CN1231698A - DNA-constructs comprising intergenic ribosomal DNA and methods to produce proteins using these DNA-constructs - Google Patents

Abstract

Provided are a DNA-construct, comprising the following fragments: ribosomal DNA, promoter region and heterologous coding region, a method for the production of proteins and for enhancing copy number or expression using these DNA-constructs, as well as host-organisms, particularly plants, comprising these constructs.

Description

DNA construct and these DNA construct method for synthesizing protein of use

The invention provides the DNA construct that comprises the intergenic region dna fragmentation between rrna rRNA gene redundancy sequence, also be provided at simultaneously eukaryotic cell, especially in vegetable cell, use the method for these DNA construct method for synthesizing protein and increase copy number or expression.

Background of invention

The genomic integral part of higher eucaryote body is rDNA.Many transcription units (with the Arabidopis thaliana is example, is about 600) are arranged in series one by one.On cytogenetics, these unit forms become nucleolus organizer region, and they are positioned near the telomere of limited quantity karyomit(e) (for example karyomit(e) 2 of Arabidopis thaliana and karyomit(e) 4).The RNA polymerase I is responsible for ribosome-RNA(rRNA) (rRNA) gene transcription specifically.Because their high copy number, ribosomal RNA gene is first gene that obtains analysis of molecules.The repetitive nature of these genes has hindered the further analysis to them considerably.

The progress that the understanding rDNA is transcribed mainly uses the vitro system that is derived from zooblast to obtain.In plant, after deliberation (between gene) zone between the many genus transcription unit, but to the explanation of function mainly relatively based on sequence.

The controlling element of transcribing be positioned at intergenic region (intergenic region, IGR), be also referred to as intergenic region (intergenic spacer, IGS).IGR is the DNA zone between the 18S RNA coding region of the 25S RNA coding region of previous rRNA gene unit and a back rRNA gene unit.It generally is made up of the nontranscribed domain between transcriptional domain outside 3 ' outer transcriptional domain (from the Transcription Termination site that the encoding mature 25S RNA zone end of previous rRNA gene unit extends to) and 5 ' (extending to the section start in encoding mature 18S RNA zone from the transcription initiation site of next rRNA gene unit) and these two zones.For higher plant, in most of the cases, IGR comprises the tumor-necrosis factor glycoproteins element.Since the accurate base sequence of these elements has only less mostly between relevant kind of system or do not have sequence similarity, they may be under low selective pressure so.This is the good conservative opposite of peripheral region, transcription initiation place with another part of IGR.

Use the moment expression system of Arabidopis thaliana recently, transcribe at rrna and to obtain sizable progress (Doellling on the understanding, J.H. and Pikaard, C.S. (1995) " the minimum ribosomal RNA gene promotor of Arabidopis thaliana comprises critical elements at transcription initiation site ", the phytology magazine, 8,683-692; Doellling, J.H., Gaudino, R.J. and Pikaard, C.S. (1993) " carries out functional analysis to Arabidopis thaliana ribosomal RNA gene and transcribed spacer promotor " in vivo and by the moment expression, institute of NAS newspaper, 90,7528-7532).In addition, in-vitro transcription system (Yamashita, J., the Nakajima of Kidney bean and tobacco have also been obtained, T., Tanifuji, 5. and Kato, A. (1993) " the correct transcription initiation of broad bean rDNA in the full cell extract of plumular axis ", the phytology magazine, 3,187-190; Fan, H., Yankura, K., Miyanishi, M., Sugita, M. and Sugiura, M. (1995) " it is species specific that the rRNA gene in vitro of plant RNA polymerase I-dependence is transcribed ", the phytology magazine, 8,295-298).

Summary of the invention

According to the present invention, DNA construct is provided, comprise the following continuous dna fragmentation of operating:

-comprise the dna fragmentation of ribosomal dna sequence, preferably derive from plant, preferably derive from the intergenic region of plant ribosome DNA; The upstream Sal I tumor-necrosis factor glycoproteins that comprises the intergenic region of Arabidopis thaliana rDNA specifically, or the similar area of another kind of plant;

-comprise the fragment of effable promoter region, especially plant-effable promoter region, preferably by the promotor of RNA polymerase II identification;

-allos coding region; And optionally

-Transcription Termination and polyadenylic acid zone, preferably activated zone in vegetable cell.

Particularly preferred ribosomal dna sequence comprises the dna sequence dna that is selected from following dna sequence dna: from the dna sequence dna of 486 Nucleotide to 5212 Nucleotide of SEQID NO:1, dna sequence dna from 1263 Nucleotide to 3003 Nucleotide of SEQ ID NO:1, dna sequence dna from 569 Nucleotide to 2862 Nucleotide of SEQ ID NO:1, dna sequence dna from 1263 Nucleotide to 2862 Nucleotide of SEQ ID NO:1, from the dna sequence dna of 486 Nucleotide to 5212 Nucleotide of SEQ ID NO:1, from the dna sequence dna of 596 Nucleotide to 5373 Nucleotide of SEQ ID NO:1.

Method for synthesizing protein also is provided, comprises the steps:

-will import in the suitable host organisms according to DNA construct of the present invention;

-under the condition of the protein expression that allows structural gene coding, cultivate host organisms; And

-results expressed protein.

In addition, be provided at the method that increases Stability of Transgenic, copy number or expression in the plant, comprise the steps:

-will import in the vegetable cell according to DNA construct of the present invention;

-from plant transformed cell regeneration plant.

According to the present invention, provide to comprise the host organisms that is integrated in their nuclear gene groups, particularly plant and vegetable cell equally according to DNA construct of the present invention.

The accompanying drawing summary

Fig. 1: the restriction map of Arabidopis thaliana rDNA;

Fig. 2: the sequence in the Arabidopis thaliana 25S rRNA V1 zone of (left side) and back (right side) before (A) insertion sequence imports

(B) be used to detect the oligonucleotide sequence of 25S rRNA;

Fig. 3: transgenosis confluence analysis;

Fig. 4: the primer extension analysis that is used to detect transgenosis rRNA;

Fig. 5: the mensuration of Arabidopis thaliana 25S rRNA 5 ' end;

Fig. 6: the continuous silver that contains the cell of transforming gene R4 dyes section;

Fig. 7: dystopy ribosomal gene transcription analysis;

Fig. 8 (a)-(e): binary vector R4 to R8;

Fig. 9-A/B: the diagram that before chimeric CaMV35S-gus gene, comprises or lack the binary vector of upstream Sal I tumor-necrosis factor glycoproteins.

Detailed Description Of The Invention:

An aspect of of the present present invention is to provide and makes the especially improved DNA construct of external source protein expression in plant cell of eukaryotic. Comprise the following DNA fragmentation that is operatively connected according to DNA construct of the present invention at reading direction:

-comprising the DNA fragmentation of ribosomal dna sequence, it preferably derives from plant;

-comprise the fragment of effable promoter region, especially plant-effable promoter region,

-allos code area; And optionally

-tanscription termination and Polyadenylation zone, preferably activated zone in plant cell.

Surprisingly use such construct can obtain the transformant of higher quantity, the stability that the more transformed gene of high copy number, and transgenosis strengthens and the expression of enhancing. Since those skilled in the art do not expect, by being combined and can obtaining more effective expression system with general DNA fragmentation of by the RNA polymerase II, being transcribed by effective DNA fragmentation (rDNA) in RNA polymerase I identification and/or transcription initiation, so this or even more unforeseen.

Although preferably by the promoter of RNA polymerase II identification, promoter also can be included in rDNA according to the promoter region in recombinant DNA of the present invention, it is easier according to the structure of recombinant DNA of the present invention that this can make. As used herein, term " plant-effable promoter region " expression can start the promoter of transcribing in plant cell. It comprises any promoter that originates from plant, but also comprise any promoter that can instruct the non-plant origin of transcribing in plant cell, be promoter such as CaMV35S or the T-DNA gene promoter of some virus or bacterium origin, and come from bacteriophage and can be by promoter region such as T7 or the special promoter of T3 RNA polymerase of the single subunit of specificity RNA polymerase identification. Under latter event, host cell must comprise the functional r NA polymerase of identification specificity promoter.

This is not, when mentioning rDNA and should derive from plant, just means that the sequence of that rDNA fragment should be same or similar with the sequence found in plant. Certainly, the DNA fragment can be cloned in middle organism such as Escherichia coli, or is completely or partially synthesized.

