CN1315999A - Genetic method for expression of polyproteins in plants - Google Patents

Abstract

A method of expressing or improving expression levels of one or more proteins in a transgenic plant comprising inserting into the genome of said plant a DNA sequence comprising a promoter region operably linked to two or more protein encoding regions and a 3'-terminator region wherein said protein encoding regions are separated from each other by a DNA sequence coding for a linker propeptide, said propeptide providing a cleavage site whereby the expressed polyprotein is post-translationally processed into the component protein molecules. In particular, a signal sequence is also included such that the post-translational processing is effected in the secretory pathway of plants. Suitable linker sequences and DNA constructs for use in the method are also described.

Description

In plant, express the genetic method of polyprotein

The present invention relates to a kind of method that strengthens protein expression level, especially strengthen proteic expression by the two or more albumen of coexpression in one of plant independent transcriptional units.The invention still further relates to two or more proteic coexpressions and secretion in the plant, relate to applied joint sequence in the inventive method, relate to the DNA construct used in the invention and with this construct institute plant transformed in the invention.

Because many application all change based on the plant genetics of being undertaken by transgenosis, the two or more transgenosiss of coordinate expression are desirable.For example, in plant, the genetically modified coexpression that coding has the antibacterial protein of different biochemical target spots can produce the disease-resistant level of enhanced, produces the pathogen resistance than broad range, perhaps produces the more difficult resistivity that is overcome by pathogenic agent variation adaptation.Other examples comprise, are included in a plurality of transgenosiss in the biosynthetic pathway by coexpression, produce a kind of special metabolite in transgenic plant.Obtain to express a plurality of genetically modified transgenic plant, have diverse ways.A kind of selection scheme commonly used is, imports each transgenosis respectively by independent transformation, and with different single transgene expression incrosses.The shortcoming of this method is that in the offspring who produces, different transgenosiss will be inserted into different positions.This can make breeding process subsequently complicated.And this method is not suitable for the crop that nourishes and generates, for example potato, many ornamental plants and fruit tree.

Second kind of possibility be, different transgenosiss is imported as the expression cassette that connects in the independent conversion carrier, and each expression cassette has promotor and the terminator of oneself.In this case, such one group of transgenosis will be separated as a single gene locus.Yet have been found that the existence of a plurality of copies of identical promoters in the transgenic plant, often suppress genetically modified and transcribe (Matzke, M.A. and Matzke, A.J.M., 1998, cell and molecular biology 54,94-103).In an experiment that carrier is imported plant, carrier wherein comprises four transgenosiss that are connected, each transgenosis is by a CaMV35S promoters driven, (Phil.Trans.Soc.Lon.B. such as Van denElzen P.J., 1993, find that 342:271-278) none can be in four whole transgenosiss of quite high horizontal expression in the transgenic strain of being analyzed.For fear of this problem, can in applied each expression cassette, use different promotors.Yet, have only limited alternative promotor group at present, in expression level, cell type and evolution specificity and reaction, have similar characteristic to environmental factors.

The 3rd option is to separate different coding regions with so-called internal ribosome entry site, produces a plurality of albumen from a transcriptional units.Internal ribosome entry site makes rrna repeat translation at the interior location of mRNA.Although internal ribosome entry site the animal system obtained good proof (Kaminiski A. etc., 1994, genet.Eng.16 115-155), does not know also whether these sites also work at present in the nuclear-encoded gene from plant.The gene of polycistron when inserting the plant chloroplast genome, can be expressed (Daniell H. etc., 1998, Nature Biotechnol 16,345-348), but gene product in this case is limited at chloroplast(id), and chloroplast(id) not always foreign protein preferably deposit the site.

At last, the 4th kind of strategy is based on polyprotein precursor by single transcriptional units coding of hydrolytic cleavage, and produces a plurality of albumen.For example, marmor upsilon group (Potyviruses) is translated into a single polyprotein precursor to their geneome RNA, the proteolysis structural domain that this precursor comprises can cis fracture polyprotein precursor (Dougherty, W.G. and Carrington, J.C., 1988, plant pathology summary annual 26,123-143).Beckvon Bodman, S.et al., (1995, biology/technology 13, the biosynthetic two kinds of enzymes of participation mannopine that 587-591) utilized marmor upsilon group of methods coexpression.These two kinds of biosynthetic enzymes are merged in an open reading frame with a proteolytic enzyme that derives from marmor upsilon group polyprotein precursor, and neighboring region by the transcribed spacer of 8 amino acid longs separately, and transcribed spacer has been represented the special cracking site of proteolytic enzyme.With the phytosynthesis of this thaumatropy glycosides reveal alkali, show that these two kinds of enzymes have produced the form that has partial function at least in some way, although in plant (inplanta), do not propose direct evidence for the splitting action of inferring.The shortcoming of this method is, viral protein need with the protein of interest coexpression, this is always undesirable.Recently, Urwin P.E. etc. (1998, phytology 204,472-479) verified can coexpression with two different protease inhibitors that engage from the responsive propetide of the proteic proteolytic enzyme of plant metallothionein sample.Have been found that in transgenic arabidopsis (Arabidopsis thaliana) suppressing son (oryzacystatin from vice), one by L-Cysteine HCL Anhydrous derives from the proteic propetide of pea metallothionein(MT) sample and serine protease to suppress a molecular polyprotein precursor cleaved.Yet only be the part cracking, do not have cleaved polyprotein precursor because in transgenic plant, also can detect.Because this polyprotein precursor does not comprise a guiding peptide, the prediction translation product will be deposited in the cytosol.The metallothionein(MT) that derives propetide does not comprise a guiding peptide (Evans IM 1990, European biological chemistry association associating proceedings (FEBS Lett.262,29～32)) yet, so its processing must be carried out in kytoplasm.

For some application, kytoplasm processing and deposition are defectives.Many albumen, especially glycosylated protein or the albumen of a plurality of disulfide linkage is arranged must be at Secretory Pathway synthetic (comprising endoplasmic reticulum and Golgi's organs), to be folded into a kind of form (Bednarek and Raikhel1992 that function is arranged, molecular biology of plants 20,133-150).In addition, use for some, for example expression of antibacterial protein, ECS is preferably to deposit the site because most of microbe at least the early infection process occur in ECS.The albumen that is assigned to ECS also is by the Secretory Pathway synthetic, but except the guiding peptide, also lack extra target information (Bednarek and Raikhel 1992, molecular biology of plants 20,133-150).Other of this method used example and described to some extent in WO 95/24486 and WO95/17514.

The applicant is unexpected to be found, in a polyprotein precursor construct plant transformed, the expression level of plant defense element is far above its expression in the plain construct plant transformed with single plant defense.

Therefore, the invention provides the method that improves a kind of protein expression level in transgenic plant, the genome that this method is included in described plant inserts a dna sequence dna.This dna sequence dna comprise a promoter region and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment and becomes component (component) protein molecular in this site.

System of processing described herein not only can be used for the two or more different albumen of coexpression, and can be used for obtaining the especially high expression level of small protein of an albumen.The reason of viewed promotion translation efficiency is not clear at present.May belong to the influence of mRNA length or original translation product length.

A preferred signal sequence is connected between the protein-coding region effectively.

" signal sequence " of being explained here is a sequence that is used for defining coding guiding peptide, and this guiding peptide makes newborn polypeptide enter endoplasmic reticulum, and the guiding peptide is cut after this transposition effect.

Signal sequence can and may naturally be united from any suitable source has it to want connected promotor.We have found that, from signal sequence (Broekaert et a1,1995 plant physiology 108, the 1353-1358 of vegetable-protein defensin family; Broekaertet al, 1997, Crit, Rev, Plant Sci 16 297-323), is particluarly suitable for using in the inventive method.

Like this, in an embodiment that is more preferably, a kind of method that improves protein expression level in transgenic plant is provided, and the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.

The especially suitable length of this method of the present invention is at 100 amino acid or be less than 100 amino acid whose proteic expression.

The invention provides a kind of method of simple and effective, promptly in plant two or more albumen as an independent transcriptional units coexpression, in transcriptional units, two kinds of albumen are connected by a kind of connector of cleavable.This structure of design so that in the Secretory Pathway of plant splitting action takes place, is discharged into albumen outside the born of the same parents like this.

According to another aspect of the present invention, provide a kind of a plurality of proteic methods of expressing in transgenic plant, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence is connected to two or more protein-coding regions and 3 a ＇-terminator, and wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.

According to all aspects of the present invention, two or more protein-coding regions identical albumen of preferably not encoding, that is, method of the present invention allows to produce different protein in a single transcriptional units.The method according to this invention, the dna sequence dna that express are not the natural sequences that is used for producing polyprotein in the plant, and promptly one or more components of this dna sequence dna are xenogeneic for plant host.

Polyprotein expression method described herein does not comprise that resembling international patent application discloses SEQ ID NO.14 among the No.WO95/24486,15,16, described in 17 or 18, use as the joint propetide of Ib-AMP genetic expression, three each proteic coding regions of own coding Rs-AFP2 are separated and be inserted into Plant Genome.Adaptably, do not use SEQ ID NO.14 among the WO95/24486 in the method for the present invention, the joint propetide of 15,16,17 or 18 described natural Ib-AMP genes.

In yet another aspect, the invention provides a kind of a plurality of proteic methods of expressing in transgenic plant, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.Precondition is, when the joint propetide derives from SEQ ID NO.14 among the open No.WO95/24486 of international patent application, during 15,16,17 or 18 described Ib-AMP genes, it does not separate three each proteic coding regions of own coding Rs-AFP2.

The sequence of Rs-AFP2 has complete description in the open No.WO93/05153 of the international patent application of issue on March 18th, 1993.

Promoter sequence may be connected with signal sequence naturally, and also/or may unite the albumen coded sequence that has it to connect naturally, perhaps it may be any other transcripting promoter in the plant.Promotor may be the promotor of a composition or may be an inducible promoters.

The present invention as described herein in all respects with all embodiment in a kind of preferred joint propetide that uses, in the vegetable cell Secretory Pathway that polyprotein coding-DNA is expressed, cracking from described dna encoding polyprotein precursor.Preferred such design or the selection of joint propetide, so that make propetide by the natural proteasome degradation that is present in the vegetable cell Secretory Pathway, the DNA of the polyprotein of wherein encoding expresses at Secretory Pathway.The special promoter of cauliflower mosaic virus is the Penh 25S promotor of 35SRNA for example, and such examples of proteases comprises subtilisin sample proteolytic enzyme.

Therefore, in an embodiment preferred of the present invention, a kind of a plurality of proteic methods of expressing in transgenic plant are provided, and the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.Described joint propetide in the vegetable cell Secretory Pathway of expressible dna coding polyprotein, cracking from described dna encoding polyprotein precursor.

Polyprotein expression method as described herein does not comprise that resembling international patent application discloses SEQ ID NOs 14 among the No.WO95/24486,15,16, described in 17 or 18, use the joint propetide that derives from the Ib-AMP gene separately and be inserted into Plant Genome with three each proteic coding regions of own coding Rs-AFP2.

In some embodiments of the present invention, the joint propetide does not get from virus.

In an embodiment preferred of the present invention, a kind of a plurality of proteic methods of expressing in transgenic plant are provided, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence is connected to two or more protein-coding regions and 3 a ＇-terminator, and wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is transcribed post-treatment in this site and become the component protein molecular.Described joint propetide in the vegetable cell Secretory Pathway of expressible dna coding polyprotein, cracking from described dna encoding polyprotein precursor.Wherein the cracking of propetide is to be undertaken by the natural proteolytic enzyme that is present in the plant secretion approach.

The joint propetide may be a kind of natural peptide that comprises the proteolytic enzyme processing site that occurs in the plant secretion approach, for example here with the inside propetide of the Ib-AMP gene source that further describes.Joint propetide or may be the peptide that proteolytic enzyme processing site has all been designed in a kind of one or both ends in office, it is beneficial to the cracking of sequence.Have the place that such proteolytic enzyme is processed the site at propetide, can add a proteolytic enzyme processing site again.If desired or expectation, can comprise multiple processing site, for example comprise six above multiple processing sites.

For example, as here fully describe, another proteolytic enzyme processing site has been added to 3 ' of dna sequence dna-end.This dna sequence encoding is from the C-CICP of Garden Dahlia and Amaranthus, and it is in the terminal natural proteolytic enzyme processing site with a unknown Secretory Pathway of N-, and these peptides are particluarly suitable for using in the method for the present invention.Describe some and comprised the Garden Dahlia sequence of C-CICP sequence, and claimed in common unsettled English Patent No.9818003.7.

Yet another strategy is based on using the virus for example sequence of picornavirus, for example 20 aminoacid sequences that are known as the 2A sequence of foot and mouth disease virus (FMDV) RNA, cracking (the Ryan and Drew 1994 that causes polyprotein, the EMPO magazine, 13,928-933).Yet in this example, for fear of the delay of unwanted amino acid in protein product, unite a sequence that produces the N-end, for example the sequence of a plant origin or fragment form a chimeric propetide.

In the present invention, we have set up new strategy and have been manufactured on the cleaved artificial polyprotein precursor of Secretory Pathway.First kind of strategy is based on the propetide that uses an IbAMP gene source.IbAMP is a gene from Flower of Garden Balsam (Impatiens balsamina), the polyprotein precursor of a kind of uniqueness of encoding, the peculiar guiding peptide of this precursor and 6 successive antibacterial peptides, each antibacterial peptide side (Tailor R.H.et al. that is connected with the propetide that 16 to 28 amino acid lengths change, 1997, journal of biological chemistry 272,24480-24487).And do not know the IbAMP precursor in the plant in its source how and process wherein.An inner propetide from IbAMP is used to separate two plain coding regions of different plant defenses, and these of two coding regions derive from radish seed (RsAFP2, Terras F.R.G.etal., 1992, biological chemistry 267,15301-15309; Terras et al, 1995, plant cytology, 7,573-588), one from the Garden Dahlia seed (DmAMP1, Osborn R.W.et al., 1995, European biological chemistry association associating proceedings 368,257-262).

Another strategy based on use from DmAMP1 precursor or AcAMP2 precursor or their segmental C-CICP (De Bolle M.F.C.ET AL., 1993, molecular biology of plants 22,1187-1190).Select the basis of these C-CICPs to be, we observed they can not influenced the maturation protein that they are connected by obvious cracking in precursor in transgene tobacco extracellular deposition (R.W.Osborn and S.Attenborough, personal communications in the past; De Bolle M.F.C.et al., 1996, molecular biology of plants 31 993-1008), shows that this fracture is undertaken by a kind of proteolytic enzyme in the Secretory Pathway that is present in except the vacuole.To be converted into inner propetide to these C-CICPs, design a subtilisin sample proteolytic enzyme processing site at the C-of propetide terminal portions.

