice at least.If see that insolubles is arranged after the dialysis, the centrifugal 10min of 4 degree 10000g sucks supernatant in the one new pipe.With carrying out SDS-PAGE metaprotein electrophoresis, the proteic purity of Rhizoma Arisaematis ah-Lectin of Detection and Extraction behind rEK enzyme (Novagen) the excision fusion rotein.Protein band at about 26kDa place is Rhizoma Arisaematis ah-Lectin maturation protein.
Rhizoma Arisaematis ah-Lectin albumen or polypeptide carry out the insect-resistance of eukaryotic cell expression and transfer-gen plant in tobacco cell identifies
The structure that contains goal gene (Rhizoma Arisaematis ah-Lectin gene) expression vector
According to the complete encoding sequence (SEQ ID NO.3) of Rhizoma Arisaematis ah-Lectin, design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively on positive anti-primer, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, Rhizoma Arisaematis ah-Lectin cDNA is cloned into intermediate carrier (as pBluescript), further be cloned into binary expression vector (as pBI121 and pCAMBIA2200), under the prerequisite that guarantees reading frame, identify good expression vector, again it is changed in the Agrobacterium, utilize leaf dish law technology transformation mode plant tobacco.
Utilize leaf dish method transformation of tobacco
1. select positive bacterium colony on the flat board with aseptic toothpick picking YEB, be inoculated in 2ml YEB liquid (Sm+, Kan+), 28 degree, 200rpm shaking culture 24-36 hour;
2. under the room temperature 4, the centrifugal 10min of 000g;
3. abandon supernatant, thalline suspends with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes about the OD600=0.5 of bacterium liquid;
4. get the aseptic blade of the tobacco about two weeks of growth, remove its main lobe arteries and veins, it is cut into about 1 square centimeter of square vanelets;
5. blade is put into the bacterium liquid for preparing, soaked 2-5min, on aseptic filter paper, blot bacterium liquid;
6. the blade through infecting is put on the MS substratum 28 ℃ of dark cultivations 48 hours;
7. blade is forwarded on the callus substratum (MS+6-BA 1.0mg/L+NAA 0.1mg/L+Kan 50mg/L+cb250mg/L), the formation of 7-15 days visible callus is cultivated in 25-28 ℃ of illumination down;
8. visible differentiation bud grows after about 20 days, treat that bud is grown up after, downcut, place on the root media (1/2MS+NAA0.5mg/L+Kan 25mg/L) and carry out root culture, take root about 2-7 days;
9. after waiting well developed root system, plant is taken out, clean the solid medium that adheres to, move in the soil, just begun to treat to take off lens again behind the robust plant, cultivate in the greenhouse with lens cover several days with sterilized water.
Utilize western blot to detect the expression of AHA albumen in transgenic tobacco plant
1. proteic extraction: a) get the 50mg blade, add 100ul 1*PBS (KH
2PO
40.2g/l, Na
2HPO
41.15g/l, KCl 0.2g/l, NaCl 8g/l) in 1.5ml Eppendorf pipe, grind; B) 13000rpm, 4 ℃ are centrifugal 10 minutes; C) get supernatant, standby.Annotate: above process is in carrying out on ice.
2. proteic quantitative: with reference to Bradford method (Bradford, 1976).Get the 2ul protein sample, add 1ml Bradford reagent, behind the mixing, spectrophotometric instrumentation OD
595Protein content calculates: 1OD
595=28.57 μ g.
3.SDS-PAGE the preparation of protein isolate: SDS-PAGE is with reference to " molecular cloning " (Sambrook etc., 1989); A) before the application of sample, sample is placed sample loading buffer (the 2* sample loading buffer: glycerine 2.4g, 1MTris-HCl pH6.81ml that contains 50mmol/L DTT; Bromjophenol blue 0.01%, H
2O is settled to 20ml), boiled 10 minutes; B) polyacrylamide gel electrophoresis under the room temperature 100V voltage reaches the gel bottom up to indicator (bromjophenol blue) forward position.