The rDNA fragment that the present invention is fit to can instruct the chromatin structure, method is to be incorporated into being positioned at the vicinity of kernel or transformed gene according to DNA construct of the present invention and integrating wherein genome area in the karyogene group, adopts the chromatin structure similar to the chromatin structure found in kernel. Determine that in cell, the sterically defined method of DNA fragmentation is known to those skilled in the art, and describe in an embodiment a kind of such method in detail. Measure chromatin structure feature, especially the method for DNA and the synergistic easy degree of interacting molecule, also known to those skilled in the art (for example, by measuring the neurological susceptibility of micrococcal nuclease or crosslinked (Ausubel etc. (1994)) by chromosomal protein. Referring to as follows, Dammann etc. (1995) " transcribing of yeast rRNA gene loci: the distribution of active flank regulating and controlling sequence ", molecule and cell biology, 15,5294-5303.

In preferred embodiments, the special rDNA of selecting to be suitable for will importing according to recombinant DNA of the present invention host living beings body wherein; For example be derived from the rDNA of the very near kind of that host living beings body or affiliation.

rDNA is preferably from the intergenic region of rDNA. as an example of arabidopsis example (intergenic region from kind Co10 has from the DNA sequence dna of 486 nucleotides to 5212 nucleotides of SEQ ID NO:1) herein, especially preferred is the DNA fragmentation that comprises from " the upstream Sal I repetitive sequence " of arabidopsis rDNA intergenic region. these upstreams Sal I repetitive sequence is arranged in three modules [so-called Sal I subassembly wrapper 1 (from 1263 nucleotides to 1557 nucleotides of SEQ ID NO:1), Sal I subassembly wrapper 2 (from 1883 nucleotides to 2177 nucleotides of SEQ ID NO:1) and Sal I subassembly wrapper 3 (from 2503 nucleotides to 3003 nucleotides of SEQ ID NO:1)], and think comprise contain these Sal I repetitive sequences DNA fragmentation (for example, contain from the fragment of the DNA sequence dna of nucleotides position, SEQ ID NO:1263 nucleotides position to 3003) be enough to obtain according to effect of the present invention. in fact, do not need the existence of the 3rd complete Sal I repetitive sequence, because can use, only comprise Sal I subassembly wrapper 3 and upwards reach similar effects to the fragment (containing from the DNA sequence dna of 569 nucleotides to 2862 nucleotides of SEQ ID NO:1) of the part in EcoR I site. therefore, in preferred embodiments, rDNA comprises the fragment that has from the DNA sequence dna of 1263 nucleotides to 2862 nucleotides of SEQ ID NO:1. in addition, can use the larger fragment that comprises upstream Sal I repetitive sequence, such as the fragment that comprises the complete genome spacer region (having from the DNA sequence dna of 486 nucleotides to 5212 nucleotides of SEQ ID NO:1), perhaps even comprise part coding rRNA transcript, the especially fragment of the coding region of 18S transcript (as having from the fragment of the DNA sequence dna of 596 nucleotides to 5373 nucleotides of SEQ ID NO:1). in addition, also may use the fragment that comprises complete ribosomal gene unit (its DNA sequence dna can by merging and obtain from the overlapping DNA sequence that EMBL database typing X16077, X52320 and X15550 obtain), although when using this DNA construct, may due to screen effect makes effect of the present invention may be more not obvious.

for other host plant, specially suitable DNA fragmentation comprises those domains corresponding to the rDNA of those domains in arabidopsis, especially comes from their those domains own. the DNA fragmentation that preferably comprises the intergenic region between the DNA zone in coding 25S and 18S zone, particularly comprise the DNA fragmentation of the non transcribed intergenic region between the transcription initiation site of the tanscription termination site of previous rRNA gene unit and a rear rRNA gene unit. in the art, [14:4953-4968 (1986) for McMullen etc., nucleic acids research for corn at least, Toloczyki and Feix, nucleic acids research, 14:4969-4986 (1986)], rye [Appels etc., Canada's cytogenetics magazine, 28:673-685 (1986)], wheat [Barker etc., the molecular biology magazine, 201:1-17 (1988)], radish [Delcasso-Tremousaygue etc., european journal of biological chemistry, 172:767-776 (1988)], paddy rice [Takaiwa etc., molecular biology of plants, 15:933-935 (1990)], mung bean [Gerstner etc., genome, 30:723-733 (1988), Schiebel etc., molecular genetics and General Genetics, 218:302-307 (1989)], potato [Borisjuk and Hemleben, molecular biology of plants, 21:381-384 (1993)], tomato [Schmidt-Puchta etc., molecular biology of plants, 13:251-253 (1989)], broad bean [Kato etc., molecular biology of plants, 14:983-993 (1990)], pea [Kato etc., the same, and barley [Procunier etc. (1990)], molecular biology of plants, 15:661-663 (1990)] other rRNA intergenic region be known. and use oligonucleotides corresponding to 5 ' end of 3 ' end of the ripe rRNA code area of conservative 25S and the conservative ripe rRNA code area of 18S, can be in the PCR reaction amplification gene spacer region from plant simply.

Have been reported (Gruendler etc., 1991, molecular biology magazine 221,1209-1222), the number of upstream Sal I repetitive sequence may be different between different separators of the same race or mutually not of the same race (take arabidopsis as example). The present invention also comprises this variation.

Also may use by variation, insert or disappearance comes from the rDNA of the modification of original rDNA, as long as rDNA basic features relevant (for example for the DNA binding protein dna such as polymerase) or be that basic topology is learned characteristic and because of modification, do not lost at least particularly.

As used herein, " coding region " or " encoding sequence " is meant when the appropriate regulation zone is provided, especially can be transcribed into the DNA zone of the RNA of biologically active during promoter region, promptly it can interact with another nucleic acid such as sense-rna or ribozyme and maybe can translate into the polypeptide or the protein of biologically active.

As used herein, the term " allogenic " about the coding region is meant any and used according to chimeric DNA of the present invention different coding region, the natural bonded rRNA of rDNA fragment coding region.

For the coding region, can use all known natural or coding regions of modifying, this coding region and host organisms are compatible, in other words, expression product is expressed in such a way, before collecting product, if collect, product does not have too big toxicity to the host when promptly particularly causing significantly expressing.Especially, the coding region vaccine of may encoding, antibody, treatment albumen, insecticidal protein such as Bt toxin or its minimum toxic fragment, used protein, sense-rna or ribozyme in the food technology.

DNA construct can prepare according to the mode that is adapted to specific host or conversion system.In preferred embodiments, be with carrier format according to DNA construct of the present invention.Particularly, DNA construct is the T-DNA carrier.

Although in according to DNA construct of the present invention, the rDNA fragment is preferably located in before promoter region and the allos coding region, but when the promoter region that expection can be operatively connected when the rDNA fragment is positioned at and the downstream of coding region, similar effect will be obtained.

On the other hand, the present invention also provides method for synthesizing protein, comprises the steps:

-will import in the host organisms that is fit to according to DNA construct of the present invention,

-under the condition of the protein expression that allows structural gene coding, cultivate host organisms,

-optionally, collect expressed protein.

On the other hand, the invention provides especially enhancing transforming gene stability in plant, the method for copy number and/or expression comprises the steps:

-will be according to DNA construct transfered cell of the present invention, the preferred plant cell,

-from cell transformed regeneration biological body, preferred plant.

Method of the present invention is particularly suitable for strengthening the stability of recombination and expressing, this recombination at least with host cell, especially the sequence that exists in the vegetable cell has portion homologous, as the transforming gene of multiple copied or comprise similar sequences such as the difference of identical promoters transforms group.Known such transforming gene usually has homologous recombination and reduces the tendency of expressing by for example methylation or inhibition incident altogether.Although, deliberately do not limit the invention to a kind of binding mode, but thought by transform according to DNA construct of the present invention the transforming gene that causes in kernel or the reinforcement of integrating the kernel sample chromatin Structure on wherein the chromosomal region of the contiguous location of kernel or transforming gene reduced reorganization (therefore having strengthened stability), the methylation between the homologous sequence and caused any incident of such transforming gene expression decreased.

Can depend on used host organisms and import DNA by any method or mode and the different technologies that uses those of skill in the art to grasp.The method of particularly preferred importing DNA is T-DNA conversion, electroporation or particle bombardment, and plasmid or virus conversion.

Optimal culture condition depends on the character of used host organisms and expressed proteins.These conditions are also understood (for known host organisms) by those skilled in the art and/or can be used known biotechnological means to determine or optimization at an easy rate.

Protein is collected also preferably according to carrying out for the standard method of relevant host organisms, and (for example, protein is secretion or drains to substratum, still at the host living beings cylinder accumulation also to depend on the mode of marking protein, or be included in the special space, or the like).The same with culturing step, experiment that need not be too much herein, those skilled in the art just can determine and/or optimize the collection condition for specific host/structural protein system.