Subtilisin sample proteolytic enzyme is that in the end two residues are enzyme (Barr, P.J., 1991, cell 66, the 1-3 of the special fracture of recognition site of alkalescence; Park C.M.etal., 1994, molecular microbiology, 11,155-164).Although subtilisin sample proteolytic enzyme is proved in fungi (for example Kex2 sample proteolytic enzyme) and higher animal (for example furin) well, but recent evidence shows these enzymes and also is present in (Kinal H.et al. in the plant, 1995, vegetable cell 7,677-688; Tornero P.et al., 1997, journal of biological chemistry 272,14412-14419), comprise the Arabidopsis plant (Ribeiro A.et al., 1995, vegetable cell 7,785-794).

We have found that, comprise the polyprotein precursor of a guiding peptide and two different plant defense elements of separating mutually by any above-mentioned inner propetide, can be processed in transgenic plant, discharge two kind of plant defensins simultaneously.This cracking has taken place really, and the major part of plant defense element is deposited on ECS so at least.Therefore, the processing of precursor takes place at Secretory Pathway or ECS.Different propetides cleaved in transgenic plant do not demonstrate the original series homology.As if yet all sequences all are rich in p1 amino acid A, V, and S and T, and all comprise by two acidic residues, two alkaline residues or an acidic residues and two peptide sequences that alkaline residue is formed.Although Zheng Ming propetide cracking does not occur in the vacuole significantly in embodiments of the present invention,, may also can use inside propetide in vacuole from vacuole protein (for example albumin of 2S) if expectation albumen deposits.Used two different plant defense elements in the coexpression experiment described here, but prediction will obtain identical result when using the albumen of other type or use plural maturation protein territory in the polyprotein front body structure.

To in Secretory Pathway, point to a special organoid to polyprotein, can be added to one or more polyprotein coding regions to a suitable target sequence.For example, an endoplasmic reticulum target sequence, the sequence of encoded K DEL (SEQ ID NO 65) for example, can be added to one or more mature protein coding regions, the perhaps sequence of a vacuole target (Chispeel and Raikhel 1992, cytology 68 613-616) can be added to 3 ' of one or more protein-coding regions-or 5 '-end.A recent example is to prove that the C-terminal propetide of barley lectin element specifies heterologous protein to be secreted into (Bednarek and Raikhel 1991, plant cytology 3,1195-1206 in the vacuole by different way; De Bolle etc., 1996, molecular biology of plants 31,933-1008).

According to described here all aspects of the present invention and method, the sequence of preferred employed joint propetide has at least 40% to comprise the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or comprise 2 to 5 extensions that are selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.

The preferred L-Ala of described hydrophobic residue, Xie Ansuan, leucine, methionine(Met) and/or Isoleucine, and the preferred aspartic acid of described hydrophilic residue, L-glutamic acid, Methionin and/or arginine.

More preferably, the joint propetide has a sequence with 2 to 5 continuous acidic residues, 2 to 5 continuous alkaline residues or 2 to 5 mixing acid alkaline residues in 7 residues of its N-or the terminal cracking site of C-.

According to all aspects of the present invention described here, the sequence of especially preferred employed joint propetide has at least 40% to be made up of the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or the extension that is selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.And the joint propetide has one to have 2 to 5 continuous acidic residues, 2 to 5 continuous alkaline residues or 2 to 5 successive mixing acid alkaline residue sequences in 7 residues of its N-or the terminal cracking site of C-.

Also preferred the use is rich in p1 amino acid A, V, S and T, and the joint propetide that comprises two peptide sequences of forming by two acidic residues, two alkaline residues or acidic residues and alkaline residue, a cracking site is provided in translation, and the polyprotein of expression is translated post-treatment in this site becomes the component protein molecule.

Terminology used here " be rich in " expression A, V, A and T residue exist beguine according to the amino acid stochastic distribution estimated more frequent.

More preferably the joint propetide has one two peptide sequence in 7 amino acid of its N-end and/or C-end, this two peptide sequence is made up of two acidic residues, two alkaline residues or an acidic residues and an alkaline residue, and wherein said two peptide sequences are identical or different in each terminal possibility of joint propetide.

In a preferred embodiment, described two peptide sequences are selected from EE, ED and/or KK.

Special expectation joint propetide has distance two (or a plurality of) albumen territories enough far away, so that they can be suitably folding independently.For this purpose, the joint propetide is fit at least 10 amino acid longs and more preferably at least 15 amino acid longs.Even more advantageously, any secondary building unit that the joint propetide is not connected with it interacts and therefore self does not have special secondary structure or form independent secondary structure, as the α spiral.

Among this and other all aspects and the embodiment of the present invention that here describes, the joint propeptide sequence of cracking site is provided, preferably include one from natural resources such as plant or virus, or its variant, perhaps the separable joint sequence that arrives in the fragment of any in these.Particularly, the joint propetide can be from a kind of vegetable-protein, or fragment, or separates in its variant or the derivative, and this provides the cracking site that is fit to.Special example comprises the joint propetide of a cleavable, from the propetide district of Garden Dahlia gene C-end, as described in the common unsettled UK Patent Application No.9818003.7 and requirement.

When using virus sequence, a preferred chimeric propetide element.

Here " variant " of Biao Daing be meant aminoacid sequence inside one or more amino acid by other amino acid replacement, the basic sequence that makes aminoacid sequence be different from them to originate.Amino acid replacement can be considered to " guarding ", and amino acid is wherein replaced by a different aminoacids with extensive similar characteristics.It is that amino acid is replaced by dissimilar amino acid that non-conservation substitutes.Briefly say, do not change the biological activity of polypeptide, nonconservative substituting can not be arranged.Adaptably, have 85% at least between variant and basic sequence, preferred at least 90% similarity.

In the scope of the invention, two aminoacid sequences that have at least 85% similarity each other have the amino-acid residue of 85% similar (same or conservative alternate) at least a similar position, when optimal arrangement, provide 3 breach.Precondition is with regard to breach, to be no more than 15 amino-acid residues altogether and to be affected.Similarly, two aminoacid sequences that have at least 90% similarity each other have 90% same or conservative alternate amino-acid residue at least a similar position, when optimal arrangement, provide 3 breach.Precondition is with regard to breach, to be no more than 15 amino-acid residues altogether and to be affected.

For this purpose of the present invention, stipulate a conserved amino acid, do not compare with changing albumen, do not change protein-active/function.Can between the amino acid in following each group, guard especially and substitute.

(ⅰ) L-Ala, Serine, glycine and Threonine

(ⅱ) L-glutamic acid and aspartic acid

(ⅲ) arginine and Methionin

(ⅳ) Isoleucine, leucine, Xie Ansuan and methionine(Met)

(ⅴ) phenylalanine, tyrosine and tryptophane

The similarity of sequence can be measured with series arrangement algorithm known in the art, the series arrangement algorithm for example, Myers and the Miller (computing application 4 of bio-science, 11-17 (1988)) and Wilbur and Lipman (institute of NAS periodical 80,726-30 (1983)) described Clustal method, also have Watterman and Eggert method (molecular biology magazine (1987) 197,723-728).Can be from Wisconsin, USA, 53715, Madison, the paired method of MegAlign Lipman Pearson (use default parameters) that the DNAstar company in 1228 Selfpark street obtains also can be used because of the part of method as Lasergene.

Especially, the joint propetide be from a kind of can be from the isolating sequence of vegetable-protein, and more preferably from the proteic precursor of plants antimicrobial, separate.The antibacterial peptide of described plants antimicrobial albumen such as a kind of defensin or a kind of hevein (hevein) type (Broekaert etc. 1997, plant science summary comment 16,297-323).Most preferably from the antibacterial peptide of a kind of defensin and/or a kind of hevein type, especially from the C-CICP of Dm-AMP1 and Ac-AMP2, its sequence is (SEQ ID NO 5 and SEQ ID NO 8) as described in Figure 2 for the joint propetide.

Also preferred the use from the joint propetide in the antibacterial peptide source of impatiens.The Ib-AMP gene comprises 5 propetide districts, and all propetide districts all are applicable to the present invention, and fully describes at the 29th page of international patent application bulletin WO95/24486 and 40-42 page or leaf, and its content is hereby incorporated by.Can use all or part of C-CICP from Dm-AMP and Ac-AMP gene.

In an especially preferred embodiment, the joint propetide of use comprises a joint propeptide sequence that exists naturally, and it has been carried out modification, so that after making its cracking, the remnants minimizing of amino acid on protein product from this sequence preferably do not have remnants.Suitable change can be measured with described ordinary method hereinafter.The simplest in form, separate protein product of the present invention and analyze them whether comprise any residual amino acid from the propetide connector.Joint sequence can be modified then removes some or all of these residues, and precondition is the function that can keep post-translational cleavage.

" fragment " this term is meant the sequence that amino acid has therefrom been deleted, preferably from an one end region deletion amino acid.Such fragment comprises the modification of native sequences above-mentioned.

If the cracking site of a joint propetide of the present invention after as a translation may comprise the one or more this fragment that the source is different.The example of joint propeptide sequence is the SEQ ID NOs3 shown in here, 4,6,7,21,22,23,24,25,26,27,28 and 29 with and serve as the variant of propetide.Wherein concrete example is SEQ IDNOs3,4,6,7,21,22,23,24,25,26,27,28 and 29 self.

Propeptide sequence especially comprises SEQ ID NOs3,4,6 or 7.

Preferred embodiment according to the present invention further provides a kind of a plurality of proteic methods of expressing in transgenic plant, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Its center tap propetide is from the antibacterial peptide of a kind of defensin and/or a kind of hevein type, and this propetide provides a cracking site, and the polyprotein of expression is translated post-treatment and becomes each component protein molecule in this site.

Here, preferred especially use as Fig. 2 described in Dm-AMP1 and the Ac-AMP2 C-CICP of originating as the connector of cleavable, the connection site of a cleavable promptly is provided.Have necessary selection according to propetide, an extra specific proteins enzyme recognition site is all designed in one or both ends in office, is beneficial to the cracking of sequence.Suitable specific proteins enzyme recognition site comprises subtilisin sample proteolytic enzyme recognition site, discern two peptide sequences of forming by two alkaline residues, or tetrapeptide array of being made up of a hydrophobic residue, arbitrary residue, alkaline residue and alkaline residue or one are by an alkaline residue, arbitrary residue, an alkaline residue and the tetrapeptide that alkaline residue is formed.Especially preferably use subtilisin sample proteolytic enzyme recognition site in the method for the invention.

According to a preferred embodiment of the present invention, a kind of a plurality of proteic methods of expressing in transgenic plant further are provided, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment and becomes each component protein molecule in this site.Wherein designed an extra specific proteins enzyme recognition site, be beneficial to the cracking of sequence in the one or both ends of appointing of this joint propetide.

According to a preferred embodiment of the present invention, a kind of a plurality of proteic methods of expressing in transgenic plant further are provided, the genome that is included in described plant inserts a dna sequence dna, and this dna sequence dna comprises a promoter region and connects a signal sequence.This signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Its center tap propetide is from the antibacterial peptide of a kind of defensin and/or a kind of hevein type, and this propetide provides a cracking site, and the polyprotein of expression is translated post-treatment and becomes each component protein molecule in this site.Wherein designed an extra specific proteins enzyme recognition site, be beneficial to the cracking of sequence in the one or both ends of appointing of this joint propetide.

The present invention further provides use isolating propetide from plant origin albumen, as the cleavable connector that passes through in the transgenic plant in the Secretory Pathway synthetic polyprotein precursor.Preferably separation from the antibiotic propeptide of a kind of plant defense element or a kind of hevein type of propetide (Broekaert etc. 1997, plant science summary comment 16,297-323).Propetide may be also preferably separates from a kind of antibacterial peptide that derives from impatiens.

In another aspect of the present invention, provide use a kind of propetide as transgenic plant by the cleavable connector in the Secretory Pathway synthetic polyprotein precursor.Wherein at least 40% propeptide sequence is made up of the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or the extension that is selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.

More preferably the joint propetide has a sequence with 2 to 5 continuous acidic residues, 2 to 5 alkaline residues or 2 to 5 successive mixing acid alkaline residues in 7 amino acid of its N-end or the terminal broken site of C-.

The sequence of especially preferred joint propetide has at least 40% to be made up of the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or the extension that is selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.And the joint propetide has one to have 2 to 5 continuous acidic residues, 2 to 5 alkaline residues or 2 to 5 successive mixing acid alkaline residue sequences in 7 residues of its N-or the terminal cracking site of C-.

Of the present invention one deep aspect, provide and use the joint sequence of a kind of peptide sequence as a cleavable, this peptide sequence is rich in p1 amino acid A, V, S and T, and comprise by two acidic residues, two alkaline residues or an acidic residues and two peptide sequences that alkaline residue is formed.Wherein said sequence is isolating from the antibacterial protein of a kind of plant defense element or a kind of hevein type.

Method of the present invention can be used for obtaining any desired proteic effective expression and secretion, and especially being fit to express those must be synthetic naturally so that be folded into a kind of protein of functional form at Secretory Pathway, and for example glycosylated protein has the albumen of disulfide linkage with those.In addition, the albumen that is very beneficial for participating in the defense function that pathogenic agent invasion and attack plant causes is secreted into ECS effectively, because this initial site of attacking of pathogenic agent normally.These methods of invention are transported a kind of effective means that provide for a plurality of proteic extracellulars.

Method of the present invention also especially is fit to produce little peptide, and the little peptide of production can be used for the resist printing processing intent then.That is, the seed in transgenic plant or its source can directly be used as food, thus the passive immunization receptor.

The method according to this invention can comprise antifungal protein by the expressed proteins example, and it announces WO92/15691 at international patent application, WO92/21699, WO93/05153, WO93/04586, WO94/11511, WO95/04754, WO95/18229, WO95/24486 describes among WO97/21814 and the WO97/21815 to some extent, and antifungal protein comprises Rs-AFP1, Rs-AFP2, Dm-AMP1, Dm-AMP2, Hs-AFP1, Ah-AMP1, Ct-AMP1, Ct-AMP2, Bn-AFP1, Bn-AFP2, Br-AFP1, Br-AFP2, Sa-AFP1, Sa-AFP2, Cb-AMP1, Cb-AMP2, Ca-AMP1, Bm-AMP1, Ace-AMP1, Ac-AMP1, Ac-AMP2, Mj-AMP1, Mj-AMP2, Ib-AMP1, Ib-AMP2, Ib-AMP3, Ib-AMP4, the albumen of PR-1 type, chitinase for example, dextranase such as β-3 and β-1,6 dextranase, chitin bonded lectin, zeatin, infiltration albumen, the nonactive albumen of thionine and rrna and from the peptide of the performance 85% sequence identity in they or antifungal protein source preferably surpasses 90% sequence identity, the any described albumen of sequence identity, wherein the row identity of preface such as top defined more preferably greater than 95%.

The joint of cleavable is used for connecting two or more protein of interest and provides cracking site to make polyprotein translation back be processed into each component protein molecule at this.

Provide a DNA construct at one of the present invention aspect deep, an included dna sequence dna of this construct includes the signal sequence that a promoter region connects a plant origin, described signal sequence be connected to two or more protein-coding regions and one 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Wherein said joint propetide provides a post-translational cleavage site.