4. protein is to shifting on the nitrocellulose membrane: before a) shifting, use transfering buffering liquid (39mmol glycine, 48mmol Tris Base, 0.037% SDS, 20% methyl alcohol) balanced gel and nitrocellulose membrane 30 minutes; B) shift 1h with the half dry type electroporation under the room temperature, 3 layers of Whatman filter paper are respectively filled up in the gel both sides;
5. proteic detection on the film: a) nitrocellulose membrane is immersed in the confining liquid, 37 ℃ are slowly shaken, seal 60 minutes (confining liquids: get the 5g skim-milk and be dissolved in 100ml 1*PBS (containing the 0.5g sodium azide); B) again filter membrane is immersed in the lavation buffer solution 37 ℃ of washed twice, each 15 minutes; C) add first antibody (antibody of anti-Rhizoma Arisaematis ah-Lectin), 37 ℃ of incubations 30 minutes; Same step b) is washed three times; D) add second antibody (avidin-alkaline phosphatase enzyme complex), 37 ℃ of incubations 30 minutes; Same step b), washed twice; E) add the substrate colour developing and observe protein band.
The insect-resistance that contains the transfer-gen plant of arisaema agglutinant gene (aba) is identified
In view of the gene of the coding single-minded bonded phytohemagglutinin of seminose such as GNA (GNA) has been proved to be to disease and pest such as piercing-sucking mouthparts class insect such as aphid (Hilder etc., Transgenic Res, 1995,4:18～25), plant hopper (Tang etc., Plant Breeding, 2002,121:93～95) and chewing mouthparts class insect such as tomato moth Lahanohia olerucea (Gatehouse etc., Molecular Breeding, 1997,3:49～63) all has resistance, and arisaema agglutinant (AHA) also has higher homology for the single-minded bonded lectin of seminose and with GNA (GNA), and the lectin that is purified into from arisaema tuber also is proved to be in to the test of the feeding of aphid (cotten aphid) cotten aphid is had very strong resistance (Mao Xue etc., J Shanxi Agric Univ, 1999,19:122～124), so the gene (aha) of the arisaema agglutinant of encoding is similar with GNA gene (gna), also should have pest-resistant function.We further carry out insect-resistance to the transfer-gen plant that contains arisaema agglutinant gene (aha) and identify.Press (Transgenic Res such as Hilder, 1995,4:18～25) method is carried out the evaluation of aphid (black peach aphid) resistance to the transgenic tobacco plant (20-30 centimetre high) that contains arisaema agglutinant gene (aha), studies its influence to aphid survival rate and development progress.In the research to the aphid survival rate, per 2 days mensuration aphid survival numbers (measuring 14 days altogether) behind 10 aphid nymphs in one age of inoculation on every strain plant.In the research to the aphid development progress, 5 aphid nymphs in one age of inoculation on every strain plant, the aphid of measuring survival after 13 days becomes borer population and nymph number.The result shows, in the aphid quantity of surviving on the transgenic tobacco plant that contains arisaema agglutinant gene (aha) with comparing remarkable minimizing (P＜0.05) in the non-transgenic contrast, simultaneously, significantly slowed down (P＜0.05) at its development progress of the aphid that survives on the transgenic tobacco plant that contains arisaema agglutinant gene (aha) with comparing also in the non-transgenic contrast, its quantity that develops into adult after 13 days also significantly reduces (P＜0.05).Therefore the insect-resistance result proves that arisaema agglutinant gene (aha) truly has resistance to insect (as aphid), and arisaema agglutinant gene (aha) will can be used for utilizing in the research and industrialization of transgenic technology controlling plant insect pest.