Preferred host living beings, particularly plant are the systems of well-known and the fine characteristic of tool, such as Arabidopis thaliana or important cash crop such as tobacco, corn, wheat, potato, paddy rice, soybean, barley, rye, wild cabbage vegetables, beet kind or cassava.

The present invention also provides the host organisms that comprises DNA construct of the present invention.Preferably, this host organisms is an eukaryotic cell, vegetable cell particularly, and comprise the organism of these cells or from the organism of these cell regenerationes.

On the other hand, the invention provides the reproductive material that comprises the cell that contains with good grounds DNA construct of the present invention, especially plant propagation material.

The present invention and advantage thereof are further specified in non-restrictive example and accompanying drawing.

At embodiments of the invention with in describing, the following sequence of reference sequences table:

SEQ ID No.1: the dna sequence dna of the intergenic region of Arabidopis thaliana rRNA gene redundancy sequence

SEQ ID No.2: oligonucleotide

SEQ ID No.3: oligonucleotide

SEQ ID No.4: oligonucleotide E

SEQ ID No.5: oligonucleotide Q

SEQ ID No.6: the dna sequence dna experiment in Arabidopis thaliana zone between 5.8S rDNA 3 ' end and 25S rDNA 5 ' end

Unless explanation is arranged in an embodiment in addition, all recombinant DNA technologies are all according at (1989) molecular clonings such as Sambrook: experiment guide, second edition, cold spring harbor laboratory publishes, NY and Ausubel etc. (1994) molecular biology common method, the first roll and second volume, common method, the standard method described in the USA is carried out.The standard material of plant molecular work and method are described in BIOS scientific publication thing limited liability company (UK) and Blackwell scientific publication thing (UK) combined publication at the molecular biology of plants Labfax (1993) that R.D.D. showed.These publications also comprise the catalogue of explaining abbreviation commonly used.1. vector construction

Will be from Sal I fragment [Grundler, P., Unfried, I., Pointner, R. and Schweizer, D. (1989) " nucleotide sequence of Arabidopis thaliana 25S-18S ribosomal gene transcribed spacer ", nucleic acids research, 17, the 6395-6395 of lambda particles phage ATR3; Unfried, I., Stocker, U. and Grundler, P. (1989) " from the nucleotide sequence of the 18S rRNA gene of Arabidopis thaliana Col-0 ", nucleic acids research, 17,7513; Unfried, I., and Grundler, P. (1990) " nucleotide sequence of Arabidopis thaliana 25S and 18S rRNA gene and internal transcription transcribed spacer ", nucleic acids research, 18,4011], insert the Sal I restriction site that border (1400 Nucleotide of sequence X 16077) extends to 1263 Nucleotide of sequence X 15550 from carrier, insert binary vector pBIB Hyg[Becker, D. (1990) " allows the binary vector of plant selectable marker and reporter gene exchange ", nucleic acids research, 18,203] in the Sal I restriction site.Direction of insertion is that the 18S gene location is near the Asp718 restriction site.The carrier R2 of gained cuts with the Asp718 enzyme, handles with the Klenow enzyme, uses the Sfi I partially digested then.The sequence of rrna transcription unit is inserted and finished to the fragment that will extend to Sfi I restriction site (sequence X 16077 1490) from (mending flat) Xho I restriction site (569 Nucleotide of sequence X 15550) of ATR3 in carrier R3.

In order to insert oligonucleotide, at first will go into pSK+ (plasmid pBlu/SP) from 305 Sal I restriction site to the largest portion that comprises the 18SrDNA sequence of 1425 the Pst I restriction site that is positioned at sequence X 52320 and the fragment cloning of 25S rDNA sequence 5 ' end that is arranged in sequence X 16077.After cutting with Xba I and Srf I enzyme, mend flat sticking terminally and connect with the Klenow enzyme, connect product and cut, handle and connection again with the Klenow enzyme with Xho I and EcoN I enzyme.In the carrier that forms single Aat II restriction site is arranged now.Make oligonucleotide CCAAGGTAACCTTCGACGT (SEQ ID NO 2) and CGAAGGTTACCTTGGACGT (SEQID NO 3) annealing and insert Aat II restriction site.After measuring sequence, change BamH I/BstB I fragment over to carrier pBlu/SP (carrier pBlu/SP+E).To insert carrier R3 (carrier R4 from sfi I/srf I fragment of carrier pBlu/SP+E; Fig. 8 a).2. Plant Transformation

Except using the Totomycin selection, according at Valvekens, D., Van Montagu, M. with Van Lijsebettens, M. (1988) " carries out the conversion of the Arabidopis thaliana root explant of Agrobacterium tumefaciems mediation by using kantlex to select ", institute of NAS newspaper, 85, carry out Plant Transformation described in the 5536-5540.Selective marker among embodiment 1 and the embodiment 2 in the used plasmid is actually hygromix phosphotransferase.3.DNA separate

According at (Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) " the method II for preparing DNA of plants on a small quantity ", molecular biology of plants report, 1, the DNA that carries out 19-21) separates, and following modification is arranged: after the phenol extracting, add the ethidium bromide (10g/l) and the CsCl (0.9g/ml) of 1/10 volume in dna solution.Solution was placed on ice 30 minutes.Centrifugal (5 minutes, 5000RPM) after, supernatant liquor with CsCl with Auto-regulating System of Density of Heavy Medium to 1.6g/ml, in Beckman NVT90 rotor centrifugal 3 hours in 80000RPM.Use standard method to collect DNA[Ausubel from gradient, F.M, Brent, R., Kingston, R.F., Moore, D.O., Seidman, J.G., Smith, J.A. and Struhl, K. (editor), (1987), molecular biology common method, John Wiley ﹠amp; Sons company, New York].4. carry out DNA analysis by the gel trace

From the purify DNA of callus material with Restriction Enzyme such as the digestion of BstE II and be used for carrying out gel engram analysis (Ausubel etc., 1987).5.RNA preparation

According at Logemann, J., Schell, J. and Willmitzer, L. (1987) " isolation of RNA improves one's methods from plant tissue " biochemical analysis, 163, method 16-20) prepares RNA, and following modification is arranged: in liquid nitrogen, grind the callus material, every gram callus material and 2ml extraction buffer (8M chlorination guanidine, 20mM MES, 20mM EDTA, the 50mM 2 mercapto ethanol, 0.5% sarcosyl, pH is about 3) mix.The suspension liquid that melts is transferred to centrifuge tube, with long-pending phenol/chloroform/primary isoamyl alcohol (50: 49: the 1) extracting of monoploid.Behind the thermal agitation, mixture placed on ice about 15 minutes and centrifugal 10 minutes in 10000g.Move on to water in the new pipe and add the sodium acetate (1M) of 1/10 volume and the long-pending ethanol of monoploid with precipitate nucleic acids.Solution mixing back is centrifugal in 10000g.With 3M sodium acetate and 70% washing with alcohol precipitation.After residual alcohol volatilization, will precipitate water-soluble, in 60-65 ℃ of incubation 3 hours.6. primer extension analysis

With 25ng oligonucleotide E[GAAGACGTCGAAGGTTACCTTGG (SEQ ID NO 4); Single-minded combination has the oligonucleotide of the rRNA of 19 Nucleotide insertions] or oligonucleotide Q[CCCGGTTCGCTCGCCGTTACTAAG (SEQ ID NO 5); The Nucleotide 950 of the noncoding strand of sequence X 52320 can combine with all 25SRNA molecules of Arabidopis thaliana to Nucleotide 927] in 10 μ l volumes with 10 μ Ci ³²P-γ-ATP phosphorylation 90 minutes, add 1mg tRNA after, with phenol/chloroform extracting, use sodium acetate and alcohol precipitation then.The 2.5 μ g Arabidopis thalianas total RNA incubation of oligonucleotide in 11 μ l water to make it hybridization, is cooled to 63 ℃ from 90 ℃, then at about one and a half hours time range internal cooling to 52 ℃.For extension, add 5 μ l5 * RT damping fluid (manufacturer provides by enzyme), 2 μ l dNTPs (2mM) and 0.5 μ l (12 unit) AMV reversed transcriptive enzyme (Bao Lingman).38 ℃ of incubations added the DNA enzyme (5mg/ml that 1 μ l does not have RNA enzyme A after one hour; Bao Lingman), with mixture in 37 ℃ of further incubations 30 minutes.Reaction mixture is separated in the used denaturing polyacrylamide gel of dna sequencing.7. cell section and dyeing

The callus material is fixed and is embedded among the LR White Harz with the 0.1M phosphoric acid buffer that contains 2.5% Paraformaldehyde 96 and 0.5% glutaraldehyde.Use Reichert Ultracut E microtome to carry out continuous servo-actuated section (each nucleus approximately generates 5-6 and opens section) with 800nm thickness.Section is dyeed and analyze with the Richardson dyestuff with Leitz Dialux microscope.EXAMPLE Example 1 imports DNA construct Arabidopis thaliana and analyzes transformant

Relatively disclosed the existence in some tool highly variable zones from the sequence of ribosome-RNA(rRNA) not of the same race.In many cases, Variable Area has hairpin structure, this hairpin structure or longer or shorter or even disappearance fully.A kind of such variable region (being called V1) among the big rRNA can be used for inserting specific nucleic acid squences.This zone is enough near from the section start of ripe 25S rRNA, can extend experiment like this.