Compatibly, the protein-coding region different albumen of encoding.The preferred embodiment of propetide joint sequence as detailed above.

In an embodiment preferred of this respect of the present invention, provide a kind of DNA construct, inside propetide of the dna sequence encoding of wherein said coding joint propetide from the Ib-AMP gene.Provide a kind of DNA construct in an embodiment that is more preferably of this respect of the present invention, the dna sequence encoding of wherein said coding joint propetide is from the C-CICP of Dm-AMP or Ac-AMP gene.

In a particularly preferred embodiment of the present invention, provide aforesaid a kind of DNA construct, wherein when the dna sequence dna of coding joint propetide derived from Dm-AMP gene or Ac-AMP gene, it all additionally comprised one or more proteolytic enzyme recognition sites at arbitrary end points or two end points.

Provide a kind of DNA construct at one of the present invention aspect going deep into, an included dna sequence dna of this construct includes a promoter region and is connected to two or more protein-coding regions and 3 a ＇-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide, and this dna sequence encoding derives from the C-CICP of Dm-AMP gene or Ac-AMP gene.Wherein said joint propetide provides a post-translational cleavage site.

Aforesaid a kind of DNA construct is provided in a particularly preferred embodiment of the present invention, wherein derive from the dna sequence dna of the coding joint propetide of Dm-AMP gene or Ac-AMP gene, all additionally comprise one or more proteolytic enzyme recognition sites at its arbitrary end points or two end points.

Aspect of the present invention another go deep into, provide a kind of according to the present invention the DNA construct institute transgenic plant transformed of above-mentioned any aspect.

A kind of transgenic plant that transform by a dna sequence dna are provided aspect going deep at one of the present invention, this dna sequence dna includes a promoter region and is connected to a signal sequence, described signal sequence is connected to two or more protein-coding regions and 3 a ＇-terminator, and wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide.Wherein said joint propetide provides a cracking site in translation.

A preferred embodiment in this respect, described joint propetide have at least 40% sequence to be made up of the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or the extension that is selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.

The preferred L-Ala of described hydrophobic residue, Xie Ansuan, leucine, methionine(Met) and/or Isoleucine, and the preferred aspartic acid of described hydrophilic residue, L-glutamic acid, Methionin and/or arginine.

More preferably the joint propetide has a sequence with 2 to 5 continuous acidic residues, 2 to 5 alkaline residues or 2 to 5 successive mixing acid alkaline residues in 7 amino acid of its N-end or the terminal broken site of C-.

Preferred especially joint propetide has at least 40% sequence to be made up of the extension of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or the extension that is selected from 2 to 5 hydrophilic residues of aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.And the joint propetide has one to have 2 to 5 continuous acidic residues, 2 to 5 alkaline residues or 2 to 5 successive mixing acid alkaline residue sequences in 7 residues of its N-or the terminal cracking site of C-.

In a preferred embodiment of this respect of the present invention, peptide sequence of dna sequence encoding of cracking site is provided, this peptide sequence is rich in p1 amino acid A, V, S and T, and comprise by two acidic residues, two alkaline residues or an acidic residues and two peptide sequences that alkaline residue is formed.

In a particularly preferred embodiment of this respect of the present invention, peptide sequence that derives from the Ib-AMP gene of dna sequence encoding of cracking site is provided, for example Fig. 2 is described.At one of this respect of the present invention more in the particularly preferred embodiment, the dna sequence encoding that cracking site is provided is from as described in Figure 2 Dm-AMP1 and the C-CICP of Ac-AMP2, and this C-CICP may be contained the dna sequence dna of a coding subtilisin-sample proteolytic enzyme recognition site by random design packet.

One of the present invention is goed deep into the aspect carrier that comprises above-mentioned DNA construct is provided.

Some joint sequence as described herein is new, and these joint sequences and encoding sequence thereof have formed a deep aspect of the present invention.Therefore, provide a kind of nucleic acid especially, a joint propetide or joint propetide and the variant thereof shown in Figure 34 of its coding SEQ ID NO 4,6,7,29,21,22,23,24,25,26,27,28.Special variant is connected with SEQ ID NO 77 at the C-end.

As those skilled in the art will obviously see, the sequence of each component of dna sequence dna, promptly, can predict that according to the used signal sequence of the inventive method, joint sequence, protein sequence, terminator sequence the DNA of proteins encoded can make with a kind of nucleic acid synthesizer of standard from its known amino acid sequence.Alternately, code book invent the DNA of each component can be from natural resources appropriate separation produce.

Following by reference unrestriced embodiment and accompanying drawing are further illustrated the present invention.Wherein

Fig. 1: nucleotide sequence (SEQ ID NO 1) and the amino acid sequence corresponding (SEQ ID NO 2) of showing the DmAMP1 gene coding region.Corresponding to the amino acid of ripe DmAMP1 by underscore.Corresponding to the following stroke two-wire of the nucleic acid of intron.

Fig. 2: the sketch that shows coding region (SEQ ID NO 3-8) from carrier structure.The propeptide sequence that on behalf of the joint propetide, the aminoacid sequence below inner propetide originated.

Fig. 3: the diagram that has shown plant conversion carrier pFAJ3105.

Fig. 4: the diagram that has shown plant conversion carrier pFAJ3106.

Fig. 5: the diagram that has shown plant conversion carrier pFAJ3107.

Fig. 6: the diagram that has shown plant conversion carrier pFAJ3108.

Fig. 7: the diagram that has shown plant conversion carrier pFAJ3109.

Fig. 8: shown the nucleotide sequence (SEQ ID NO 9) and the amino acid sequence corresponding (SEQID NO 10) that are included in the open reading frame zone between pFAJ3105 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.

Fig. 9: shown the nucleotide sequence (SEQ ID NO 11) and the amino acid sequence corresponding (SEQID NO 12) that are included in the open reading frame zone between pFAJ3106 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.

Figure 10: shown the nucleotide sequence (SEQ ID NO 13) and the amino acid sequence corresponding (SEQID NO 14) that are included in the open reading frame zone between pFAJ3107 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.

Figure 11: shown the nucleotide sequence (SEQ ID NO 15) and the amino acid sequence corresponding (SEQID NO 16) that are included in the open reading frame zone between pFAJ3108 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.

Figure 12: shown the nucleotide sequence (SEQ ID NO 17) and the amino acid sequence corresponding (SEQID NO 18) that are included in the open reading frame zone between pFAJ3109 plasmid Nco I and the Sac I site.At amino acid underscoring corresponding to ripe DmAMP1.

Figure 13: shown the Dm-AMP1 expression level (as the per-cent of total soluble proteins) that some use the transgenosis individuality of pFAJ3105 transgenic plant transformed that makes up and the pFAJ3109 conversion that some use structure.

Figure 14: shown that anti-phase-high pressure liquid chromatography (RP-HPLC) that the C8-silica column to the leaf crude extract that transforms with pFAJ3105 (A) or pFAJ3109 (B) carries out analyzes.Prepare described in extract such as material and the method.Come wash-out pillar (0-35 minute, the acetonitrile of 15%-50% is dissolved in 0.1% the trifluoroacetic acid) with the acetonitrile of finite concentration gradient in 0.1% trifluoroacetic acid.Eluate by at-once monitor measuring the photoabsorption (trajectory diagram on top) of 214nm, fractional separation, and carry out enzyme-linked immunosorbent assay and detect DmAMP1 (histogram of bottom, solid post) and RsAFP2 (histogram of bottom, open tubular column).With arrow at A ₂₁₄Express the definite wash-out position of DmAMP1 and RsAFP2 on the color atlas.

Figure 15: shown reverse-phase chromatography (RPC) result who makes Arabidopsis plant (transformant 14) the extracellular fluid body portion of conversion with 3105 structures.(Microsorb-MV, 4.6 * 250mm Rainin) go up implementation to reverse-phase chromatography at a usefulness 0.1% trifluoroacetic acid equilibrated C8-silica column.Behind the dress post, with 0.1% trifluoroacetic acid wash-out pillar 20 minutes, be applied in the acetonitrile wash-out 35 minutes of 15% to 50% linear gradient in 0.1% trifluoroacetic acid then with the flow velocity of 1ml/min.The instant concentration (dotted line) of measuring the photoabsorption (solid line) of 280nm and measuring acetonitrile with a conduction monitor immediately.Collect each several part and detect DmAMP1-CRP and RsAFP2-CRP with enzyme-linked immunosorbent assay.Use the existence of the peak number expression DmAMP1-CRP of runic, number represent the existence of RsAFP2-CRP with the peak of italic.

Figure 16: reverse-phase chromatography (RPC) result who has shown the extract of the Arabidopsis plant (transformant 14) that transforms with 3105 constructs.Sample is two distinct portions from ion exchange chromatography, and the existence of indication DmAMP1-CRPs or RsAFP2-CRPs that is to say that those parts are wash-out between the NaCl of the NaCl of 0.17-0.33M (A) and 0.33-0.49M.Reverse-phase chromatography is carried out according to the caption of Figure 14.The instant concentration (dotted line) of measuring the photoabsorption (solid line) of 280nm and measuring acetonitrile with a conduction monitor immediately.Collect each several part and detect DmAMP1-CRP and RsAFP2-CRP with enzyme-linked immunosorbent assay.Use the existence of the peak number expression DmAMP1-CRP of runic, number represent the existence of RsAFP2-CRP with the peak of italic.

Figure 17: shown aminoacid sequence by the polyprotein precursor of pFAJ3105, pFAJ3106 and pFAJ3108 construct coding.Dotted line is represented to have done omission from complete sequence for simplicity.The sequence that italic is write is the guiding peptide of DmAMP1, and the sequence of underscore is sophisticated DmAMP1, and the sequence of black matrix is the joint peptide.The sequence of drawing two-wire down is sophisticated RsAFP2.Arrow refers to the processing site that the mass spectroscopy according to the DmAMP1-CRP of N-end sequencing and purifying and RsAFP2-CRP obtains.

Figure 18: reverse-phase chromatography (RPC) result who has shown Arabidopsis plant (transformant 9) the extracellular fluid body portion that transforms with the pFAJ3106 construct.The execution of reverse-phase chromatography and the analysis of each several part are as described in the caption of Figure 15.Use the existence of the peak number expression DmAMP1-CRP of runic, number represent the existence of RsAFP2-CRP with the peak of italic.

Figure 19: reverse-phase chromatography (RPC) result who has shown Arabidopsis plant (transformant 9) extract that transforms with 3108 constructs.Sample is a part from ion exchange chromatography, and the existence of indication DmAMP1-CRPs or RsAFP2-CRPs that is to say, those parts are the wash-out and the existence of indicating DmAMP1-CRPs between the NaCl of 0.17-0.33M.The execution of reverse-phase chromatography and the analysis of each several part are as described in the caption of Figure 15.Use the existence of the peak number expression DmAMP1-CRP of runic.

Figure 20: the coding region synoptic diagram that is pFAJ3105, pFAJ3343, pFAJ3344, pFAJ3345, pFAJ3346 and pFAJ3369 construct.Complete arrow represents to test definite cracking site.Open arrow is represented the cracking site supposed.Abbreviation: the signal peptide district (referring to Fig. 1) of SP DmAMP1:DmAMP1; The maturation protein district (referring to Fig. 1) of DmAMP1:DmAMP1; The maturation protein district of RsAFP2:RsAFP2 (Terras etc., 1995, vegetable cell, 7,573-588).Fully shown joint peptide sequence (SEQ ID NO 3,29,21-24 shows respectively).

Figure 21: be synoptic diagram with pFAJ3367 construct coding region of SEQ ID NO 24 joint peptides.Abbreviation: the signal peptide district (referring to Fig. 1) of SP DmAMP1:DmAMP1; The maturation protein district (referring to Fig. 1) of DmAMP1:DmAMP1; The maturation protein district of RsAFP2:RsAFP2 (Terras etc., 1995, vegetable cell, 7,573-588); The maturation protein district of HsAFP1:HsAFP1 (Osborn etc., 1995, European biological chemistry association associating proceedings 368,257-262); The maturation protein district of AceAMP1:AceAMP1 (Cammue etc., plant physiology 109,445-455).

Figure 22: the synoptic diagram that is pFAJ3106-2, pFAJ3107-2 and pFAJ3108-2 construct coding region.Abbreviation: the signal peptide district (referring to Fig. 1) of SP DmAMP1:DmAMP1; The maturation protein district (referring to Fig. 1) of DmAMP1:DmAMP1; The maturation protein district of RsAFP2:RsAFP2 (Terras etc., 1995, vegetable cell, 7,573-588); The recognition sequence of RSKex2p:Kex2 proteolytic enzyme (IGKR) (Jiang and Rogers, 1999, plant physiology 18,23-32); The maturation protein district of AcAMP1:AcAMP1 (De Bolle etc., molecular biology of plants 31,997-1008).Distinguish fully show such in joint propeptide sequence such as SEQ ID NOs 25,26 and 27.

Figure 23: the synoptic diagram that is pFAJ3368 and pFAJ3370 construct coding region.The cracking site of open arrow indication supposition.Abbreviation: the signal peptide district (referring to Fig. 1) of SP DmAMP1:DmAMP1; The maturation protein district (referring to Fig. 1) of DmAMP1:DmAMP1; The maturation protein district of RsAFP2:RsAFP2 (Terras etc., 1995, vegetable cell, 7,573-588); 2A sequence: the cracking recognition site of foot and mouth disease virus polyprotein.The joint propeptide sequence such as among the SEQ IDNO 28 difference fully shows.

Figure 24: shown the nucleotide sequence (SEQ ID NO 30) and the amino acid sequence corresponding (SEQID NO 31) that are included in the open reading frame zone between pFAJ3343 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO29) corresponding to the internal connection peptide.

Figure 25: shown the nucleotide sequence (SEQ ID NO 32) and the amino acid sequence corresponding (SEQID NO 33) that are included in the open reading frame zone between pFAJ3344 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO21) corresponding to the internal connection peptide.

Figure 26: shown the nucleotide sequence (SEQ ID NO 34) and the amino acid sequence corresponding (SEQID NO 35) that are included in the open reading frame zone between pFAJ3345 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO22) corresponding to the internal connection peptide.

Figure 27: shown the nucleotide sequence (SEQ ID NO 36) and the amino acid sequence corresponding (SEQID NO 37) that are included in the open reading frame zone between pFAJ3346 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO23) corresponding to the internal connection peptide.

Figure 28: shown the nucleotide sequence (SEQ ID NO 38) and the amino acid sequence corresponding (SEQID NO 39) that are included in the open reading frame zone between pFAJ3369 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO24) corresponding to the internal connection peptide.

Figure 29: shown the nucleotide sequence and the amino acid sequence corresponding that are included in the open reading frame zone between pFAJ3347 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1, ripe RsAFP2, ripe HsAFP1 and ripe AceAMP1, drawing single line, two-wire, dotted line and dotted line respectively.Amino acid runic (SEQID NO 24) corresponding to the internal connection peptide.