Table 2
89％ identity in 771 nt overlapAHA：83 tcatggcctccaagctcctcctcttcctcctcccggccatcctcggcctcgtcattcctc 142
||||||||||||||||||||||||||||||||||||||||||||||||||||||| || |PTA：37 tcatggcctccaagctcctcctcttcctcctcccggccatcctcggcctcatcatcccgc 96AHA：143 gggcagccgcggcagtgggcaccaaccacctgctgtccggcgaaaccctggacaccaacg 202
|| |||||| ||| |||||||||||| |||| ||||||||||||||||| ||||| |||PTA：97 ggccagccgtggcggtgggcaccaactacctactgtccggcgaaaccctagacacggacg 156AHA：203 gccatctcaggaacggcgacttcgacttgatcatgcaggaagactgcaacgccgtcctgt 262
||||||| | ||| |||||||||||||| |||||||||||||||||||||||||||||||PTA：157 gccatctgaagaatggcgacttcgactttatcatgcaggaagactgcaacgccgtcctgt 216AHA：263 acaatggcaactggcagtccgacacggccaacaaaggacgggactgcaagctcaccctca 322
|||| ||||||||||||||| ||||||||||||||||||| |||||||||||||||||||PTA：217 acaacggcaactggcagtccaacacggccaacaaaggacgagactgcaagctcaccctca 276AHA：323 ccgaccgcggcgagctcgtcatcaaaaatggagacagatccatcgtctttaggagcggct 382
||||||||||||||||||||||||| || || || ||||| ||||| |||||||||||PTA：277 ccgaccgcggcgagctcgtcatcaacaacggcgagggatccgccgtctggaggagcggct 336AHA：383 cccagtccgacgtgaggggcaactacgccttggtcgtccatccggaggggaggctggtca 442
||||||||| || ||||||||||||| ||| ||||||||||||||| ||||||||PTA：337 cccagtccgc---gaagggcaactacgccgccgtcctccatccggaggggaaactggtca 393AHA：443 tctacggcccatccgtgttcaagatcaacccttgggtccccggccgccacagcctgcggc 502
|||||||||||||||| |||||||||||||||||||||||||||| | ||||||||||||PTA：394 tctacggcccatccgtcttcaagatcaacccttgggtccccggcctcaacagcctgcggc 453AHA：503 agctcggcaacatccctgtcaccgacaacatgctcttctctggccaggtcctctacggcg 562
||||||||| ||||| |||| ||||||||||||||| |||||||||||||||||||PTA：454 ---tcggcaacgtccctttcacgtgcaacatgctcttctccggccaggtcctctacggcg 510AHA：563 acggcaagctcagggcgaggaaccacatgctcgtgatgcaggatgactgcaacctggttc 622
|||||||| ||| ||||||||||||||||| || |||||| ||||||||||||||| |PTA：511 acggcaagatcactgcgaggaaccacatgctggtcatgcagggcgactgcaacctggtcc 570AHA：623 tgtacggcggcaagtacggctggcagtccaacacccacggcaacggcgagaactgcttcg 682
|||||||||| |||| || ||||||||||||||||||||||||||||||| |||||||||PTA：571 tgtacggcgggaagtgcgactggcagtccaacacccacggcaacggcgagcactgcttcc 630AHA：683 tgaggctgagccacaagggcgagctcatcatcaaggacgacgacttccagaccatctgga 742
| |||||| ||||||||||||||||||||||||||| ||||||||| ||| ||||||||PTA：631 tccggctgaaccacaagggcgagctcatcatcaaggatgacgacttcaagagcatctgga 690AHA：743 gcagccgatccggttccaagcagggtgactacgtcttcatcctccaggaggacggctttg 802