To contain dna fragmentation insertion binary vector pBIB Hyg (Becker, D., 1990) sometimes more than the rDNA of a tumor-necrosis factor glycoproteins unit (transcription unit).This construct comprises the transcription termination signal of supposition at tumor-necrosis factor glycoproteins unit two ends.The restriction map of structure of this construct (being called R4) and Arabidopis thaliana rrna tumor-necrosis factor glycoproteins unit is in Fig. 1 and Fig. 8 (a) expression.

Be expressed as 18S, the zone of 3 ribosome-RNA(rRNA)s of hollow frame representative coding of 5.8S and 25S.Three modules of repetition DNA wherein comprise many Sal I restriction sites in " upstream Sal I tumor-necrosis factor glycoproteins " expression intergenic region.The thick line at chart two ends is represented carrier DNA.Inserting the BstE II restriction site that imports by Nucleotide represents with " B ".The difference of carrier R5 and carrier R4 is the disappearance of " upstream Sal I tumor-necrosis factor glycoproteins " and Kpn I restriction site (K) is arranged on the border of rrna unit.B,BstEⅡ；E,EcoRⅠ；F,FspⅠ；H,HindⅢ；K,KpnⅠ；P,PstⅠ；Pc,PacⅠ；S,SalⅠ；Sc,ScaⅠ；Sf,SfiⅠ；Sr,SrfⅠ；X,XbaⅠ；Xh,XhoⅠ。

This regional nucleotide sequence can be from EMBL database typing X16077, and X52320 and X15550 obtain.Also can be from EMBL database typing X52631, X52637 and X52636 obtain variant [Griundler etc., 1989 of the rDNA of part; Unfried etc., 1989; Unfried etc., 1990; Griundler, P., Unfried, I., asch, R. and Schweizer, D. (1991) Arabidopis thaliana rDNA intergenic region: structural analysis, variation and functional meaning between kind.The molecular biology magazine, 221:1209-1222].

As mentioned above, in order to discern it specifically and it to be distinguished with other ribosomal gene unit of being present in the genome, be inserted in greatly on (25S) ribosome-RNA(rRNA) with mark ribosomal gene unit (Fig. 1) with little Nucleotide.Fig. 2 represents that Nucleotide inserts and allow to detect the oligonucleotide that this (mark) ribosomal gene unit transcribes.Insertion sequence has BatE II restriction site, and this site can be used for specific recognition gene unit (a), the sequence in the front (left side) that the oligonucleotide sequence is inserted and the Arabidopis thaliana 25S rRNA V1 zone on back (right side).Inserting segmental secondary structure and suppose, only is to compare for the structure when not inserting.Insertion sequence is at DNA (b), allow by reverse transcription to the 25S ribosome-RNA(rRNA) detect with quantitative oligonucleotide sequence in BstE II restriction site is arranged.

The further construct [seeing Fig. 8 (b)] that is called R5 is to remove " upstream Sal I tumor-necrosis factor glycoproteins " with the R4 difference.

Use Agrobacterium tumefaciems construct R4 and R5 to be imported the root explant of arabidopsis thaliana.The assignment of genes gene mapping is transcribed by the RNA polymerase II near the rrna unit, and has the resistance (hygromycin phosphotransferase gene to hygromycin B; Becker, D., 1990).According to they resistances, select cell transformed/callus to Totomycin.To be used to carry out the southern blotting technique experiment from the DNA of these callus.

Fig. 3 represents that the copy of rrna transforming gene and contiguous marker gene can be incorporated into genomic different positions.For digested genomic dna, use BstE II enzyme, this enzyme does not cut in the ribosomal gene of the environmental Col-0 of Arabidopis thaliana.Therefore, can see that rDNA is the high molecular band or even partly is trapped in the gel groove.On the contrary, because near the genome area the integration position, BstE II restriction site should exist with statistical random fashion, so the rrna transforming gene of integrating between natural rrna repetitive dna sequence can not produce little band.In the callus of analyzing, can see a kind of result in back exactly.

The DNA hybrid experiment that Fig. 3 represents proves that at least in most of the cases the integration of transforming gene does not occur in the rDNA zone.The DNA that is derived from the callus material cuts with BstE II enzyme, and is transferred on the nylon membrane after size classification on the sepharose separates.Swimming lane 1 and swimming lane 2 contain the DNA from the callus material of the DNA of unconverted callus material and the DNA construct R4 conversion of using by oneself respectively.With the DNA of hygromycin phosphotransferase gene as probe.Fragment one end that detects has the BstE II restriction site that is positioned 25S rRNA transforming gene, has at the other end to be positioned at the BstE II restriction site that DNA integrates the position.Since endogenous rDNA does not comprise BstE II restriction site, the existence of so this restriction site in the host DNA of next-door neighbour's transforming gene constitutes the evidence that transforming gene is integrated outside rDNA.The dna digestion that 4 expressions of swimming lane 3 and swimming lane and swimming lane 1 and swimming lane 2 are identical still uses rDNA as probe here.Be derived from the DNA of callus or stay in the gel groove, perhaps under the restriction of gel separation ability, move as polymer DNA.Swimming lane 5-swimming lane 8 comprises from three further DNA of the independent callus that transform, wherein swimming lane 8 with the rrna probe hybridization.

Prepare RNA from the callus that transforms, and as primer extension analysis.Therefore, the design with variable region V1 in the insertion fragment combine but not with the RNA bonded oligonucleotide that does not contain this insertion.The synthetic DNA fragment that is expressed as oligonucleotide E in Fig. 2 is specially suitable.In the callus that transforms, obtain using the reverse transcription product of this oligonucleotide, but in unconverted callus, can't obtain as primer.In most of the cases, can detect the activity (Fig. 4) of rrna transforming gene.

Carry out reverse transcription reaction with oligonucleotide E as primer and produce the product that is about 150 bases (representing) with asterisk.Collect several callus with preparation RNA[(a) swimming lane 1: the callus material that rrna transforming gene DNA of no use transforms; Swimming lane 2: with the callus material of rrna transforming gene R4 conversion; (b) gel groove (indicating) and molecular weight size marker with arrow; In many experiments, can see the have higher molecular weight precursor of (asterisk 2 or asterisk 3); Swimming lane 1: molecular weight size marker; Swimming lane 2: with swimming lane 1 in (a); Swimming lane 3: with swimming lane 2 in (a)).

Obtain from preference pattern, in all cases, contiguous hygromycin gene all is activated.

The processing of rrna transforming gene can be identical with natural ribosomal gene; can determine the 5 ' end (Fig. 5) of identical 25S rRNA therein: use oligonucleotide Q to measure the 5 ' end of normal gene group 25SrRNA, use the 5 ' end of oligonucleotide E mensuration with the transforming gene of insertion sequence mark.The order-checking band relatively turn out to be same end.And the RNA end is different with supposition up to the present, and wherein 25S rRNA is than longer (Fig. 5) of up to the present being admitted.Equally, also can detect the processing intermediate of 25SrRNA, show that again unaltered rRNA is similar with the processing of the rRNA that contains insertion sequence.

Expression Arabidopis thaliana 25S rRNA 5 ' terminal determining in Fig. 5.Fig. 5 (a) shows the primer extension analysis that uses oligonucleotide Q to carry out, and this oligonucleotide Q can be in conjunction with all 25SrRNA transcripts.The right side represents to use identical oligonucleotide as the resulting order-checking band of primer.Fig. 5 (b) shows the reverse transcription that uses oligonucleotide E to carry out, and this oligonucleotide E is special to transgenosis rrna copy.The left side expression uses identical oligonucleotide as the resulting order-checking band of primer.The last base of asterisk mark reverse transcription thing.Genetically modified and endogenous rrna 25S rRNA has 5 ' identical end.Fig. 5 (c) is similar to the swimming lane 3 of Fig. 4 (b), but has the order-checking ladder.With 5 ' the terminal indication of 25S rRNA, the sequence of Fig. 5 (d) (with SEQ IDNO 6) expression rDNA has the indication (representing with an asterisk and two asterisks respectively) of 25S rRNA 5 ' end and precursor.5 ' the end that the some representative was inferred in the past.The part of 25S and 5.8S RNA represented to be present in respectively in black matrix.