Figure 30: shown the nucleotide sequence (SEQ ID NO 42) and the amino acid sequence corresponding (SEQID NO 43) that are included in the open reading frame zone between pFAJ3106-2 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO 4) corresponding to the internal connection peptide.

Figure 31: shown the nucleotide sequence (SEQ ID NO 44) and the amino acid sequence corresponding (SEQID NO 45) that are included in the open reading frame zone between pFAJ3107-2 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO 6) corresponding to the internal connection peptide.

Figure 32: shown the nucleotide sequence (SEQ ID NO 46) and the amino acid sequence corresponding (SEQID NO 47) that are included in the open reading frame zone between pFAJ3108-2 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO 7) corresponding to the internal connection peptide.

Figure 33: shown the nucleotide sequence (SEQ ID NO 48) and the amino acid sequence corresponding (SEQID NO 49) that are included in the open reading frame zone between pFAJ3370 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.The joint peptide is represented (SEQ ID NO 28) with bold, and has the amino acid of representing with bold Italic corresponding to the 2A sequence.

Figure 34: shown the nucleotide sequence (SEQ ID NO 50) and the amino acid sequence corresponding (SEQID NO 51) that are included in the open reading frame zone between pFAJ3368 plasmid Nco I and the Sac I site.Below the amino acid of ripe DmAMP1 and ripe RsAFP2, drawing single line and two-wire respectively.Amino acid runic (SEQ ID NO24) corresponding to the internal connection peptide.The joint peptide is represented with bold, and is had the amino acid of representing with bold Italic corresponding to the 2A sequence.

The present invention will be described for the following example.

Embodiment 1

Clone DmAMP1 cDNA and DmAMP1 gene

Clone's program and polymerase chain reaction,PCR (PCR) program is pressed standard schedule and is carried out (Sambrook etc., 1989, molecular cloning: laboratory manual, the 2nd edition, the publication of Cold Spring Harbor laboratory, Cold Spring Harbor, New York).Set up a cDNA library with what collect near the exsiccant seed from smooth Garden Dahlia (Dahliamerckii).(1991, molecular biology of plants report 9 131-138), is purified into total RNA from these seeds with people's such as Jepson I method.Smooth Garden Dahlia from 2g

Obtain total RNA of 0.6mg in the seed.From the total RNA of 0.2mg, isolate the poly-A+RNA of 2 μ g nearly with PolyATract magnetic bead (Promega).

Utilize a ZAP-cDNA synthetic agent box (Stratagene), make up a cDNA library with poly-A+RNA.After first and second strands of chains are synthetic, cDNA and carrier DNA are linked together.After the assembling of the phage that carries out with Gigapack Gold (Stratagene) package pickup thing, nearly obtain 1 * 10 ⁵Individual plaque-forming unit (pfu).Use is based on oligonucleotide AFP-5 (5 '-TG (T of the N-end sequence CEKASKTW (SEQ ID NO.14) of DmAMP1, C) GANAANGCN (A, T) (G, C) NAA (A, G) (SEQ ID NO.13) (Osborn R.W. etc. ACNTGG), 1995, Europe biological chemistry association associating proceedings 368,257-262) with based on AFP-3EX (5 '-CA (A of the C-end sequence MCFCYFNC (SEQ ID NO.53) of DmAMP1, G) AANTANCANAAA (A, G) CACAT) (SEQ ID NO.52) and from smooth Garden Dahlia leaf isolating genomic dna, generate a kind of PCR product of 144 base pairs, and from sepharose, separate.The PCR product is cloned into pBluescript.10 transformant inserting are checked order.Sequence is represented 3 intimate homologous DmAMP1-genoids, the viewed ripe DmAMP1 of PCR clone 4 codings wherein.Be used to survey from containing cDNA library altogether 6 * 10 with the PCR product mixtures of 144 base pairs of 32P CTP mark ⁴Hybond N (Amersham) filter disc that makes in the culture plate of individual plaque-forming unit.30 possible positive signal have been obtained.22 plaques of picking also carry out the screening of two-wheeled again.After the excision, 13 clones characterize by dna sequencing in the body.4 class DmAMP1 related peptides are by 13 cDNA clones codings.Embody in 4 types in the DmAMP1 maturation protein district of three kinds of forms.One type (Dm2.5 type) comprise one may corresponding to the maturation protein district of DmAMP2 (OsbornR.W. etc., 1995, European biological chemistry association associating proceedings 368,257-262).None cDNA a kind of maturation protein district that is equivalent to observed ripe DmAMP1 peptide sequence of encoding.

Use PCR clone 4 sequence (above-mentioned) and from the peptide N-of cDNA inference terminal and C-final word, designed the gene that two pairs of oligonucleotides are used for amplification coding DmAMP1.Genomic dna and oligonucleotide MATAFP-5P (5 '-ATGGC (C from smooth Garden Dahlia, G) AAN (A, C) (A, G) NTC (A, G) GTTGCNTT) (5 '-AAACACATGTGTTTCCCATT) (SEQ ID NO.54) is applied to PCR reaction together, and the PCR product is cloned into pBluescript and to cloning and sequencing for (SEQ ID NO.66) and MATAFP-5.A clone who contains DmAMP1 gene 5 ' part is identified.((5 '-TAAAGAAACCGACCCTTTCACGG) (SEQ ID NO.56) is used to the PCR reaction, and the PCR product is cloned into pBluescript and to cloning and sequencing for 5 '-AGCGTGTCATGTGCGTAAT) (SEQ ID NO.55) and DM25MAT-3 with MATAFP-3 from the genomic dna of smooth Garden Dahlia.A clone who contains DmAMP1 gene 5 ' part is identified.5 ' and 3 ' zone of ripe gene is combined the coding region sequence (Fig. 1) of assembling DmAMP1 gene.

The precursor of DmAMP1 genes encoding contains one 28 amino acid whose guiding peptides, 50 amino acid whose maturation proteins and 40 amino acid whose C-CICPs.The intron that open reading frame is positioned in 92 base pairs of guiding peptide intra-zone interrupts.In order from the DmAMP1 gene order, to delete intron, and allow into expression cassette carrier is cloned in a coding region of containing or do not conform to the DmAMP1 in C-CICP district, carried out two PCR reactions.It uses primer DMVEC-3 (5 ' ATGCATCCATGGTGAATCGGTTGCGTTCTCCGCGTTCGTTCTGATCCTTTCGTGCT CGATCTCAGATATCGCATCCGTTAGTGGAGAACTATGCGAGAAA) (SEQ ID NO.57) and DMVEC-2 (5 ' AAACCGACCGAGCTCACGGATGTTCAACGTTTGGAAC) (SEQ ID NO.58) respectively, and DMVEC-3 and DMVEC4 (5 '-AGCAAGCTTTTCGGGAGCTCAATTGAAGTAA) (SEQ ID NO.59).The top chain of DMVEC-3 guiding DmAMP1 gene imports a Nco I site corresponding to the guiding peptide district that does not have intron and at transcriptional start point.The C-CICP district of DMVEC-2 gene guiding DmAMP1 gene bottom chain 3 '-end is also transcribing the Sac I site of importing, back that stops codon.The maturation protein district of DMVEC-4 gene guiding DmAMP1 gene bottom chain 3 '-end, and, import a Sac I site in the back of terminator codon at termination codon of this back, district fusion.

Two PCR products are all used Nco I and the cutting of Sac I, and they are cracked into two fragments to the PCR product owing to the existence in an inner Nco I site in maturation protein district.Nco I-Sac the I and the Nco I-Nco I fragment that produce sequentially are cloned into the pMJB1 plasmid.The pMJB1 plasmid is an expression cassette carrier, contain a Hind III site successively, enhanced flower coconut palm Cauliflower leaf disease 35S RNA (CaMV35S) promotor (Kay R etc., 1987, science 236,1299-1302), an Xho I site, tobacco mosaic virus (TMV) (TMV) 5 ' the untranslated homing sequence (Gallie D.R. and Walbot V., 1992, nucleic acid correlative study 20,4631-4638), a poly connector comprises the Nco I, the Sma I, Kpn I and Sac I site, root nodule bacterium 3 ' untranslated terminator (the Bevan M.W. etc., 1983, nature 304,184-187) and an EcoR I site.The plasmid that produces is hereinafter referred to as pDMAMPE (guiding peptide district, maturation protein district and C-CICP district) and pDMAMPD (guiding peptide district and maturation protein district).Identify by dna sequencing the coding region.

Embodiment 2

Make up plant conversion carrier

In order to study the possibility of in plant, expressing the polyprotein gene, make the plain coexpression of four different plant conversion carriers plant defense different with two.These two plant defense elements that have antifungal property and be rich in halfcystine are exactly a RsAFP2 and DmAMP1. guiding peptide from DmAMP1 cDNA, the maturation protein district of DmAMP1, the maturation protein district of inner propetide district and RsAFP2 all is the feature of the polyprotein prosoma of these constructions.Four kinds of constructs have only inner propetide difference (Fig. 2).

3105 constructs are separated DmAMP1 and RsAFP2 with the inner propetide of IbAMP.

3106 constructs have a propetide at the C-end, are made up of the part of DmAMP1 propetide and the subtilisin of a supposition-sample proteolytic enzyme processing site (IGKR) (SEQ ID NO.67).

3107 constructs are except adopting complete DmAMP1 propetide, and other is identical with 3106 structures.

3108 constructs have a propetide at the C-end, are made up of the subtilisin-sample proteolytic enzyme processing site (IGKR) of a DmAMP1 propetide and a supposition.

3106, the theoretical basis of 3107 and 3108 constructions is based on our discovery, when in tobacco, expressing as Ac-AMP21-and DmAMP1-preproprotein respectively, the C-CICP of AcAMP2 and DmAMP1 cleavedly falls in that N-is terminal, and this processing does not reduce distribution (the De Bolle etc. of maturation protein to the non-protogenous plastid, 1996, molecular biology of plants 31,993-1008; R.W.Osborn and S.Attenborough, person-to-person communication).This expression processive enzyme is at Secretory Pathway or in the non-protogenous plastid.On the other hand, the cracking of the inner propetide C-of these constructs end may be carried out by the proteolytic enzyme of a subtilisin sample, such proteolytic enzyme member (Kex2) in the known yeast is at Golgi's organs crack protein (Wilcox C.A and Fuller R.S., 1991, cytobiology magazine 115,297), and a member in the tomato carries out cracking (Tornero P. etc. at apoplast, 1997, journal of biological chemistry 272,14412-14419).Synthetic by the Secretory Pathway that comprises Golgi's organs usually at the sedimentary albumen of non-protogenous plastid.The non-protogenous plastid be the deposition site preferred of the antibacterial protein of design in transgenic plant (Jongedijk E etc., 1995, Euphytica 85,173-180; De Bolle etc., 1996, molecular biology of plants 31,993-1008).Also make up a construct of only expressing DMAMP1 (3109 constructs, Fig. 7).

Fig. 3-7 has shown the synoptic diagram of prepared plant conversion carrier pFAJ3105, pFAJ3106, pFAJ3107, pFAJ3108 and pFAJ3109 respectively.Be included in the nucleotide sequence between these plasmids Xho I and the Sac I site, comprise the antibacterial protein coding region, in Fig. 8-13, introduce.Be included in the zone (shown in Figure 8) between pFAJ3105 plasmid Xho I and the Sac I site, (1994, biotechnology 16 1010-1011) makes up according to the two-step pcr program of Pont-Kindom G.A.D..Primer OWB175 (5 ' AGGAAGTTCATTTGG) and (SEQ ID NO.68), OWB278 (5 ' GCCTTTGGCACAACTTCTGTCCTGGCTCCACGTCCTCTGGGGTAGCCACCTCGTCA GCAGCGTTGGAACAATTGAAGTAACAAACAC) (SEQ ID NO.60) is employed in first PCR reaction, and with plasmid pDMAMPE as template.Plasmid pFRG4 (Terras F.R.G. etc. are used in second PCR reaction, 1995, vegetable cell 7,573-588) as template, with the mixture of first PCR reaction product, primer OWB175 and primer OWB172 (5 ' TTAGAGCTCCTATTAACAAGGAAAGTAGC (SEQ ID NO.61), Sac I site underscore) as primer.The PCR product that forms is with Xho I and the digestion of Sac I and be cloned into expression cassette carrier pMJB1 (referring to top).The plasmid that is produced is known as pFAJ3099, expression cassette wherein digests with EcoR I (side is connected 3 ' end of rouge alkali synthetase terminator) with Hind III (side connects 5 ' end of flower coconut palm Cauliflower mosaic virus 35S promoter), and be cloned into corresponding site (the Becker D etc. of plant conversion carrier pGPTVbar, 1992, molecular biology of plants 20 is 1195-1197) to produce the pFAJ3105 plasmid.

The structure of plasmid pFAJ3106, pFAJ3107 and pFAJ3108 is similar, except the primer OWB278 in the PCR reaction first time replaces with following primer respectively:

OWB279(??5’GCCTTTGGCACAACTTCTGCCTCTTTCCGATGAGTTCGGCTTTAAGTTTGTC)(SEQ?ID?NO.62)；

OWB303(????5’GCCTTTGGCACAACTTCTGCCTCTTTCCGATCGGAGTTCAACGTTTGGAACC)(SEQ?ID?NO.63)；

OWB304(5’GCCTTTGGCACAACTTCTGCCTCTTTCCGATAGTTTTGGTGGCAGCAACATCAGGCTTGGTGATCCACAGTAGTAGTACTGGCACAATTGAAGTAACAGAAACAC)(SEQ?ID?NO.64)；

Plasmid pFAJ3109 makes up by the corresponding site (referring to top) of the Hind of plasmid pDMAMPD III-EcoR I fragment (referring to top) being cloned into plant conversion carrier pGPTVbar.

Embodiment 3

Plant Transformation

According to the inflorescence infiltration method of Bechtold N. etc. (1993, C.R. science association 316,1194-1199), with the Columbia-O ecotype of the Agrobacterium tumefaciems arabidopsis thaliana transformation (Arabidopsisthaliana) of reorganization.Transformant is selected in sand/perlite mixture, is that the water sub-irrigation of weedicide Basta (Agrevo) of 5mg/l is there to be active phosphinothricin composition with containing final concentration.

Embodiment 4

Measure target protein, comprise enzyme linked immunological absorption (ELISA) mensuration and analysis of protein

With RsAFP2 (according to Tetras F.R.G. etc., 1992, journal of biological chemistry 267, description purifying among the 15301-15309) or DmAMPl according to Osborn R.W. etc., 1995, Europe biological chemistry association associating proceedings 368, the description purifying among the 257-262) causes antiserum(antisera) in the rabbit body of injection.Basic as Pennninckx I.A.M.A. etc. (1996, vegetable cell 8,2309-2323) described like that, enzyme linked immunological is adsorbed (ELISA) mensuration and sets up as a kind of state of conflict analysis.Handle the ELISA microtiter plate with the 50ng/mlRsAFP2 or the DmAMPl that are dissolved in the coating damping fluid.Elementary antiserum(antisera) is diluted to 1000-and 2000-solution doubly in the PBS that contains 3% (mass/volume) gelatin and 0.05% (volume ratio) Tween20 (DmAMP1 and RsAFP2 use respectively).