|||||| ||| | |||||||||||||||||||||||||||||||||||| ||||||| |PTA：691 gcagccagtccagctccaagcagggtgactacgtcttcatcctccaggacaacggctacg 750AHA：803 ccgtcatctacggccccgccatctgggccaccagctcgaagcgccccattg 853
|| || || || || || || | || || || || || | || || || || || || || | || || | PTA:751 gcgtcatctacggccctgccatctgggcgaccagctcgaagcgctccgttg 801 is above-listed: the proteic nucleotide sequence of arisaema agglutinant (AHA) is following: the proteic nucleotide sequence of pinellia agglutinin (PTA)
Table 378% identity in 258aa overlap, 81% similarity in 258aa overlapAHA:1 MASKXXXXXXXXXXXXVIPRAAAAVGTNHLLSGETLDTNGHLRNGDFDLIMQEDCN AVLY 60
MASK +IPR A AVGTN+LLSGETLDT+GHL+NGDFD IMQEDCNAVLYPTA：1 MASKLLLFLLPAILGLIIPRPAVAVGTNYLLSGETLDTDGHLKNGDFDFIMQEDCNAVLY 60AHA：61 NGNWQSDTANKGRDCKLTLTDRGELVIKNGDRSIVFRSGSQSDVRGNYALVVHPEGRLVI 120
NGNWQS+TANKGRDCKLTLTDRGELVI NG+ S V+RSGSQS +GNYA V+HPEG+LVIPTA：61 NGNWQSNTANKGRDCKLTLTDRGELVINNGEGSAVWRSGSQS-AKGNYAAVLHPEGKLVI 119AHA：121 YGPSVFKINPWVPGRHSLRQLGNIPVTDNMLFSGQVLYGDGKLRARNHMLVMQDDCNLVL 180
YGPSVFKINPWVPG +SLR LGN+P T NMLFSGQVLYGDGK+ ARNHMLVMQ DCNLVLPTA：120 YGPSVFKINPWVPGLNSLR-LGNVPFTCNMLFSGQVLYGDGKITARNHMLVMQGDCNLVL 178AHA：181 YGGKYGWQSNTHGNGENCFVRLSHKGELIIKDDDFQTIWSSRSGSKQGDYVFILQEDGFA 240
YGGK WQSNTHGNGE+CF+RL+HKGELIIKDDDF++IWSS+S SKQGDYVFILQ++G+PTA：179 YGGKCDWQSNTHGNGEHCFLRLNHKGELIIKDDDFKSIWSSQSSSKQGDYVFILQDNGYG 238AHA：241 VIYGPAIWATSSKRPIVA 258
VIYGPAIWATSSKR+ APTA:239 VIYGPAIWATSSKRSVAA 256AHA: Rhizoma Arisaematis ah-Lectin Argine Monohydrochloride sequence PTA: pinellia agglutinin p-Lectin Argine Monohydrochloride sequence
Table 439% identity in 108aa overlap, 63% similarity in 108aa overlapa.pep:146 VTDNMLFSGQVLYGDGKLRARNHMLVMQDDCNLVLYG-GKYGWQSNTHGNGENCFV RLSH 204
++DN+++SG+ L L +++ +MQ+DCNLVLY K W +NT G +C+++g.pep：19 LSDNIMYSGETLSTGEFLNYGSYVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCYLNMQT 78a.pep：205 KGELIIKDDDFQTIWSSRSGSKQGDYVFILQEDGFAVIYGPAIWATSS 252
G L++++ IW+S+G+G+YV ILQ+D VIYGPA WAT+g.pep:79 DGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGPARWATGT 126a.pepp: Rhizoma Arisaematis ah-Lectin Argine Monohydrochloride sequence g.pep: snowdrop g-Lectin Argine Monohydrochloride sequence (GenBank Accession No.AAA33349.1)