The processing that genetically modified with natural ribosome-RNA(rRNA) is identical shows that the necessary composition of this process (generally occurring in the kernel) can arrive the ectopic integration gene.The processing of further analyzing natural ribosome-RNA(rRNA) is to be positioned in the kernel, still is positioned near the kernel.Positive findings and fruit bat (Karpen, G.H., Schaefer, J.E. and Laird, C.D. (1988) " transcribing when forming with kernel, it is activated being positioned euchromatic fruit bat rRNA gene ", the gene development, 2, situation about finding in 1745-1763) conforms to, and situation about finding in negative findings and the Saccharomyces cerevisiae is similar.Silver dyes the transgenic arabidopsis nucleus and prepares continuous thin section.Fig. 6 and table 1 show that the arabidopsis cell that comprises aforesaid transforming gene on average has an extra kernel.

Callus cell that table 1:R4 transforms and the average kernel number in the unconverted control cells (in each case, as shown in Figure 6, analyzing 25 cells) by serial section.

Average kernel is counted R4 transgenic cell 2.8 ± 0.5 control cells 2.0 ± 0.3

These pass the serial section of the cell that contains transforming gene R4 and silver and dye and shown the existing [(a) to (j): pass the section that does not have the Arabidopis thaliana of dystopy rDNA callus cell of extra kernel; (k), to (j), still use the Arabidopis thaliana callus material (pointing out tangible cell) that contains the dystopy rDNA with asterisk as (a) to (t); Visible extra kernel in (k) to (t).]。

Callus cell nuclear through selecting on average has 3 kernels, and is about 2 in unconverted callus material; Can not illustrate that one in them only comprises the dystopy copy by the size of 3 kernels.Can suppose that equally the effective active ribosomal gene divides in 3 all copies.Further experiment shows the position of the assignment of genes gene mapping of the hygromycin resistance of encoding at contiguous at least kernel.

In further a series of experiments, the construct R5 that analyze to lack upstream Sal I tumor-necrosis factor glycoproteins whether also obtain transcribing with and transcriptional level whether different with the transcriptional level of construct R4.

Therefore, to only with carrier sequence (swimming lane 1 in Fig. 7 two boards), or with the rrna copy (swimming lane 2 in the two boards) that lacks upstream Sal I tumor-necrosis factor glycoproteins or copy (swimming lane 3 in the two boards) plant transformed material with complete rrna and carry out reverse transcription and analysis.In Fig. 7 (a), use oligonucleotide E (special) to the transgenosis copy; In Fig. 7 (b), use can be in conjunction with the oligonucleotide Q (as the contrast of similar amt RNA) of all 25S rRNA molecules.

Fig. 7 demonstration does not detect any difference.For the position effect on the transforming gene of getting rid of some integration, the mixture that uses several independent callus that transform is as parent material.And it seems that rRNA processing is not subjected to the influence of upstream Sal I tumor-necrosis factor glycoproteins disappearance.Therefore it seems that the sequence that is derived from what is called " upstream Sal I tumor-necrosis factor glycoproteins " (see figure 1) is to not obviously influence of ribosomal gene unit's expression level in the fresh transformed calli material.Fig. 7 shows the qualitative assessment to the genetic expression of ribosomal gene unit.

Yet, confirm that the dna fragmentation that comprises " upstream Sal I tumor-necrosis factor glycoproteins " is stable influential to the DNA's that imports.Fig. 8 (a) to (e) shows that [R4-R8 is the intestinal bacteria-edaphic bacillus shuttle vectors with RK2 replication orgin to the construct that compares mutually, and wherein " Br " and " Bl " represents a left side and the right margin of T-DNA respectively; (a) binary vector R4: the complete rrna unit that comprises the Arabidopis thaliana adjacent with hygromycin gene (HPT); (b) binary vector R5: similar to R4, there be not " upstream Sal I tumor-necrosis factor glycoproteins " (shortage) from the dna sequence dna of 1269 Nucleotide to 3002 Nucleotide of SEQ ID NO:1; (c) binary vector R6: the intergenic region (dna fragmentation of 596 to 5373 bit sequences by will having SEQ ID NO:1 inserts and obtains among the pBIB Hyg making up) that only comprises the rDNA of contiguous hygromycin gene; (d) binary vector R7: similar to R6, but lack " upstream Sal I tumor-necrosis factor glycoproteins " (disappearance from the dna sequence dna of 1269 to 3002 Nucleotide of SEQ ID NO:1); (e) binary vector R8: the RNA (similar, but not from the dna sequence dna of 1269 to 5075 Nucleotide of SEQ IDNO:1) that only comprises the encoding part rDNA of contiguous resistant gene] to R1.The size of plasmid is about (pressing base pair) 23810 (R4), 21810 (R5), 16010 (R6), 14010 (R7), 19960 (R8).

Make the transforming gene stabilization according to all constructs of the present invention, this stabilization shows as the existence, particularly structure gene of high copy number more and/or higher transformation efficiency especially such as the more stable expression of the marker gene of hygromycin phosphotransferase gene.

The hypothesis of explaining this effect is that this sequence protection rrna tumor-necrosis factor glycoproteins is avoided reorganization and/or methylated.This sequence also is responsible for the location of rDNA in the territory, special subprovince of nuclear (kernel).Since experiment shows that the contiguous gene of transcribing by the RNA polymerase II allows to be positioned the kernel adjacent domain and confirmed activity, can reach a conclusion so, promptly stabilising effect can expand this genoid to.Embodiment 2: (contain " upstream Sal I tumor-necrosis factor glycoproteins ", USR) (do not contain " upstream Sal I tumor-necrosis factor glycoproteins ", comparison USR) is especially about genetically modified copy number and expression level with the conventional construction body according to construct of the present invention.

2.1 as described in the experiment part, also digest with restriction enzyme BamH I and Hind II from the callus material preparation DNA that transforms.Two kinds of enzymes all cut in the DNA that shifts, and like this, when carrying out size separation with gel electrophoresis, are integrated into genomic all copy swimmings and run into single band.By Southern hybridization, use the Totomycin drug resistant gene as probe, by determine the relative number of different callus transfer gene copies by means of Phospho imaging instrument comparison hybrid belt intensity.In contrast, use the known DNA that in genome (chlorata 42), exists southern blotting technique to be hybridized as probe with two copy numbers.Measure the band relative intensity as mentioned above, be used for estimating the quantity of different swimming lane DNA.Band intensity by two kinds of probes compares, and can determine that the transforming gene of being analyzed exists with following copy number:

Construct Fig. 8-A (contains USR; According to of the present invention; At present the applicant thinks that this structure is to implement best mode of the present invention) and design of graphics 8-B (do not contain USR; Prior art) comparison:

The construct that contains USR: the genomic copy number of every monoploid is 7 in 6 examples; 5.5; 0.5; 3.5; 3; 0.5, obtain average 3.3 ± 2.7 copies.

The construct that does not contain USR: the genomic copy number of every monoploid is 2 in 5 examples; 1; 2; 1.5; 1.5, obtain average 1.6 ± 0.5 copies.

Construct Fig. 8-C (contains USR; According to of the present invention) and construct Fig. 8-D (do not contain USR; Prior art) comparison:

The structure that contains USR: the genomic copy number of every monoploid is 1.5 in 4 examples; 1; 2.5; 2, obtain average 1.8 ± 1.2 copies.

The structure that does not contain USR: the genomic copy number of every monoploid is 2 in 5 examples; 1; 1; 1.5; 0.5, obtain average 1.2 ± 0.5 copies.

The dispersity that should be mentioned that the construct that contains USR is obviously higher.This explanation can guarantee that the expression of transforming gene for example also exists with suitable height or quite low copy number with wideer transgenosis configuration scope in competent mode.

Also confirm, use, particularly when they are incorporated in the genome with the appropriate location, can realize the increase (seeing the result of construct 8-A) of copy number according to construct of the present invention.

2.2 make up two DNA construct, these two DNA express reporter gene beta-glucuronidase gene (gus) under the control of the CaMV35S promotor of fully identifying.Contiguous in addition this gene, USR sequence or existence or disappearance (seeing Fig. 9 A and B).