Total protein concentration according to Bradford (1976, biological chemistry annual 72, method 248-254) is measured as standard substance with bovine serum albumin.

The leaf of arabidopsis thaliana in liquid nitrogen homogenate and with a kind of by 10mM NaH ₂PO ₄, 15mMNa ₂HPO ₄, the damping fluid extracting formed of 100mM KCl, 1.5mM NaCl.Tissue homogenate is 85 ℃ of heating 10 minutes and in cooled on ice.The extract of heat treated was centrifugal 15 minutes of 15000 * g and be injected into an anti-phase high pressure liquid chromatography post (RT-HPLC), and (0.46 * 25cm Rainin) forms this post by the C8 silicon-dioxide of crossing with 0.1% (volume ratio) trifluoroacetic acid (TFA) balance.Pillar with the flow velocity of 1ml/min with the acetonitrile wash-out of 15% to 50% (volume ratio) linear gradient in 0.1% (volume ratio) trifluoroacetic acid 35 minutes.Measure the photoabsorption of eluate at 214nm, collect each fraction of 1ml, fractional separation is also in the end dissolved in the water again.These fractions are used for enzyme linked immunological absorption (ELISA) and measure.

Embodiment 5

Extract in the preparation cell

Contain extraction damping fluid (10mMNaH by the leaf of Arabidopsis plant being immersed one ₂PO ₄, 15mM Na ₂HPO ₄, 100mM KCl, 1.5mM NaCl) beaker in, liquid in the collecting cell from plant leaf.The beaker that fills leaf is placed in the vacuum vessel and continuous six takes turns and vacuumize 2 minutes, and vacuum discharges suddenly then.The leaf that soaks into is put in the centrifuge tube lightly, separates with pipe end scale.1800 * g after centrifugal 15 minutes in the collecting cell of bottom liquid.Vacuum impregnation is also centrifugal again with leaf, and (extracellular) liquid that obtains combines with the part of the first step vacuum gained.After this step, back and forth in the oscillator leaf is carried out extracting at a Phastprep (BI0101/Savant), and purify extract by centrifugal (10,000 * g10 minute).The supernatant that produces is considered to extract in the born of the same parents.

In the leaf of the T1 transgenic arabidopsis platymiscium of the above-mentioned thaumatropy of a series of usefulness, analyze the expression level of DmAMP1 and RsAFP2.In table 1, provide above-mentioned expression analysis result based on enzyme-linked immunosorbent assay.

Table 1:Dm-AMP1 and Rs-AFP2 belong to the expression level of planting among the dwarf at transgenic arabidopsis

Construct	Plant	?Expression ?level?of?Dm- ???AMP1(％)	expression level?of?Rs- ??AFP2(％)	Construct	Plant	Expression level?of?Dm- ?AMP1(％)	?expression ?level?of?Rs- ???AFP2(％)
								??3105	???1	????0,77	????0,29	???3107	??1	????0,04	????0,04
	???2	????1,13	????0,22		??2	????0,75	????0,42
									???3	????0,48	????0,20		??3	????0,14	????0,13
	???4	????0,005	??＜0,001		??4	????0,01	????0,01
									???5	????0,36	????0,05		??5	????0,27	????0,29
	???6	????0,99	????0,25	???3108	??1	????0,47	????0,10
									???7	????0,60	????0,09		??2	????3,00	????0,53
	???8	????0,13	??＜0,001		??3	????0,91	????0,24
									???9	????0,25	????0,08		??4	????2,04	????0,22
	???10	????4,15	????0,85		??5	????0,17	????0,04
									???11	????1,35	????0,35		??6	????0,55	????0,05
	???12	????0,24	????0,07		??7	????0,16	????0,11
									???13	????4,43	????0,91		??8	????0,05	????0,02
	???14	????1,18	????0,24		??9	????0,45	????0,02
									???15	????0,68	????0,17	???3109	??1	????0,19	????nd
	???16	????0,49	????0,07		??2	????0,05	????nd
								??3106	???1	????0,10	????0,001		??3	????0,02	????nd
	???2	????1,82	????0,008		??4	????0,20	????nd
									???3	????0,68	????0,20		??5	????0,10	????nd
	???4	????1,15	????0,38		??6	????0,06	????nd
									???5	????0,20	????0,10		??7	????0,07	????nd
	???6	????0,10	????0,05		??8	????0,003	????nd
									???7	????0,40	????0,17		??9	????0,18	????nd
	???8	????2,64	????0,50
									???9	????0,40	????0,15
	???10	????0,21	????0,07
									???11	????0,06	????0,03
	???12	????0,24	????0,09

Last table not " nd " refer to test.

DmAMP1-CRPs (DmAMP1-cross-reacting protein) and RsAFP2-CRPs (PsAFP2-cross-reacting protein) are obviously expressed in the test strain that transforms with 3105,3106,3107 and 3108 polyprotein constructs mostly.A good dependency is arranged between DmAMP1-CRP and the RsAFP2-CRP expression level basically.Yet the level of RsAFP2-CRP is lower 2-5 times than the level of DmAMP1-CRP substantially.And the enzyme-linked immunosorbent assay of RsAFP2-CRPs is credible not as the enzyme-linked immunosorbent assay of DmAMP1-CRPs in the detection extract.In the enzyme-linked immunosorbent assay of RsAFP2, the dose-response curve that transgenic plant extract diluent produces has departed from those and has contained the dose-response curve of the standard diluent of real RsAFP2, shows that the most of Rs-AFP2-CRPs in the extract are different from RsAFP2 self on immunology.With 3106,3107 with the extract of 3108 construct plant transformed with compare with the extract of 3105 construct plant transformed, the former is more obvious from departing from of RsAFP2 standard dose-response curve.

Do not have a kind of extract in the enzyme-linked immunosorbent assay of Dm-AMP1, on dose-response curve, depart from the standard substance of Dm-AMP1.Compare with single albumen construct 3109 plants transformed with those with 3105,3106,3107 or 3108 polyprotein construct plants transformed, the former DmAMP-CRP level is far above the latter.This is also in Fig. 3 illustrated, wherein to comparing with the DmAMP1-CRP expression level of polyprotein construct 3105 plant transformed with the DmAMP1-CRP expression level of single albumen construct 3109 plant transformed.Up to now, also there is not bibliographical information to say that the expression level of a kind of peptide of transgenic plant can be up to 4% (for example, the DmAMP1-CRP level among plant 3105-15 and the 3105-18 be referring to table 1) of total protein.As if therefore, use the polyprotein construct and caused obvious enhanced expression level, this is unexpected a discovery.

Embodiment 6

Separation is from the albumen of polyprotein precursor processing

Transfer-gen plant is selected from the colony that transforms with 3105 structures (plant 1) or 3106 structures (plant 2).And selected plant is further bred to obtain transgenosis homozygote plant.Whether correctly processed in these plant in order to analyze DmAMP1 and RsAFP2, prepare the extract of plant as described in example 1 above and separate by the anti-phase-high pressure lipuid chromatography (HPLC) of on the C8-silica column, carrying out.Collect each fraction and, identify whether there is the compound that cross reaction is arranged with DmAMP1 antibody or RsAFP2 antibody with enzyme-linked immunosorbent assay as described in the embodiment 4.As shown in figure 15, transform with 3105 constructs and with 3106 construct transformed plants in, DmAMP1-CRPs one with the real identical or very close position wash-out of DmAMP1.Equally, 3105 with the transformant of 3106 constructs in all detect RsAFP2-CRPs one with the real identical or very close position wash-out of RsAFP2.Do not have a kind of component and anti--DmAMP1 all to react with the antibody of anti--RsAFP2, this shows not exist in the extract does not have cleaved fusion rotein.In non-plants transformed, do not find the compound of cross reaction.

So, 3105 constructs (the inner propetide of IbAMP is as the joint peptide) and the original translation product of 3106 constructs (the part DmAMP1 C-CICP with subtilisin-sample protease site is as the joint peptide) transcriptional units are processed in some way, produce isolating DmAMP1-CRPs and RsAFP2-CRPs, as if identical with DmAMP1 and RsAFP2 respectively or very close in nature in chromatogram.

Embodiment 7

The Subcellular Localization analysis of the plant defense element of coexpression

For the plant defense element that detects coexpression is secreted into the extracellular or is deposited in the cell, from belonging to the leaf of plant, the homozygote transgenosis Ah cloth who transforms with 3105 constructs (plant 2), 3106 constructs (plant 2) or 3108 constructs (plant 12) obtains to extract in extracellular fluid and the cell component.Cytosolic glucose-6-phosphate dehydrogenase (G6PD) is used as marker, detects the situation that extracellular fluid is polluted by the cell interior component.As shown in table 2, glucose-6-phosphate dehydrogenase (G6PD) extracts in cell between part and the extracellular fluid body portion with about 80/20 ratio and distributes.On the contrary, most of DmAMP1-CRP of the discovery transgenic plant that detect and RsAFP2-CRP content are in the extracellular fluid body portion.These results show, the two kind of plant defensins that discharge from the polyprotein precursor all major sedimentary at the non-protogenous plastid.Therefore, to fix on non-protogenous plastid or Secretory Pathway be to take place in endoplasmic reticulum, Golgi's organs or the vesicle that transports between Golgi's organs and non-protogenous plastid to all polyprotein structure cracked procedure of processings one.

Table 2: from extracellular fluid (EF) and the interior extraction of cell (IE) composition that the transgenic arabidopsis platymiscium obtains, the relative abundance of glucose-6-phosphate dehydrogenase (G6PD) (GPD) activity, DmAMP1 and RsAFP2.Construct relative abundance (%) ¹

GPD????????DmAMP1?????RsAFP2

EF????IE????EF????IE???EF????IEpFAJ3105????17????83????93????7????92????8pFAJ3106????17????83????94????6????60????4pFAJ3108????20????80????98????2????75????25

¹Relative abundance is represented with the percentage ratio that extracts part content summation in extracellular fluid and the cell.

Embodiment 8

The albumen that purifying comes from the processing of polyprotein precursor 3105 constructs

The transfer-gen plant 14 that transforms colony from 3105 constructs is further bred, to obtain genetically modified homozygote plant.In the extracellular fluid of this transformed plant leaf preparation, by reverse-phase chromatography technology purifying DmAMP1-CRPs and RsAFP2-CRPs.In order to reach this purpose, leaf is with a kind of damping fluid and a kind of protease inhibitor mixture (1mM phenylmethylsulfonyl fluoride that contains 50mM MES (pH6), the 1mM N-ethylomaleimide, 5mM EDTA and 0.02mM Pepstatin A) at vacuum impregnation, the outer liquid of centrifugal collecting cell.Avoided DmAMP1-CRPs and RsAFP2-CRPs are exposed to the proteolytic enzyme that separates with this homogenized process.The extracellular fluid of collecting is by (Microsorb-MV on the C8-silica column, 4.6 * 250mm, anti-phase-the high pressure liquid chromatography of Rainin) carrying out is analyzed, use the antibody of anti-DmAMP1 and RsAFP2 respectively, there are situation in the DmAMP1-CRPs and the RsAFP2-CRPs that detect each several part by enzyme-linked immunosorbent assay.Figure 15 has shown this analytical results of the Arabidopsis transfer-gen plant 14 that transforms with 3105 constructs.DmAMP1-CRPs is at two peak wash-outs, and the position of a back elution peak is very near the wash-out position of real DmAMP1.Find RsAFP2-CRPs at fine isolating peak, peak wash-out with DmAMP1-CRPs, and the wash-out position of the very approaching real RsAFP2 in wash-out position.Do not have a part and anti--DmAMP1 all to react with the antibody of anti--RsAFP2, this shows not exist in the extracellular fluid does not have cleaved fusion rotein.Based on the peak area of DmAMP1-CRPs and RsAFP2-CRPs respectively with the comparison of the peak area of other a series of standard substances of forming by real DmAMP1 and RsAFP2, can conclude that the extract of 3105 construct transformants comprises the DmAMP1-CRPs and the RsAFP2-CRPs of about equivalent.By analyzing from the extracellular fluid of transgenosis strain preparation, obtain closely similar color atlas (result does not show), show that the chromatogram form of DmAMP1-CRPs and RsAFP2-CRPs and the transgenosis strain that is detected are irrelevant.

In order to detect the true composition whether purge process based on extracellular fluid preparation has reflected DmAMP1-CRPs and RsAFP2-CRPs in the transgenic arabidopsis plant leaf, developed an alternate purge process, this process is from the crude extract of leaf.For this purpose, leaf homogenate and extract in liquid nitrogen with the MES (pH6) that 50mM contains a kind of protease inhibitor cocktail (1mM phenylmethylsulfonyl fluoride, 1mM N-ethylomaleimide, 5mM EDTA and 0.02mM Pepstatin A).Homogenate purifies by centrifugal (10000 * g, 10 minutes).Supernatant is then by ion exchange chromatography (ICE) and reversed phase chromatography separation subsequently (RPC).At each after separating, collect each several part and also detect DmAMP1-CRPs and RsAFP2-CRPs respectively by two kinds of different enzyme-linked immunosorbent assays with the antibody of anti-DmAMP1 and RsAFP2.(Mono S, 5 * 50mm Pharmacia), carry out ion exchange chromatography through the cationic exchange coloum of a pH6 by making extract.When with the NaCl wash-out pillar of 0-0.5M linear gradient in the 50mM N-morpholino ethyl sulfonic acid (MES) of pH 6, the part of wash-out detects between 0.17-0.33MNaCl DmAMP1-CRPs, and RsAFP2-CRPs wash-out between 0.24-0.49M NaCl.Each fraction that contains DmAMP1-CRPs or RsAFP2-CRPs is concentrated into two portions (0.17-0.33M NaCl; 0.24-0.49M NaCl), wherein each part all (Microsorb-MV, 4.6 * 250mm Rainin) carry out the reverse-phase chromatography analysis, with the acetonitrile wash-out (Figure 16) of a linear concentration gradient on the C8-silica column.DmAMP1-CRPs is at two peak wash-outs, and the position of a back elution peak is very near the wash-out position of real DmAMP1.Find RsAFP2-CRPs at fine isolating peak, peak wash-out with DmAMP1-CRPs, and the wash-out position of the very approaching real RsAFP2 in wash-out position.Still do not have a part and anti--DmAMP1 all to react with the antibody of anti--RsAFP2, showing not exist in the extract does not have cleaved fusion rotein.