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 20bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
CCAGCATCAHAGGGAAGAAG
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 20bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.2
TATGCAACTCAGGGTAGCCA
(3) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 1169bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: Nucleotide
(iii). sequence description: SEQ ID NO.3
1 ccagcatcac agggaagaag aagaagaatt agctagggtt ttgacttagt agccagccag
61 ctagcagcaa cctcgttcct gatcatggcc tccaagctcc tcctcttcct cctcccggcc
121 atcctcggcc tcgtcattcc tcgggcagcc gcggcagtgg gcaccaacca cctgctgtcc
181 ggcgaaaccc tggahaccaa cggccatctc aggaacggcg acttcgactt gatcatgcag
241 gaagactgca acgccgtcct gtahaatggc aactggcagt ccgahacggc caahaaagga
301 cgggactgca agctcaccct caccgaccgc ggcgagctcg tcatcaaaaa tggagahaga
361 tccatcgtct ttaggagcgg ctcccagtcc gacgtgaggg gcaactacgc cttggtcgtc
421 catccggagg ggaggctggt catctacggc ccatccgtgt tcaagatcaa cccttgggtc
481 cccggccgcc ahagcctgcg gcagctcggc aahatccctg tcaccgahaa catgctcttc
541 tctggccagg tcctctacgg cgacggcaag ctcagggcga ggaaccahat gctcgtgatg
601 caggatgact gcaacctggt tctgtacggc ggcaagtacg gctggcagtc caahacccac
661 ggcaacggcg agaactgctt cgtgaggctg agccahaagg gcgagctcat catcaaggac
721 gacgacttcc agaccatctg gagcagccga tccggttcca agcagggtga ctacgtcttc
781 atcctccagg aggacggctt tgccgtcatc tacggccccg ccatctgggc caccagctcg
841 aagcgcccca ttgtcgcgta ggatcagaag atgatcggcg ccgtcggtgg ggtactgtct
901 gagaaggtgt acctgtgcgg aagcgaaatg aagttggcaa ctatatatat aagccttctc
961 caccccactg caaaataata aataatcgtg cgccatgtcg ggcatgcggt tatgtcgatc
1021 ctgtactcct ggtctttctt cgtatcgtta agaggctttg atctgccgta gttctggctc
1081 ctgctggtac gtactgtaaa gtggtccttt gaataaatat ggctaccctg agttgcataa
1141 aaaaaaaaaa aaaaaaaaaa aaaaaaaa
(4) information of SEQ ID NO.4
(i) sequence signature:
(A) length: 258 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description: SEQ ID NO.4
1 MASKLLLFLL PAILGLVIPR AAAAVGTNHL LSGETLDTNG HLRNGDFDLI
51 MQEDCNAVLY NGNWQSDTAN KGRDCKLTLT DRGELVIKNG DRSIVFRSGS
101 QSDVRGNYAL VVHPEGRLVI YGPSVFKINP WVPGRHSLRQ LGNIPVTDNM
151 LFSGQVLYGD GKLRARNHML VMQDDCNLVL YGGKYGWQSN THGNGENCFV
201 RLSHKGELII KDDDFQTIWS SRSGSKQGDY VFILQEDGFA VIYGPAIWAT
251 SSKRPIVA
Sequence table
＜110〉Shanghai Fudan Di'en Biological Techn. Co., Ltd.