Carrier shown in Fig. 9 B passes through at the no of pBIB Hyg terminator and (Becker etc., 1990) importing contains (1993) " high level expression expression carrier in dicotyledons and monocotyledons " such as coming from pRTgus[Topfer in the polynucleotide joint between the hyg gene, Enzymology method, 217, the dna fragmentation of chimeric CaMV35S-gus gene 66-78) obtains making up.Carrier shown in the design of graphics 9A obtains making up by containing the carrier that imports Fig. 9 B from EcoR I-Sal I dna fragmentation of the dna sequence dna of 596 to 2862 Nucleotide of SEQ ID NO1, and inserted mode is to locate the contiguous segmental end of rDNA of Pol I promotor at first to be positioned now near the CaMV35S promotor.

The enzymic activity that transforms genes encoding in the transgenic calli material is assessed, with transgenic calli material (2 to 25mg) at 200 μ l extraction buffer [50mM sodium sulfate (PH7), 10mM 2 mercapto ethanol, 10mM EDTA, 0.1%SDS, 0.1%Triton X-100] middle homogenate.Centrifugal (10 minutes, 4 ℃, 18000rpm) after, measure active.Then, add right-amino-benzene-beta-glucuronic acid (pNPG; 2mM) and in 37 ℃, incubation 10 to 30 minutes.By adding 0.2M yellow soda ash termination reaction (Gallagher compiles, GUS scheme, AcademicPress, 1992 modification protocols).

Contain the comparison (the relative unit of enzyme with the transformed calli of every gram fresh weight is represented) of USR and the construct that does not contain USR:

The construct that contains USR: the activity of measurement is 2.3; 6.9; 11.2; 0.3; 1.2; 0.3; 1.0; 2.3; 2.0; Average 3.1 ± 2.8.

The construct that does not contain USR: the activity of measurement is 0.1; 1.6; 0.0; 1.1; 0.6; 0.2; 0.6; 1.5; 0.8; Average 0.7 ± 0.4.

Very obvious, contain the not only active mean level (ML) of USR higher (the difference factor 4), and active dispersity is also bigger.(for example 0.0 or 0.1) callus of utmost point low activity level does not appear showing with the construct that contains USR furthermore.Reach a conclusion thus, can obtain wider transgenosis activity with stable manner.

Also prove at this, by the integration in a certain position can reach higher expression level (according to Fig. 9 A, seeing the activity from 6.9 to 11.2 that contains the USR construct) according to construct of the present invention.This obvious enhanced activity may be because r-DNA transcribes to have created satisfied chromatin environment.

The more wide region of transgenosis configuration is stablized and allowed another reason of genetic expression obviously is the following fact, and promptly r-DNA has hindered for example normal regulation mechanism that methylates and suppress by existing in the pairing inhibition of homology zone, homologous recombination inhibition, the euchromatin of chromatin.

When some units exist with tandem repetitive sequence or some sites exist on genome, under the situation that some sequence of rDNA exists, can guarantee that the transgenosis activity causes higher expression with higher copy number.Embodiment 3: analyze using the maize plant that transforms according to DNA construct of the present invention

Use is corresponding to 5 ' of 3 ' of the ripe rRNA of conservative 25S the coding region terminal and conservative ripe rRNA of 18S coding region terminal oligonucleotide (sequence that use can obtain from EMBL database typing X03990), by the rRNA intergenic region of pcr amplification corn.The design oligonucleotide comprises suitable restriction site with the end at amplified fragments.

As described in the WO92/09696, with the CaMV35S promoter region upstream that fragment cloning can be operatively connected to the zone with coding phosphinothricin acetyl transferase (PAT of bar coding), direction is the contiguous chimeric now bar gene of intergenic region of contiguous ripe 18S rRNA coding region.The DNA construct that will contain rDNA between gene and chimeric bar gene is inserted between the border of T-DNA carrier pGSV5 (describing in WO97/13865).

According to the method described in WO92/09696 or the EP0469273 DNA construct is integrated in the corn nuclear gene group then.

Transgenic corns kind system is carried out expression level (by the mensuration of PAT activity level) and transgenosis copy number (by Southem hybridization) analysis.Be higher than with the contrast kind that DNA construct the transformed system that does not contain tumor-necrosis factor glycoproteins between gene with the average copy number of the kind that DNA construct the transformed system that contains tumor-necrosis factor glycoproteins between gene and the average expression level of chimeric bar gene.Embodiment 4: analyze using the oilseed rape plant that transforms according to DNA construct of the present invention

Use is corresponding to 5 ' of 3 ' of the ripe rRNA of conservative 25S the coding region terminal and conservative ripe rRNA of 18S coding region terminal oligonucleotide (using the relevant sequence that is of planting as a sequence that can obtain from EMBL database typing X60324), by pcr amplification oilseed rape rRNA intergenic region.The design oligonucleotide comprises suitable restriction site with the end at amplified fragments.

As described in the WO92/09696, with the CaMV35S promoter region upstream that fragment cloning can be operatively connected to the zone with coding phosphinothricin acetyl transferase (PAT of bar coding), direction is the contiguous chimeric now bar gene of intergenic region of contiguous ripe 18S rRNA coding region.The DNA construct that will contain rDNA between gene and chimeric bar gene is inserted between the border of T-DNA carrier pGSV5 (describing in WO97/13865).

According to the method described in the WO97/13865 DNA construct is integrated in the seed oil rape nuclear gene group then.

Transgenic rape seed rape kind system is carried out expression level (by the mensuration of PAT activity level) and transgenosis copy number (by Southern hybridization) analysis.Be higher than the contrast kind that DNA construct the transformed system that does not contain tumor-necrosis factor glycoproteins between gene with the average copy number of the kind that DNA construct the transformed system that contains tumor-necrosis factor glycoproteins between gene and the average expression level of chimeric bar gene.

Sequence table (1) essential information:

(ⅰ) applicant:

(A) title: Plant Genetic System N.V.

(B) street name: Jozef Plateaustraat 22

(C) city: Gent

(D) country: Belgium

(E) postcode (ZIP): B-9000

(F) phone: 32-9-2358454

(G) fax: 32-9-2231923

(ⅱ) denomination of invention: DNA construct and these DNA construct synthetic proteins of use

Method

(ⅲ) sequence number: 6

(ⅳ) computer-reader form

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, version #1.30 (EPO) (2) are about the information of SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 5373 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linear

(ⅱ) molecule type: DNA (genome)

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅵ) primary source

(A) biology: Arabidopis thaliana

(ⅸ) feature:

(A) title/key :-

(B) position: 1..485

(D) out of Memory :/remarks=" 25S rDNA 3 ' end "

(ⅸ) feature:

(A) title/key :-

(B) position: 486..5211

(D) out of Memory :/remarks=" intergenic region "

(ⅸ) feature:

(A) title/key :-

(B) position: 1263..1557

(D) out of Memory :/remarks=" Sal I subassembly wrapper 1 " be feature (ⅸ):

(A) title/key :-

(B) position: 1883..2177

(D) out of Memory :/remarks=" Sal I subassembly wrapper 2 " be feature (ⅸ):

(A) title/key :-

(B) position: 2503..3003

(D) out of Memory :/remarks=" Sal I subassembly wrapper 3 " be feature (ⅸ):