(program such as Cammue etc. are 1992 to be subjected to the N-terminal sequence analysis from the different DmAMP1-CRPs of extracellular fluid purifying and RsAFP2-CRPs, journal of biological chemistry, describe among the 2228-2233) also be subjected to substance assistant laser desorpted loss ionization time (Matrix-assistedtaser desorption ioniration-time of flight) mass spectroscopy (Mann and Talbo (MALDI-TOF), 1996, current biotechnology comment 7,11-19).The mass spectral best approximation of prediction theory (table 3) that the mensuration of C-end amino acid records based on experiment.Less DmAMP1-CRPs, p3105EF1 and bigger DmAMP1-CRP, p3105EF2 (encoding histone such as Figure 15 and table 3) has identical N-end sequence with ripe DmAMP1.P3105EF1 and p3105EF2 compare with real DmAMP1, and they have and the terminal extra corresponding to chromatogram of serine residue that exists of C-.Yet, when the chromatogram (in experimental error) of p3105EF2 during fully corresponding to the chromatogram predicted for DmAMP1 (after this being called DmAMP1+S) with the terminal Serine of a C-, the chromatogram of p3105EF1 has approximately surpassed 8 dalton for the prediction chromatogram of DmAMP1+S.Therefore, albumen may be the DmAMP1+S derivative with reduction disulfide linkage.According to N-end sequence and mass-spectrometric data, the p3105EF3 of RsAFP2-CRP partly is a RsAFP2 derivative that has extra pentapeptide sequence D VEPG at the N-end.This albumen DVEPG+RsAFP2 that are called as more.Use the same method and analyze different DmAMP1-CRPs and RsAFP2-CRPs purified from total leaf extract.Analytical results shows and has identical molecular form in total leaf extract, i.e. DmAMP1+S, a kind of DmAMP1+S reduction form of inferring, and DVEPG+RsAFP2 (table 3 of the embodiment 10 that vide infra).

The purifying fraction that comprises main processed products, DmAMP1+S and DVEPG+RsAFP2, respectively according to described methods such as Cammue (1992, biological chemistry 267,2228-2233), carry out anti-microbial activity with yellow sickle spore (Fusarium culmorum) and detect, represent special anti-microbial activity with institute's needed protein concentration of detection of biological body 50% growth-inhibiting, DmAMP1+S is identical with the special anti-microbial activity of real DmAMP1.The special anti-microbial activity of purifying DVEPG+RsAFP2 is less than about 2 times of real RsAFP2 greatly.The slight decline of DVEPG+RsAFP2 anti-microbial activity is likely because terminal 5 occurrences of amino acid of N-.Yet the processing of polyprotein precursor may cause delivery of biologically active albumen in our the digital proof transgenic plant.

The AFPs that produces in the 3105 construct plant transformed is analyzed, discloses precursor and obviously process (Figure 17) by three cleavage step:

(ⅰ) precursor carries out cracking at the C-end of guiding peptide in the mode identical with acting on real DmAMP1 precursor; (ⅱ) precursor has discharged DmAMP1+S like this in the terminal cracking of first amino acid whose C-of joint peptide; (ⅲ) precursor has discharged DVEPG+RsAFP2 like this in the terminal further processing of the N-of the 5th end residue of joint peptide.Do not know also which proteolytic enzyme has caused the cracking of these observations, does not know to have related to for how much proteolytic enzyme yet.The cracking of joint peptide may include only the proteolytic enzyme inscribe, or protein incision enzyme and in the terminal synergistic result of exopeptidase who further shears of split product.The processing of joint peptide C-end is carried out between E and two acidic residues of D, and this may be the target sequence of a specific proteins restriction endonuclease to acidic residues.In the past from the arabidopsis thaliana seed purifying a kind of can between two continuous acidic residues, carry out cracked aspartic acid protein incision enzyme (D ' Hont etc., 1993, journal of biological chemistry 268,20884-20891).Be worth should be mentioned that, in six inner propetides of IbAMP1 polyprotein precursor, have five ED sequence occur in just propetide the C-end (Tailor etc., 1997, journal of biological chemistry 272,24480-24487).In the inner propetide of six IbAMP1 one, more accurate used that in 3105 constructs of saying so, its ED sequence does not occur in the C-end of propetide, and separately terminal from C-by 4 amino acid.The processing of this propetide may comprise the cracking of ED sequence and terminal local pruning of N-of with a kind of aminopeptidase generation albumen being carried out subsequently in the Flower of Garden Balsam.

Desired is, a kind ofly be similar to the IbAMP1 propetide of using in 3105 constructs but its ED dipeptides is moved to the propetide of C-end, to produce a kind of split product, this product has only one or do not have extra-terminal amino acid in the terminal localized albumen of inner propetide C-.In other words, the C-end had an ED sequence another kind of IbAMP1 propetide (Tailor etc., 1997, journal of biological chemistry 272,24480-24487) or correlated series can provide same improvement to the accuracy of processing.

Embodiment 9

Purifying is processed from polyprotein front body structure pFAJ3106 and next albumen

The transgenosis strain 9 of the Arabidopsis plant population of the pFAJ3106 thaumatropy of using by oneself is further bred, and obtains genetically modified homozygote plant.By reverse-phase chromatography purifying DmAMP1-CRPs and RsAFP2-CRPs the liquid outside leaf cells, the preparation method of extracellular fluid is identical with the preparation method of top embodiment 8 described pFAJ3105 construct transformant extracellular fluids.The color atlas that in Figure 18, has shown isolate.DmAMP1-CRPs is called pFAJ3106EF1 and pFAJ3106EF2 at two peak wash-outs, and these two portions all have identical N-end sequence (referring to the table 3 of following embodiment 10) with DmAMP1.The mass spectrum of pFAJ3106EF2 is corresponding to being a mass spectrum that the DmAMP1 derivative with extra Methionin is predicted.Therefore we conclude that it is that precursor carries out the cracked product afterwards at signal peptide cleavage site point and terminal first residue of joint peptide C-(Methionin); This albumen DmAMP1+K that are known as more.

The homing sequence of finding the RsAFP2-CRP part by the-terminal amino acid order-checking is LIGKRQK.Therefore, this albumen that is called as LIGKRQK+RsAFP2 derives from the cracking of precursor N-end at the 6th terminal residue of joint peptide (glutamine).The cleavage step of the participation processing pFAJ3106 structure of suggestion as shown in figure 17.

Embodiment 10

Purifying is processed from polyprotein precursor construct pFAJ3108 and next albumen

The transgenosis strain 9 of the Arabidopsis plant population that the pFAJ3108 construct of using by oneself transforms is further bred, and obtains genetically modified homozygote plant.According to a kind of based on the top embodiment 8 described programs that are used for the ion exchange chromatography and the reverse-phase chromatography of 3105 transformants, purifying DmAMP1-CRPs and RsAFP2-CRPs from the total crude extract of the leaf of this transformant.The color atlas that in Figure 19, has shown ion exchange chromatography and reversed phase chromatography separation.Ion exchange chromatography separates two peaks that comprise DmAMP1-CRPs of generation.Yet, all can not detect RsAFP2-CRPs in any one elutriated fraction.Because obviously there are RsAFP2-CRPs (referring to table 1 and table 2) in crude extract and extracellular fluid part with the plant of pFAJ3108 thaumatropy, so RsAFP2-CRPs has lost in sepn process.Most probable explanation is, RsAFP2-CRPs is not come out by the NaCl of 0.5M wash-out from ion exchange column, and 0.5M NaCl is the used maximum concentration of gradient.The composition that contains DmAMP1-CRPs produces two DmAMP1-CRP peaks by reversed phase chromatography separation.Detect by N-end sequencing and MALDI-TOF chromatogram and to analyze this composition, disclosing it is the DmAMP1 derivative (DmAMP1+A) that a kind of C-end has an extra L-Ala.This albumen is that precursor carries out cracked product (Figure 17) at signal peptide cleavage site point and terminal first residue of joint peptide C-(L-Ala).

Table 3: shown DmAMP1-CRP and RsAFP2-CRP composition, by the mass spectrum of MALDI-TOF-mass spectroscopy or EI-mass spectrometric determination and the N-end sequence of measuring by automatization Edman degraded as purifying as described in Figure 15,16,18 and 19.Also shown the C-end sequence that between experiment mass spectrum and theoretical mass spectrum, provides best conforming prediction.

Construct	Protein fractions is (referring to Figure 15 16,18 and 19	Mass spectrum by MALDI-TOF-MS mensuration	Mass spectrum by EI-MS mensuration	N-end sequence (the SEQ ID NOS 69-71 that detects	The C-end sequence (SEQ ID NOS 72-76) of prediction	The theoretical mass spectrum of forecasting sequence
							pFAJ3105 ?pFAJ3106 ?pFAJ3108	?D3105EF1 ?D3105EF2 ?D3105EF3 ?D3105TE1 ?D3105TE2 ?D3105TE3 ?D3106EF1 ?D3106EF2 ?p3106EF3 ?D3108TE1	?5614±5 ?5602±5 ?6223±6 ?5610±5 ?5604±5 ?6224±6 ???N.D. ?5640±5 ???N.D. ?5583±5	????5608.3±1 ????5604.9±1 ?????N.D. 1?????N.D. ?????N.D. ?????N.D. ?????N.D. ?????N.D. ?????N.D. ?????N.D.	????ELCEKAS ????ELCEKAS ????DVEPGQK ????ELCEKAS ????ELCEKAS ????DVEPGQK ????ELCEKAS ????ELCEKAS ????LIGKRQK ????ELCEKAS	????CYFNCS ????CYFNCS ????ICYFPC ????CYFNPS ????CYFNCS ????ICYFPC ????CYFNCK ????CYFNCK ????ICYFPC ????CYFNCA	??5604.25 ??5604.25 ??6225.15 ??5604.25 ??5604.25 ??6225.15 ??5645.34 ??5645.34 ??6295.38 ??5588.25
Do not measure

Embodiment 11

The transformation of pFAJ3105 construct

Can find out obviously that from the analysis that the Arabidopsis plant that the pFAJ3105 construct is transformed carries out the polyprotein precursor is cleaved really (referring to table 3, Figure 17).Yet cracking is to take place like this, keep attaching to the terminal localized maturation protein from joint peptide N-from an amino acid of joint peptide, and 5 amino acid keeps attaching to the terminal localized maturation protein (referring to Figure 17) from joint peptide C-.The amino acid that attaches to the joint peptide-source of maturation protein may disturb the function proterties of these maturation proteins, in order to reduce these amino acid whose quantity, many constructs have been designed, so that obtain the cracking that takes place near the border of (or preferably existing just) maturation protein more.

In the pFAJ3343 construct, deleted the codon of coding joint peptide N-terminal residue among the pFAJ3105.The cracking of estimating the ripe DmAMP1 of generation does not additionally have any amino acid from the joint peptide (Figure 20).In pFAJ3344, pFAJ3345 and pFAJ3346 construct, the joint peptide C-termination codon of pFAJ3105 is changed, and has deleted last 2,4 and 5 residues respectively.After estimating pFAJ3344, pFAJ3345 and the cracking of pFAJ3346 construct, the residue quantity that keeps being attached to RsAFP2 N-end will be 3,1 and 0 (Figure 20) respectively.Can make up other structure, wherein reduce the N-or the C-terminal residue number in pFAJ3105 construct center tap peptide district.

In the pFAJ3105 construct, the joint peptide from the 4th inner propetide of IbAMP precursor (Tailor R.H. etc., 1997, journal of biological chemistry 272,24480-24487).In the pFAJ3369 construct, this joint peptide is substituted (Tailor R.H. etc., 1997, the same) by first inner propetide of IbAMP precursor.In a back joint propetide, paired acidic residues occurs in the C-end.The cracking that expectation takes place has only a residue will keep being attached to the N-end (Figure 20) of RsAFP2.

Embodiment 12

Make up a kind of construct, be used for expressing a kind of polyprotein with 4 maturation protein districts

The composition in polyprotein district is in the pFAJ3367 structure: the signal peptide district of DmAMP1 cDNA, and there is the coding region of four different antibacterial peptides the back, and each is distinguished by first inner propetide of IbAMP precursor and separates.The coding region of four different antibacterial proteins is (seeing Figure 21) in proper order:

The plain DmAMP1 of plant defense (Osborn R.W. etc., 1995, European biological chemistry associating proceedings 368,257-262)

The plain RsAFP2 of plant defense (Terras F.R.G. etc., 1995, vegetable cell 7,573-588)

The plain HsAFP1 of plant defense (Osborn R.W. etc., 1995, European biological chemistry associating proceedings 368,257-262)

Lipid transfer protein-sample albumin A cAMP1 (Cammue B.P.A. etc., 1995, plant physiology 109,445-455)

This construct will produce four kinds of different ripe antibacterial proteins (DmAMP1, RsAFP2, HsAFP1 and AcAMP1), and each albumen all is secreted into ECS.

Can make up other construct, contain other mature peptide district and have above-mentioned any other joint peptide.

Embodiment 13

The transformation of construct pFAJ3106, pFAJ3107 and DFAJ3108

Polyprotein by construct pFAJ3106, pFAJ3107 and pFAJ3108 construct coding is included in the joint peptide that the C-end has Kex2 recognition site IGKR.Jiang L. and Rogers J.C. (1999, plant magazine 18,23-32) verified, in transgene tobacco, the polyprotein that contains an IGKR site is not separated or seldom is separated.The IGKR sequence by IGKRIGKRIGKR sequence (SEQ ID NO.77) alternate polyprotein in, find that cracking is improved.

Construct FAJ3106-2, pFAJ3107-2 and pFAJ3108-2 except the IGKR coding region is substituted by the zone of a coding IGKRIGKRIGKR, other all identical with construct pFAJ3106, pFAJ3107 (Figure 22) with pFAJ3108.Polyprotein by these construct codings can both effectively be ruptured at the N-of joint peptide end and C-end.

Can make up other construct, wherein reduce the N-or the C-terminal residue number in construct pFAJ3106, pFAJ3107 and pFAJ3108 center tap peptide district.

Embodiment 14

Polyprotein construct based on the heterozygosis joint peptide that contains the 2A sequence

The RNA of foot and mouth disease virus (FMDV) is translated into a polyprotein, the cracking of this polyprotein depend on 20 amino acid whose sequences that are called as the 2A sequence (Ryan and Drew, 1994, EMBO magazine 13,928-933).The cracking of the polyprotein that is connected by the 2A sequence occurs between the 19th amino acid (G) and the 20th amino acid (P) of 2A sequence, splitting action is undertaken by a course of processing that obviously has nothing to do with processive enzyme, and its generation may belong to the improper form (Halpin etc. of peptide bond between G and the P, 1999, plant magazine 17,453-459).(1999, plant magazine 17 when 453-459) the verified FMDV of comprising 2A sequence is expressed in plant as the polyprotein of a joint peptide, is ruptured Halpin etc. effectively.Yet, utilize FMDV 2A sequence to be as one of a joint peptide main shortcoming, cracking is not in the terminal generation of the N-of joint peptide.Therefore, keep depending on the C-end of maturation protein corresponding to one section of preceding 19 residues of FMDV 2A sequence 19 long amino acid whose extensions.19 extra amino acid whose extensions of this section may be disturbed the proteic function proterties that it connects.