＜120〉arisaema agglutinant protein encoding sequence and application thereof
<130>21
<160>1
<170>PatentIn version 3.1
<210>1
<211>1168
<212>DNA
<213>ah-Lectin
<220>
＜221〉Rhizoma Arisaematis
<222>(85)..(861)
<223>
<400>1
ccagcatcac agggaagaag aagaagaatt agctagggtt ttgacttagt agccagccag 60
ctagcagcaa cctcgttcct gatcatggcc tccaagctcc tcctcttcct cctcccggcc 120
atcctcggcc tcgtcattcc tcgggcagcc gcggcagtgg gcaccaacca cctgctgtcc 180
ggcgaaaccc tggahaccaa cggccatctc aggaacggcg acttcgactt gatcatgcag 240
gaagactgca acgccgtcct gtahaatggc aactggcagt ccgahacggc caahaaagga 300
cgggactgca agctcaccct caccgaccgc ggcgagctcg tcatcaaaaa tggagahaga 360
tccatcgtct ttaggagcgg ctcccagtcc gacgtgaggg gcaactacgc cttggtcgtc 420
catccggagg ggaggctggt catctacggc ccatccgtgt tcaagatcaa cccttgggtc 480
cccggccgcc ahagcctgcg gcagctcggc aahatccctg tcaccgahaa catgctcttc 540
tctggccagg tcctctacgg cgacggcaag ctcagggcga ggaaccahat gctcgtgatg 600
caggatgact gcaacctggt tctgtacggc ggcaagtacg gctggcagtc caahacccac 660
ggcaacggcg agaactgctt cgtgaggctg agccahaagg gcgagctcat catcaaggac 720
gacgacttcc agaccatctg gagcagccga tccggttcca agcagggtga ctacgtcttc 780
atcctccagg aggacggctt tgccgtcatc tacggccccg ccatctgggc caccagctcg 840
aagcgcccca ttgtcgcgta ggatcagaag atgatcggcg ccgtcggtgg ggtactgtct 900
gagaaggtgt acctgtgcgg aagcgaaatg aagttggcaa ctatatatat aagccttctc 960
caccccactg caaaataata aataatcgtg cgccatgtcg ggcatgcggt tatgtcgatc 1020
ctgtactcct ggtctttctt cgtatcgtta agaggctttg atctgccgta gttctggctc 1080
ctgctggtac gtactgtaaa gtggtccttt gaataaatat ggctaccctg agttgcataa 1140
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1168
Claims (8)
1. an isolated dna molecular is characterized in that, its coding has the nucleotide sequence of polypeptide of Rhizoma Arisaematis ah-Lectin protein active, and described nucleotides sequence is classified among the SEQ ID NO.3 nucleotide sequence from Nucleotide 85-861 position as.
2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.4.
3. an isolated Rhizoma Arisaematis ah-Lectin protein polypeptide is characterized in that it has the polypeptide of SEQ ID NO.4 aminoacid sequence.
4. a carrier is characterized in that, it comprises the described DNA of claim 1.
5. one kind with the described carrier transformed host cells of claim 4, it is characterized in that it is protokaryon or eukaryotic cell.
6. a generation has the method for the polypeptide of Rhizoma Arisaematis ah-Lectin protein active, is characterised in that its step is as follows:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Rhizoma Arisaematis ah-Lectin protein-active operationally is connected in expression regulation sequence, form Rhizoma Arisaematis ah-Lectin protein expression vector, described nucleotides sequence is classified among the SEQ IDNO.3 nucleotide sequence from Nucleotide 85-861 position as;
(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of Rhizoma Arisaematis ah-Lectin;
(3) be fit to express under the condition of Rhizoma Arisaematis ah-Lectin protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate polypeptide with Rhizoma Arisaematis ah-Lectin protein-active.
7. one kind is utilized transgenic technology that the nucleotide sequence that coding has Rhizoma Arisaematis ah-Lectin protein active polypeptide is transformed into plant to improve the method for plant to the disease and pest resistance, is characterised in that its step is as follows:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Rhizoma Arisaematis ah-Lectin protein-active operationally is connected in the expression of plants regulating and controlling sequence, formation contains the proteic plant expression vector of Rhizoma Arisaematis ah-Lectin, and described nucleotides sequence is classified among the SEQ ID NO.3 nucleotide sequence from Nucleotide 85-861 position as;
(2) change the expression vector in the step (1) over to vegetable cell;
(3), obtain transformant and final regeneration of transgenic plant and offspring thereof by antibiotic-screening.
8. energy and the described Rhizoma Arisaematis ah-Lectin of claim 3 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.
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| CN101649321B (en) * | 2009-02-16 | 2011-03-23 | 华中农业大学 | Arisaema amurense maxim agglutinin protein coding sequence and application thereof |
| CN103088035B (en) * | 2013-01-10 | 2014-04-16 | 南京农业大学 | Application of soybean agglutinin gene lec-s |
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