(A) title/key :-

(B) position: 5212..5373

(D) Other information :/ Remark = "18S rDNA 5 'end." (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCACCA AGTGTTGGAT TGTTCACCCA CCAATAGGGA ACGTGAGCTG GGTTTAGACC 60 GTCGTGAGAC AGGTTAGTTT TACCCTACTG ATGCCCGCGT CGCGATAGTA ATTCAACCTA 120 GTACGAGAGG AACCGTTGAT TCGCACAATT GGTCATCGCG CTTGGTTGAA AAGCCAGTGG 180 CGCGAAGCTA CCGTGCGCTG GATTATGACT GAACGCCTCT AAGTCAGAAT CCGGGCTAGA 240 AGCGACGCAT GCGCCCGCCG CCCGATTGCC GACCCTCAGT AGGAGCTTAG GCTCCAAAGG 300 CACGTGTCGT TGGCTAAGTC CGTTCGGCGG AACGGTCGTT CGGACCGCCT TGAATTATAA 360 TTACCACCGA GCGGCGGGTA GAATCCTTTG CAGACGACTT AAATACGCGA CGGGGTATTG 420 TAAGTGGCAG AGTGGCCTTG CTGCCACGAT CCACTGAGAT TCAGCCCTTT GTCGCTAAGA 480 TTCGACCCTC CCCTAAATCA CTCCAAAAAA AACAATCCCC AATTCTACAC AAGTGTTTCT 540 ACACTAACAA AGCAACAGCT CCTTAACGAA TTCCCAACTT TACACGAGCT CGTCTCTCGA 600 GGTTAAATGT TATTACTTGG TAAGATTCCG GACCTCGCCA AGTGTTTTGA AAACCCGCAA 660 CGCTCGCAAA GGTGGATAGT GAGAATAATA AGTGAAGAGA CAGACTTGTC CAAAACGCCC 720 ACCACGAAGG TGCATAGTGA GAAGAGTAAG TCAAGAGATA GACTTGTCCA AAAAGAAACG 780 GAAGAGAAAG CGTGGGGAGA CGCTCACGAA GGTGCATAGT GAGAAGAGTA AGTCAAGAGA 840 CAGACTTGTT CGAAAAGAAA CAGAAGAGAA TGCTTGGGGT TACACTCACG AAGGTGCATA 900 GTGAGAAGAG TAAGTCAAGA GACAGACTTG TTCGAAAAGA AACAAAAGAG AATGCTTGGG 960 GAGATAGAAG TGTGAGATAG TTCTCAAGcT AAGAAAGTTG TAAAAGCTAA GAACTAGCAT 1020 CAAATGATGG ATGAAACACA AGGTAGTTGT TGAAAAGTCA AACACTTGGT GATATGAACA 1080 CAAACGTTCA ATATGACAAA CCCATGCCAA GTAAAGAGAA AATGAAAACT GGTGATTGTT 1140 GCGGAAATCG TCCAGGATTC CTCGACCAGG ACTTGAAATC GTCGAGGGGA AAAAATCGGT 1200 TCCGAGGAAT CGTCGATCCG GACTTGGAAT CGTCGAGAAA AGTTTACCGG GTCCGAGGAT 1260 TTGTCGACCA GGAGTGGAAA TCGTCGAGAA AAATCTATCG GGTCCGAGGA ATCGTCGACC 1320 AGGACGAGGA ATCGTCGACC GGGTCCGAGG ATTTGTCGAC CAGGGGTTGA AATCGTCGAC 1380 CAGGTCCGAG ACTTCATCGA CCGGGTCCGA GGATTCGTCG ACCAGGAGTG GAAATCGTCG 1440 AGAAAAATCT ATCGGGTCCG AGGAATCGTC GACCAGGACG AGGAATCGTC GACCGGGTCC 1500 GAGGATTTGT CGACCAGGTC CGAGACTTCA TCGACCGGGT CCGAGGATTC GTCGACCAGG 1560 TCCGAGACTT CATCGACCGG GTCCAAGTAT TTGATTTTAT TTTGGAACCG GTGTCTCCTC 1620 AGACATTTCA ATGTATGTTG GTGCCAAGAG GGAAAAGGGC TATAAAGCTA TATAGGGGTG 1680 GGTGTGTGTA GTGGGAATTT TGCCCGCACG CGCGCGCGCG AATAAGAATA AGAATAAGTA 1740 ATAAGAATAA GAATAAGAAA AAGAAAAAAA AAAAAAAAAA AAAAAAACAA AAACTCCATA 1800 GAGTTGTTTT CTCAATCGGG TCCGAGGAAT CGTCGATCTG GACTTGGAAT CGTCGAGAAA 1860 AGTTTACCGG GTCCGAAAAT TTGTCGACCA GGAGTGGAAA TCGTCGAGAA AAATCTATCG 1920 GGTCCGAGGA ATCGTCGACC AGGACGAGGA ATCGTCGACC GGGTCCGAGG ATTTGTCGAC 1980 CAGGGGTTGA AATAGTCGAC CAGGTCCGAG ACTTCATCGA CCGGGTCCGA GGAATCGTCG 2040 ACCAGGAGTG GAAATCGTCG AGAAAA TCT ATCGGGTCCG AGGAATCGTC GACCAGGACG 2100 AGGAATCGTC GACCGGGTCC GAGGATTTGT CGACCAGGTC CGAGACTTCA TCGACCGGGT 2160 CCGAGGATTC GTCGACCAGG TCCGAGACTT CATCGACCGG GTCCAAGTAT TTGATTTTAT 2220 TTTGGAACCG GTGTCTCCTC AGACATTTCA ATGTATGTTG GTGCCAAGAG GGAAAAGGGC 2280 TATAAAGCTA TATAGGGGTG GGTGTGTGTA GTGGGAATTT TGCCCGCACG CGCGCGCGCG 2340 ATAAGAATAA GAATAAGTAA TAAGAATAAG AATAAGAAAA AGAAAAAAAA AAAAAAAAAA 2400 ACAAAAACAA AAACTCCATA GAGTTGTTTT CTCAATCGGG TCCGAGGAAT CGTCGATCTG 2460 GACTTGGAAT CGTCGAGAAA AGTTTACCGG GTCCGAAAAT TTGTCGACCA GGAGTGGAAA 2520 TCGTCGAGAA AAATCTATCG GGTCCGAGGA ATCGTCGACC AGGACGAGGA ATCGTCGACC 2580 GGGTCCGAGG ATTTGTCGAC CAGGGGTTGA AATCGTCGAC CAGGTCCGAG ACTTCATCGA 2640 CCGGGTCCGA GGAATCGTCG ACCAGGACGA TGAATGGTCG ATGAAAATCT ATCGGGTTCG 2700 AGGAATGGTC GACCAGGGGT TGAAATCGTC GACCAGGTCC GAGACTTCAT CGACCGTGTC 2760 CGAGGAGTGG TCGAGGGTTT GTCGACCAGG ACGAGGAATC GTCGACCGGG TCCGAGGATT 2820 TGTCGACCAG GGGTTGAAAT CGTCGACCAG GACCGAGAAT TCGTCGACCA GGACGGCGGA 2880 ACCCTCGACC AGGACGATGA ATGGGCGATG AAAATCTATC GGGTTCGAGG AATGGTCGAC 2940 CAGGGGTTGA AATCGTCGAC CAGGTCCGAG ACTTCATCGA CCGGGTCCGA GGATTCGTCG 3000 ACCAGGACGG CCGGATGTCC GAGAAAAAAA AATGTTGCCG AATAACTTTC GAAAATCATT 3060 GGATATGATG CAATGTTTTG TGATCGAATC TCTTAAAATA CATCAATAAA GAGTTTAGGA 3120 TGTCAAGTTT GCATCAAATA TGCCCACGGA GCCCCAACTA GACCATGAAA ATCCGATGTT 3180 GTATCAGGTC AAATGACCTA GCTAGAGGTG TCAAAAAATT ATGAAAATTT ACCAGAAAAT 3240 AGGATTTAGT ATCCTTATGA TGCATGCCAA AAAGAATTTT CAAATTCCAA GTATTTCTTT 3300 TTTCTTGGCA CCGGTGTCTC CTCAGACATT TCAATGTCTG TTGGTGCCAA GAGGGAAAAG 3360 GGCTATTAAG CTATATAGGG GGGTGGGTGT TGAGGGAGTC TGGGCAGTCC GTGGGGAACC 3420 CCCTTTTTCG GTTCGGACTT GGGTAGCGAT CGAGGGATGG TATCGGATAT CGGCACGAGG 3480 AATGACCGAC CGTCCGGCCG CCGGGATTTT CGCCGGAAAA CTTTTCCGGG CACTTTTCCG 3540 GCGATCGGTT TTGTTGCCTT TTTCCGAGTT TTCTCAGCAG TTCTCGGACA AAAACTGCTG 3600 AATCGTCGAG GAGAATGGGC TTGCTTGCGT GGGCTGCCAT TAGTTCTTCG AGGCGTTAGG 3660 GTGGCGGCGG TATAAAAGTG TCGGAGTTTT TTCAGCAGTT CTCGGACAAA AATTGCTGAG 3720 TGGCCGAGAA GAATGGGCGT GTCATGCGTG GGCTGACATG GATTCTTCGA GGCCTAGGGG 3780 TGGCGGTATA TAACTTGTTC GCATGATATT ACCGAGATGT CCCCACGGGC ATCTTTTCAC 3840 CTCGTCGCCG AAGAGAATGG GCGTGTCATG GCATGGGCTG ACATGGATTC TCCTAGGCCG 3900 TTTGGGTGGC GGTATAGTCG TCTTGCGCAC GAAATACCGA GATGTCCCCA TGGGCATCGA 3960 TTCCACCCGC CTAGGTTGGA TGGGCGTGCT TCGTCGGAAA GCATGGATCC GCCTAGGCTG 4020 TCCCGAGTGT GAGCGAGGTG TGAGTGTCGC CCATGGGCAT CGACACCTTG CGGCTAGGAA 4080 CTGGAACGAG ACGGGTGGCA AAGATTTCGA GTAGCACTTC ATACTACCGT GGGTTTTTTA 4140 AACCTTCCGA GTTTTGTTGA TGTTATTCCG AGAATTAGCA AACCGTAACG AAGATGTTCT 4200 TGGCAACCAT CTTTTGATGG GAGTCCGGCT GTTCGATAGC CGGCCAAGGG TGATGAACGA 4260 AATGTGAACC CTTGTCTCGC CTAGGTTGGA TGGGCGTGCT TCGTTGGAAA GCATGGATCC 4320 GCCTAGGCTG TCCCGAGTGT GAGCGAGGTG TGAGTGTCGC CCATGGGCAT CGACACCTTG 4380 CGGCTAGGAA CTGGAACGAG ACGGGTAGCA AAGATTTCGA GTAGCACTTC ATACTACCGT 4440 GGGTTTTTTA AACCTTCCTA GTTTTGTTGA TGTTATTCCG AGAATTAGCA AACCGTAACG 4500 AAGATGTTCT TGGCAACCAT CTTTTGATGG GAGTCCGGCT GTTCGAAAGC CGGCCAAGGG 4560 TGATGAACGA AATGTGAACC CTTGTCTCGC CTAGGTTGGA TGGGCGTGCT TCGTTGGAAA 4620 GCATGGATCC GCCTAGGCTG TCCCGAAGTA TCTCGCGCTT GTACGGCTTT GGCTCGGATT 4680 CGTCCGTCTT CTTTCTTCTT AGCCGAGTAC TTCGGTAGAT TAGTTGGAAC GATTGATGAT 4740 TTTGAGTTAA TTGAACGTTC GGCGTATGAG TGGTGATCGG ATAGCTAGTG TTCGTAGGCT 4800 CCATGCTCGC GCATCGAACT ACCTACCACC TATCCTTCTC AGTTAATTCA CGGGCGATGT 4860 TACGCTCGAT GATGAGTTCC GGGGCCTGTG TTTCGTACCT AATTTGAAGG AATTGTTGAG 4920 TTTGGTTTAC ACCTTTGCCC GCGGCTTCTC CTTCGTGGGG AAGTCGTGGG CTCAAACATC 4980 GGCGCTTGTT CACCTCTCGT CATCGCATTT GTTGCCTTGC TCGCATTGGT GAATGAGTTG 5040 CGGGTTGAAA TCTCGGATGC GGAAAAGTTG TCGACGGTGA CTCGAAGTGA TTCAGTCCCG 5100 CCAAAGCTCA TCCGTCCTTC GGGCAAAAGA TGACGGTCAA GACCTCGTCC TTTCTCTCTT 5160 TCCATTGCGT TTGAGAGGAT GTGGCGGGGA ATTGCCGTGA TCGATGAATG CTACCTGGTT 5220 GATCCTGCCA GTAGTCATAT GCTTGTCTCA AAGATTAAGC CATGCATGTG TAAGTATGAA 5280 CGAATTCAGA CTGTGAAACT GCGAATGGCT CATTAAATCA GTTATAGTTT GTTTGATGGT 5340 AACTACTACT CGGATAACCG TAGTAATTCT AGA 5373 (2) For SEQ ID NO: 2 of the message: ...