In order to solve the problem of not exclusively removing after the cracking of joint propetide, propose to use the joint peptide of heterozygosis, at its N-end is the part of the joint peptide described in construct pFAJ3105, pFAJ3106, pFAJ3107 or the construct pFAJ3108, in its C-end part (or part of this peptide) that is FMDV 2A sequence.Example based on this principle is pFAJ3370 construct and pFAJ3368 construct (Figure 23).The pFAJ3370 construct has a polyprotein district identical with the pFAJ3150 construct, except the joint peptide in pFAJ3370 construct polyprotein district is one 29 amino acid whose peptide, this peptide is by preceding 9 amino acid (Tailor R.H. etc. of the 4th inner propetide of IbAMP precursor, 1997, journal of biological chemistry 272 24480-24487) is formed succeeded by whole 20 amino acid of FMDV 2A sequence.The cracking of this joint peptide will discharge a C-end and have the ripe DmAMP1 of an extra Serine and the ripe RsAFP2 that N-end has an extra proline(Pro).

Construct pFAJ3368 is identical with construct pFAJ3370, except the terminal maturation protein district of C-(RsAFP2 in this case encodes) by this maturation protein district of coding and the zone (coding has the RsAFP2 of self signal peptide in this case) in previous signal peptide district replace.If occur in before polyprotein inserts to endoplasmic reticulum in the cracking between FMDV 2A sequence G and the P, estimate that so the pFAJ3368 construct compares with the pFAJ3370 construct, will make two kinds of maturation protein albumen navigate to ECS better.In this case, the excretory maturation protein will have the DmAMP1 of an extra Serine and additional amino acid whose RsAFP2 forms by the C-end.If the cracking between FMDV 2A sequence G and P occurs in after polyprotein inserts to endoplasmic reticulum, estimate that so the signal peptide that is attached to RsAFP2 can not effectively be removed, the pFAJ3370 construct will be than pFAJ3368 construct more preferably in this case.

Sequence table＜110〉Zeneca Limited

Broekaert,Willem?F

Francois,Isabelle?EJA

Evans,Ian?J

De?Bolle,Miguel?FC

Ray，John A＜120〉＜130〉PPD 50348／WO＜140〉＜141〉＜150〉GB 9818001.1＜151〉1998-08-18＜150〉GB 9826753.7＜151〉1998-12-04＜160〉81＜170〉PatentIn Vet.2.1＜210〉1＜211〉446＜212〉DNA＜213〉＜220〉＜221〉CDS＜222〉 （ 1 ） .. （ 64 ）＜220〉＜221〉CDS＜222〉 （ 157 ） .. （ 446 ）＜400〉 1atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt ctg atc ctt ttc 48Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe 1 5 10 15gtg ctc gcc atc tca g gttatcaaat ctttagttca tttattgaat atgatagtat 104Val Leu Ala Ile Ser。

20ttatattctt?ttatggtttt?atgtgttctg?acaagttgca?aatattgagt?ag?at?atc??161

Asp?Ilegca?tcc?gtt?agt?gga?gaa?cta?tgc?gag?aaa?gct?agc?aag?aca?tgg?tcg???209Ala?Ser?Val?Ser?Gly?Glu?Leu?Cys?Glu?Lys?Ala?Ser?Lys?Thr?Trp?Ser

25??????????????????30??????????????????35gga?aac?tgt?ggc?aat?acg?gga?cat?tgt?gac?aac?caa?tgt?aaa?tca?tgg???257Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys?Asp?Asn?Gln?Cys?Lys?Ser?Trp?40??????????????????45??????????????????50??????????????????55gag?ggt?gcg?gcc?cat?gga?gcg?tgt?cat?gtg?cgt?aac?ggg?aaa?cac?atg???305Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His?Val?Arg?Asn?Gly?Lys?His?Met

60??????????????????65??????????????????70tgt?ttc?tgt?tac?ttc?aat?tgt?aaa?aaa?gcc?gaa?aag?ctt?gct?caa?gac???353Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Lys?Lys?Ala?Glu?Lys?Leu?Ala?Gln?Asp

75??????????????????80??????????????????85aaa?ctt?aaa?gcc?gaa?caa?ctc?gct?caa?gac?aaa?ctt?aat?gcc?caa?aag???401Lys?Leu?Lys?Ala?Glu?Gln?Leu?Ala?Gln?Asp?Lys?Leu?Asn?Ala?Gln?Lys

90??????????????????95?????????????????100ctt?gac?cgt?gat?gcc?aag?aaa?gtg?gtt?cca?aac?gtt?gaa?cat?ccg????????446Leu?Asp?Arg?Asp?Ala?Lys?Lys?Val?Val?Pro?Asn?Val?Glu?His?Pro

105 110 115＜210〉2＜211〉118＜212〉protein＜213〉smooth dahlia＜400〉2Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe 15 10 15 Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu

20???????????????????25??????????????????30Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys

35??????????????????40??????????????????45Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His

50??????????????????55??????????????????60Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Lys?Lys?65??????????????????70??????????????????75??????????????????80Ala?Glu?Lys?Leu?Ala?Gln?Asp?Lys?Leu?Lys?Ala?Glu?Gln?Leu?Ala?Gln

85??????????????????90??????????????????95Asp?Lys?Leu?Asn?Ala?Gln?Lys?Leu?Asp?Arg?Asp?Ala?Lys?Lys?Val?Val

100?????????????????105?????????????????110Pro?Asn?Val?Glu?His?Pro

115＜210〉3＜211〉16＜212〉protein＜213〉artificial sequence＜220〉＜223〉artificial sequence description: joint propetide＜400〉3Ser Asn Ala Ala Asp Glu Val Ala Thr Pro Glu Asp Val Glu Pro Gly 15 10 15＜210〉4＜211〉20＜212〉protein＜213〉artificial sequence＜220〉＜223〉artificial sequence description: joint propetide＜400〉4Lys Lys Ala Glu Lys Leu Ala Gln Asp Lys Leu Lys Ala Glu Gln Leu 15 10 15Ile Gly Lys Arg

20＜210〉5＜21l〉40＜212〉protein＜213〉Garden Dahlia＜400〉5Lys Lys Ala Glu Lys Leu Ala Gln Asp Lys Leu Lys Ala Glu Gln Leu, 15 10 15Ala Gln Asp Lys Leu Asn Ala Gln Lys Leu Asp Arg Asp Ala Lys Lys

20??????????????????25??????????????30Val?Val?Pro?Asn?Val?Glu?His?Pro

35 40＜210〉6＜211〉44＜212〉protein＜213〉artificial sequence＜220〉＜223〉artificial sequence description: joint propetide＜400〉6Lys Lys Ala Glu Lys Leu Ala Gln Asp Lys Leu Lys Ala Glu Gln Leu 15 10 15Ala Gln Asp Lys Leu Asn Ala Gln Lys Leu Asp Arg Asp Ala Lys Lys

20??????????????????25??????????????????30Val?Val?Pro?Asn?Val?Glu?His?Pro?Ile?Gly?Lys?Arg

35 40＜210〉7＜211〉20＜212〉protein＜213〉artificial sequence＜220〉＜223〉artificial sequence description: joint propetide＜400〉7Ala Ser Thr Thr Val Asp His Gln Ala Asp Val Ala Ala Thr Lys Thr 15 10 15Ile Gly Lys Arg

20＜210〉8＜211〉3l＜212〉protein＜213〉the steady amaranth of tail＜400〉8Ala Ser Thr Thr Val Asp Mis Gln Ala Asp Val Ala Ala Thr Lys Thr, 15 10 15Ala Lys Asn Pro Thr Asp Ala Lys Leu Ala Gly Ala Gly Ser Pro

20 25 30＜210〉9＜211〉522＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: composition sequence＜220〉＜221〉CDS＜222 〉, (76) .., (513)＜400〉9ctcgagtatt tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattact 60atttacaatt acacc atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt 111

Met?Val?Asn?Arg?Ser?Val?Ala?Phe?Ser?Ala?Phe?Val

1???????????????5??????????????????10ctg?atc?ctt?ttc?gtg?ctc?gcc?atc?rca?gat?arc?gca?tcc?gtt?agt?gga????159Leu?Ile?Leu?Phe?Val?Leu?Ala?Ile?Ser?Asp?IIe?Ala?Ser?Val?Ser?Gly

15??????????????????20??????????????????25gaa?cta?tgc?gag?aaa?gct?agc?aag?acg?tgg?tcg?ggc?aac?tgt?ggc?aac????207Glu?Leu?Cys?Glu?Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn

30??????????????????35??????????????????40acg?gga?cat?tgt?gac?aac?caa?tgt?aaa?tca?tgg?gag?ggt?gcg?gcc?cat????255Thr?Gly?His?Cys?Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?45??????????????????50??????????????????55??????????????????60gga?gcg?tgt?cat?gtg?cgt?aac?ggg?aaa?cac?atg?tgt?ttc?tgt?tac?ttc????303Gly?Ala?Cys?His?Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe

65??????????????????70??????????????????75aat?tgt?tcc?aac?gct?gct?gac?gag?gtg?gct?acc?cca?gag?gac?gtg?gag????351Asn?Cys?Ser?Asn?Ala?Ala?Asp?Glu?Val?Ala?Thr?Pro?Glu?Asp?Val?Glu

80??????????????????85??????????????????90cca?gga?cag?aag?ttg?tgc?caa?agg?cca?agt?ggg?aca?tgg?tca?gga?gtc????399Pro?Gly?Gln?Lys?Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val

95?????????????????100?????????????????105tgt?gga?aac?aat?aac?gca?tgc?aag?aat?cag?tgc?att?aga?ctt?gag?aaa????447Gys?Gly?Asn?Asn?Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys

110?????????????????115?????????????????120gca?cga?cat?gga?tct?tgc?aac?tat?gtc?ttc?cca?gct?cac?aag?tgt?atc????495Ala?Arg?His?Gly?Set?Cys?Asn?Tyr?Val?Phe?Pro?Ala?His?Lys?Cys?Ile125?????????????????130?????????????????135?????????????????140tgc?tac?ttt?cct?tgt?taa?taggagctc??????????????????????????????????522Cys?Tyr?Phe?Pro?Cys

145＜210〉10＜211〉145＜212〉protein＜213〉artificial sequence＜223〉artificial sequence description: composition sequence＜400〉10Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe 15 10 15Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu

20??????????????????25??????????????????30Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys

35??????????????????40??????????????????45Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His

50??????????????????55??????????????????60Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Ser?Asn?65??????????????????70??????????????????75???????????????????80Ala?Ala?Asp?Glu?Val?Ala?Thr?Pro?Glu?Asp?Val?Glu?Pro?Gly?Gln?Lys

85??????????????????90??????????????????95Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val?Cys?Gly?Asn?Asn

100?????????????????105?????????????????110Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys?Ala?Arg?His?Gly

115?????????????????120?????????????????125Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala?His?Lys?Cys?Ile?Cys?Tyr?Phe?Pro

130 135 140Cys145＜210〉11＜211〉534＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: joint propetide＜220〉＜221〉CDS＜222, (76) .., (525)＜400〉11ctcgagtatt tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattact 60atttacaatt acacc atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt 111

Met?Val?Asn?Arg?Ser?Val?Ala?Phe?Ser?Ala?Phe?Val

1???????????????5??????????????????10ctg?atc?ctt?ttc?gtg?ctc?gcc?atc?tca?gat?atc?gca?tcc?gtt?agt?gga???159Leu?Ile?Leu?Phe?Val?Leu?Ala?Ile?Ser?Asp?Ile?Ala?Ser?Val?Ser?Gly

15??????????????????20??????????????????25gaa?cta?tgc?gag?aaa?gct?agc?aag?acg?tgg?tcg?ggc?aac?tgt?ggc?aac???207Glu?Leu?Cys?Glu?Lys?Ala?Ser?Lys?Thr?TrD?Ser?Gly?Asn?Cys?Gly?Asn

30??????????????????35??????????????????40acg?gga?cat?tgt?gac?aac?caa?tgt?aaa?rca?tgg?gag?ggt?gcg?gcc?cat???255Thr?Gly?His?Cys?Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?45??????????????????50??????????????????55??????????????????60gga?gcg?tgt?cat?gtg?cgt?aac?ggg?aaa?cac?atg?tgt?ttc?tgt?tac?ttc???303Gly?Ala?Cys?His?Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe

65??????????????????70??????????????????75aat?tgt?aaa?aaa?gcc?gaa?aag?ctt?gct?caa?gac?aaa?ctt?aaa?gcc?gaa???351Asn?Cys?Lys?Lys?Ala?Glu?Lys?Leu?Ala?Gln?Asp?Lys?Leu?Lys?Ala?Glu

80??????????????????85??????????????????90caa?ctG?atc?gga?aag?agg?cag?aag?ttg?tgc?caa?agg?cca?agt?ggg?aca???399Gln?Leu?Ile?Gly?Lya?Arg?Gln?Lys?Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr

95?????????????????100?????????????????105tgg?tca?gga?gtc?tgt?gga?aac?aat?aac?gca?tgc?aag?aat?cag?tgc?att???447Trp?Ser?Gly?Val?Cys?Gly?Asn?Asn?Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile

110?????????????????115?????????????????120???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????aga?ctt?gag?aaa?gca?cga?cat?gga?tct?tgc?aac?tat?gtc?ttc?cca?gct???495Arg?Leu?Glu?Lys?Ala?Arg?His?Gly?Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala125?????????????????130?????????????????135?????????????????140cac?aag?tgt?atc?tgc?tac?ttt?cct?tgt?taa?taggagctc?????????????????534His?Lys?Cys?Ile?Cys?Tyr?Phe?Pro?Cys

145＜210〉12＜211〉149＜212〉protein＜213〉artificial sequence＜223〉artificial sequence description: composition sequence＜400〉12Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe 15 10 15Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu

20??????????????????25??????????????????30Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys

35??????????????????40??????????????????45Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His

50??????????????????55??????????????????60Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Lys?Lys?65??????????????????70??????????????????75??????????????????80Ala?Glu?Lys?Leu?Ala?Gln?Asp?Lys?Leu?Lys?Ala?Glu?Gln?Leu?Ile?Gly

85??????????????????90??????????????????95Lys?Arg?Gln?Lys?Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val

100?????????????????105?????????????????110Cys?Gly?Asn?Asn?Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys

115?????????????????120?????????????????125Ala?Arg?His?Gly?Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala?His?Lys?Cys?Ile

130 135 140Cys Tyr Phe Pro Cys145＜210〉13＜211〉24＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotide＜220〉＜221〉misc_ feature＜222〉(6,9,12,15,21)＜223〉n is any residue＜400〉13tgyganaang cnwsnaarac ntgg 24＜210〉14＜211〉8＜212〉protein＜213〉smooth Garden Dahlia＜400〉14Cys Glu Lys Ala Ser Lys Thr Trp 15＜210〉15＜211〉606＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: composition sequence＜220〉＜221〉CDS＜222〉(76) .. (597)＜400〉15ctcgagtatt tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattact 60atttacaatt acacc atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt 111

Met?Val?Asn?Arg?Ser?Val?Ala?Phe?Ser?Ala?Phe?Val

1???????????????5??????????????????10ctg?atc?ctt?ttc?gtg?ctc?gcc?atc?tca?gat?atc?gca?tcc?gtt?agt?gga???159Leu?Ile?Leu?Phe?Val?Leu?Ala?Ile?Ser?Asp?Ile?Ala?Ser?Val?Ser?Gly

15??????????????20??????????????????25gaa?cta?tgc?gag?aaa?gct?agc?aag?acg?tgg?tcg?ggc?aac?tgt?ggc?aac???207Glu?Leu?Cys?Glu?Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn

30??????????????????35??????????????????40acg?gga?cat?tgt?gac?aac?caa?tgt?aaa?tca?tgg?gag?ggt?gcg?gcc?cat???255Thr?Gly?His?Cys?Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?45??????????????????50??????????????????55??????????????????60gga?gcg?tgt?cat?gtg?cgt?aac?ggg?aaa?cac?atg?tgt?ttc?tgt?tac?ttc???303Gly?Ala?Cys?His?Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?cys?Tyr?Phe

65??????????????????70??????????????????75aat?tgt?aaa?aaa?gcc?gaa?aag?ctt?gct?caa?gac?aaa?ctt?aaa?gcc?gaa???351Asn?Cys?Lys?Lys?Ala?Glu?Lys?Leu?Ala?Gln?Asp?Lys?Leu?Lys?Ala?Glu

80??????????????????85??????????????????90caa?ctc?gct?caa?gac?aaa?ctt?aat?gcc?caa?aag?ctt?gac?cgt?gat?gcc???399Gln?Leu?Ala?Gln?Asp?Lys?Leu?Asn?Ala?Gln?Lys?Leu?Asp?Arg?Asp?Ala

95?????????????????100?????????????????105aag?aaa?gtg?gtt?cca?aac?gtt?gaa?cat?ccg?atc?gga?aag?agg?cag?aag???447Lys?Lys?Val?Val?Pro?Asn?Val?Glu?His?Pro?Ile?Gly?Lys?Arg?Gln?Lys

110?????????????????115?????????????????120ttg?tgc?caa?agg?cca?agt?ggg?aca?tgg?tca?gga?gtc?tgt?gga?aac?aat???495Leu?Cys?Gln?ArG?Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val?Cys?Gly?Asn?Asn125?????????????????130?????????????????135?????????????????140aac?gca?tgc?aag?aat?cag?tgc?att?aga?ctt?gag?aaa?gca?cga?cat?gga???543Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys?Ala?Arg?His?Gly

145?????????????????150?????????????????155tct?tgc?aac?tat?gtc?rtc?cca?gct?cac?aag?tgt?atc?tgc?tac?ttt?cct???591Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala?His?Lys?Cys?Ile?Cys?Tyr?Phe?Pro

160 165 170tgt taa taggagctc 606Cys＜210〉16＜211〉173＜212〉protein＜213〉artificial sequence＜223〉artificial sequence description: composition sequence＜400〉16Met Val Asn Arg Ser Val Ala Phe Sar Ala Phe Val Leu Ile Leu Phe 15 10 15Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu

20??????????????????25??????????????????30Lys?Ala?Ser?Lys?Thr?Trp?Sar?Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys

35??????????????????40??????????????????45Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His

50??????????????????55??????????????????60Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Lys?Lys?65??????????????????70??????????????????75??????????????????80Ala?Glu?Lys?Leu?Ala?Gln?Asp?Lys?Leu?Lys?Ala?Glu?Gln?Leu?Ala?Gln

85??????????????????90??????????????????95Asp?Lys?Leu?Asn?Ala?Gln?Lys?Leu?Asp?Arg?Asp?Ala?Lys?Lys?Val?Val

100?????????????????105?????????????????110Pro?Asn?Val?Glu?His?Pro?Ile?Gly?Lys?Arg?Gln?Lys?Leu?Cys?Gln?Arg

115?????????????????120?????????????????125Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val?Cys?Gly?Asn?Asn?Asn?Ala?Cys?Lys

130?????????????????135?????????????????140Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys?Ala?Arg?His?Gly?Ser?Cys?Asn?Tyr145?????????????????150?????????????????155?????????????????160Val?Phe?Pro?Ala?His?Lys?Cys?Ile?Cys?Tyr?Phe?Pro?Cys

165 170＜210〉17＜211〉534＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: composition sequence＜220〉＜221〉CDS＜222〉(76) .. (525)＜400〉17ctcgagtatt tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattact 60atttacaatt acacc atg gtg aat cgg tcg gtt gcg ttc tcc gcg ttc gtt 111

Met?Val?Asn?Arg?Ser?Val?Ala?Phe?Ser?Ala?Phe?Val

1???????????????5??????????????????10ctg?atc?ctt?ttc?gtg?ctc?gcc?atc?tca?gat?atc?gca?tcc?gtt?agt?gga???159Leu?Ile?Leu?Phe?Val?Leu?Ala?Ile?Ser?Asp?Ile?Ala?Ser?Val?Ser?Gly

15??????????????????20??????????????????25gaa?cta?tgc?gag?aaa?gct?agc?aag?acg?tgg?tcg?ggc?aac?tgt?ggc?aac???207Glu?Leu?Cys?Glu?Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn

30??????????????????35??????????????????40acg?gga?cat?tgt?gac?aac?caa?tgt?aaa?tca?tgg?gag?ggt?gcg?gcc?cat???255Thr?Gly?His?Cys?Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?45??????????????????50??????????????????55??????????????????60gga?gcg?tgt?cat?gtg?cgt?aac?ggg?aaa?cac?atg?tgt?ttc?tgt?tac?ttc???303Gly?Ala?Cys?His?Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe

65??????????????????70??????????????????75aat?tgt?gcc?agt?act?act?gtg?gat?cac?caa?gct?gat?gtt?gct?gcc?acc???351Asn?Cys?Ala?Ser?Thr?Thr?Val?Asp?His?Gln?Ala?Asp?Val?Ala?Ala?Thr

80??????????????????85??????????????????90aaa?act?atc?gga?aag?agg?cag?aag?ttg?tgc?caa?agg?cca?agt?ggg?aca???399Lys?Thr?Ile?Gly?Lys?Arg?Gln?Lys?Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr

95?????????????????100?????????????????105tgg?tca?gga?gtc?tgt?gga?aac?aat?aac?gca?tgc?aag?aat?cag?tgc?att???447Trp?Ser?Gly?Val?Cys?Gly?Asn?Asn?Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile

110?????????????????115?????????????????120aga?ctt?gag?aaa?gca?cga?cat?gga?tct?tgc?aac?tat?gtc?ttc?cca?gct???495Arg?Leu?Glu?Lys?Ala?Arg?His?Gly?Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala125?????????????????130?????????????????135?????????????????140cac?aag?tgt?atc?tgc?tac?ttt?cct?tgt?taa?taggagctc?????????????????534His?Lys?Cys?Ile?Cys?Tyr?Phe?Pro?Cys

145＜210〉18＜211〉149＜212〉protein＜213〉artificial sequence＜223〉artificial sequence description: composition sequence＜400〉18Met Val Asn Arg Ser Val Ala Phe Ser Ala Phe Val Leu Ile Leu Phe 15 10 15Val Leu Ala Ile Ser Asp Ile Ala Ser Val Ser Gly Glu Leu Cys Glu

20??????????????????25??????????????????30Lys?Ala?Ser?Lys?Thr?Trp?Ser?Gly?Asn?Cys?Gly?Asn?Thr?Gly?His?Cys

35??????????????????40??????????????????45Asp?Asn?Gln?Cys?Lys?Ser?Trp?Glu?Gly?Ala?Ala?His?Gly?Ala?Cys?His

50??????????????????55??????????????????60Val?Arg?Asn?Gly?Lys?His?Met?Cys?Phe?Cys?Tyr?Phe?Asn?Cys?Ala?Ser?65??????????????????70??????????????????75??????????????????80Thr?Thr?Val?Asp?His?Gln?Ala?Asp?Val?Ala?Ala?Thr?Lys?Thr?Ile?Gly

85??????????????????90??????????????????95Lys?Arg?Gln?Lys?Leu?Cys?Gln?Arg?Pro?Ser?Gly?Thr?Trp?Ser?Gly?Val

100?????????????????105?????????????????110Cys?Gly?Asn?Asn?Asn?Ala?Cys?Lys?Asn?Gln?Cys?Ile?Arg?Leu?Glu?Lys

115?????????????????120?????????????????125Ala?Arg?His?Gly?Ser?Cys?Asn?Tyr?Val?Phe?Pro?Ala?His?Lys?Cys?Ile

130 135 140Cys Tyr Phe Pro Cys145＜210〉19＜211〉316＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: composition sequence＜220〉＜221〉CDS＜222〉(76) .. (312)

Claims (35)

1. method that in transgenic plant, improves one or more protein expression levels, the genome that is included in described plant inserts a dna sequence dna, this dna sequence dna comprises a promoter region, be operably connected two or more protein-coding regions and 3 '-terminator of promoter region wherein, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide, described propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.

2. according to the process of claim 1 wherein that said promoter region is operably connected to a signal sequence, this signal sequence is operably connected to two or more protein-coding regions and 3 '-terminator.

3. method of in transgenic plant, expressing polyprotein, the genome that is included in described plant inserts a dna sequence dna, this dna sequence dna comprises a promoter region, wherein promoter region is operably connected to a signal sequence, this signal sequence be operably connected two or more protein-coding regions and 3 '-terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide, said propetide provides a cracking site, and the polyprotein of expression is translated post-treatment in this site and becomes the component protein molecular.

4. according to the method for above-mentioned each claim, the sequence of wherein said joint propetide has at least 40% to form by one section sequence of 2 to 5 continuous hydrophobic residues that are selected from L-Ala, Xie Ansuan, Isoleucine, methionine(Met), leucine, phenylalanine, tryptophane and tyrosine or by one section sequence of 2 to 5 hydrophilic residues that are selected from aspartic acid, L-glutamic acid, Methionin, arginine, Histidine, Serine, Threonine, glutamine and l-asparagine.

5. according to the method for aforementioned each claim, wherein said joint propetide has a sequence with 2 to 5 continuous acidic residues, 2 to 5 continuous alkaline residues or 2 to 5 successive mixing acid alkaline residues in 7 residues of its N-or the terminal cracking site of C-.

6. according to the method for aforementioned each claim, the dna sequence encoding of described joint propetide of wherein encoding is a kind of can be from vegetable-protein, or the viral propetide that is separated to, or its variant, the perhaps fragment of any wherein, it provides a cracking site, becomes the component protein molecular thereby make the polyprotein of expression be translated post-treatment in this site.

7. according to the method for aforementioned each claim, a kind of propetide that can from vegetable-protein, be separated to of the dna sequence encoding of the described joint propetide of wherein encoding or its fragment.

8. according to the method for claim 6 or 7, wherein encode chimeric propetide of dna sequence encoding of described joint propetide, comprise a kind of propetide that can from one or more plants and/or a kind of virus, be separated to, perhaps its variant or the wherein fragment of any.

9. according to the method for claim 7 or 8, vegetable-protein wherein is the precursor of plant defense element, perhaps the antibacterial protein of hevein type.

10. according to the method for claim 9, vegetable-protein wherein is the antibacterial protein that derives from impatiens.

11. according to the method for claim 10, propetide wherein comprises SEQ ID NO.3,29,21,22,23 or 24.

12. method according to Claim 8, propetide wherein comprise from the C-CICP of Dm-AMP1 or Ac-AMP2 or its fragment, or wherein any one variant.

13. according to the method for claim 12, propetide wherein comprises SEQ ID NO.4,6,7,25,26 or 27.

14. according to the method for aforementioned each claim, propetide wherein is chimeric propetide.

15. according to the method for claim 13, chimeric propetide wherein comprises viral propetide or its fragment, and from isolating propetide of vegetable-protein or its fragment.

16. according to the method for claim 15, virus wherein is picornavirus.

17. according to the method for claim 15 or 16, chimeric propetide wherein comprises the SEQ ID NO.28 of viral propeptide sequence.

18. according to the method for aforementioned each claim, joint propetide has wherein all designed a proteolytic enzyme processing site at any end or two ends.

19. according to the method for claim 18, proteolytic enzyme processing site wherein is the processing site of the proteolytic enzyme of a subtilisin sample.

20. according to the method for claim 2 or 3, signal sequence wherein is from the plant defense plain gene.

21. according to the method for aforementioned each claim, wherein one or more in the polyprotein are sozinss.

22. a kind of plant joint propetide at the Secretory Pathway cleavable is applied to synthetic polyprotein precursor in the transgenic plant.

23. according to the application of the propetide of claim 22, wherein propetide is from vegetable-protein or from virus.

24. according to the application of claim 22 or claim 23 propetide, propetide wherein is a kind of precursor of plant defense element from vegetable-protein and this albumen, perhaps from the antibacterial protein of hevein type, perhaps can separate from impatiens.

25. with the cleavable joint of a kind of propetide as synthetic polyprotein precursor in the transgenic plant Secretory Pathway, wherein said preceding peptide linker is as defined in claim 4 or the claim 5.

26. use one to be rich in p1 amino acid A, V, S and T, and the propetide that comprises two peptide sequences of forming by two acidic residues, two alkaline residues or acidic residues and alkaline residue, as the joint sequence of a cleavable, wherein said sequence can be separated from the antibacterial peptide of a kind of plant defense element or a kind of hevein type.

27. DNA construct, the promoter region that comprises the signal sequence of the plant origin that is operably connected, wherein said signal sequence can be operationally connected to one or more protein-coding regions and one 3 ' terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide, and described propetide provides a cracking site after the translation.

28. DNA construct, the promoter region that comprises be operably connected two or more protein-coding regions and one 3 ' terminator, wherein said protein-coding region is separated mutually by the dna sequence dna of a coding joint propetide, this dna sequence dna is from the Dm-AMP gene or from the Ac-AMP gene and the C-CICP of encoding, and described propetide provides the cracking site after the translation.

29. according to the DNA construct of claim 27 or 28, any terminal or two end all additionally comprises one or more proteolytic enzyme recognition sites to the dna sequence dna of the joint propetide of wherein encoding at it.

30. a carrier comprises each DNA construct according to claim 19-21.

31. transgenic plant, it is to use according to each DNA construct or carrier among the claim 27-30 to transform.

32. the carrier that uses DNA construct or comprise described construct improves proteic expression level in the transgenic plant, described dna structure body comprises a dna sequence dna, this dna sequence dna comprises the promoter region of be operably connected two or more protein-coding regions and one a 3 ' terminator, wherein said protein-coding region separated mutually by the dna sequence dna of a coding joint propetide, and described propetide provides the cracking site after the translation.

33. a nucleic acid, its coding SEQ ID NO.4,6,7,29,21,22,23,24,25,26,27,28 peptide or joint peptide shown in Figure 34, the perhaps variant of any wherein.

34. according to the nucleic acid of claim 33, its coding SEQ ID NO.4,6,7,29,21,22,23,24,25,26,27,28 peptide or joint peptide shown in Figure 34.

35. according to the nucleic acid of claim 33, it is coded in the peptide that SEQ ID NO.4,6,7,29,21,22,23,24,25,26,27,28 C-end include SEQ ID NO.77, the joint peptide of perhaps encoding shown in Figure 34.

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