(ⅰ) sequence signature

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ⅱ) molecule type: other nucleic acid

(A) describe :/describe=" oligonucleotide "

(ⅹ ⅰ) sequence description: SEQ ID NO:2:CCAAGGTAAC CTTCGACGT 19 (2) information about SEQ ID NO:3:

(ⅰ) sequence signature

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: other nucleic acid

(A) describe :/description=" oligonucleotide " (ⅹ ⅰ) sequence description: SEQ ID NO:3:CGAAGGTTAC CTTGGACGT 19 (2) information about SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 23 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ⅱ) molecule type: other nucleic acid

(A) describe :/describe=" oligonucleotide E "

(ⅹ ⅰ) sequence description: SEQ ID NO:4:GAAGACGTCG AAGGTTACCT TGG 23 (2) information about SEQ ID NO:5:

(ⅰ) sequence signature

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ⅱ) molecule type: other nucleic acid

(A) describe :/describe=" oligonucleotide Q "

(ⅹ ⅰ) sequence description: SEQ ID NO:5:CCCGGTTCGC TCGCCGTTAC TAAG 24 (2) information about SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 236 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ⅱ) molecule type: other nucleic acid

(A) describe :/describe=" 5 ' end of Arabidopis thaliana 25S rDNA "

(ⅸ) feature:

(A) title/key :-

(B) position: 1..10

(D) out of Memory :/remarks=" 3 ' end of 5S rDNA "

(ⅸ) feature:

(A) title/key :-

(B) position: 198..236

(D) out of Memory :/remarks=" 3 ' end of 25S rDNA "

(ⅸ) feature:

(A) title/key :-

(B) position: 220

(D) out of Memory :/remarks=" 5 ' end of the 25S rDNA of Tui Ceing in the past "

(ⅹ ⅰ) sequence description: SEQ ID NO:6:GGTGTCACAA ATCGTCGTCC CTCACCATCC TTTGCTGATG CGGGACGGAA GCTGGTCTCC 60CGTGTGTTAC CGCACGCGTT GGCCTAAATC CGAGCCAAGG ACGCCTGGAG CGTACCGACA 120TGCGGTGGTG AACTTGATCC ATTACATTTT ATCGGTCGCT CTTGTCCGGA AGCTGTAGAT 180GACCCAAAGT CCATATAGCG ACCCCAGGTC AGGCGGGATT ACCCGCTGAG TTTAAG 236

Claims (16)

1. DNA construct, this construct comprises the following continuous dna fragmentation of operating:

-comprise the dna fragmentation of ribosomal dna sequence, preferably derive from plant;

-comprise the fragment of effable promoter region, especially plant-effable promoter region;

-allos coding region; Optionally

-Transcription Termination and polyadenylic acid zone, preferably activated zone in vegetable cell.

2. DNA construct according to claim 1, wherein said plant-effable promoter region are the promotors by the identification of RNA polymerase II.

3. DNA construct according to claim 1, wherein said rDNA comprises promotor.

4. according to any described DNA construct in the claim 1 to 3, wherein said ribosomal dna sequence derives from the plant that comprises DNA construct.

5. according to any described DNA construct in the claim 1 to 4, wherein said ribosomal dna sequence derives from the intergenic region of rDNA.

6. DNA construct according to claim 5, wherein said ribosomal dna sequence comprise from the upstream Sal I tumor-necrosis factor glycoproteins of Arabidopis thaliana rDNA intergenic region or from the similar area of other plant.

7. according to any described DNA construct in the claim 1 to 4, the included dna sequence dna of wherein said ribosomal dna sequence is selected from: from the dna sequence dna of 486 Nucleotide to 5212 Nucleotide of SEQ ID NO:1, dna sequence dna from 1263 Nucleotide to 3003 Nucleotide of SEQ ID NO:1, dna sequence dna from 569 Nucleotide to 2862 Nucleotide of SEQ ID NO:1, dna sequence dna from 1263 Nucleotide to 2862 Nucleotide of SEQ ID NO:1, from the dna sequence dna of 486 Nucleotide to 5212 Nucleotide of SEQ ID NO:1, from the dna sequence dna of 596 Nucleotide to 5373 Nucleotide of SEQ IDNO:1.

8. according to any described DNA construct in the claim 1 to 7, wherein said allos coding region coding vaccine, antibody, treatment albumen, insecticidal protein such as Bt toxin or its minimum toxic fragment, the albumen that is used for food technology, sense-rna or ribozyme.

9. DNA construct according to claim 1, wherein DNA construct is included in the T-DNA conversion carrier.

10. a method for synthesizing protein comprises the steps:

-any described DNA construct in the claim 1 to 9 is imported the host organisms that is fit to;

-under the condition that allows allos coding region encoded protein to express, cultivate host organisms; And

-collection expressed protein.

11. a method that particularly strengthens stability, copy number or the expression of transforming gene in vegetable cell comprises the steps:

-with any described DNA construct transfered cell in the claim 1 to 9, preferably in the vegetable cell; And

-from cell transformed regeneration biological body, preferred plant.

12. method according to claim 11, wherein said plant are selected from Arabidopis thaliana, potato, corn, wheat, tomato, paddy rice, soybean, barley, rye, Btassica kind, Beta kind or cassava.

13. one kind comprises the cell according to any described DNA construct in the claim 1 to 9 that is incorporated in the nuclear gene group, preferred plant cell.

14. vegetable cell according to claim 13, wherein said vegetable cell derive from Arabidopis thaliana, tobacco, corn, wheat, potato, paddy rice, soybean, barley, rye, brassica vegetable, Beta kind or cassava.

15. plant that contains cell according to claim 13.

16. comprise the purposes of the dna fragmentation of plant ribosome DNA intergenic region, be used for strengthening stability, copy number or the expression of plant transforming gene.

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