JPH07501682A - plant promoter - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 9. A claim operably linked to a heterologous gene encoding a protein A plant cell carrying the promoter according to 1 or 2.

10. A transgenic plant regenerated from the plant cell according to claim 8 or 9. .

11. A claim operably linked to a heterologous gene encoding a protein A transgenic plant having the promoter according to 1 or 2 in its cells.

12. Seeds obtained from the transgenic plant according to claim IO or ]l.

13. A method for producing a target protein in plant cells, comprising: (i) Claims Transforming plant cells with the vector described in any one of 4-7, provided that The protein encoded by the gene under the control of the promoter is the target protein. is protein: and (ii) culturing the transformed plant cells under conditions that allow expression of the protein; to nourish; A method consisting of

14. A method for producing transgenic plants capable of producing a target protein. So: (i) transforming a plant cell with the vector according to any one of claims 4-7; However, the protein encoded by the gene under the control of said promoter the protein of interest is a protein of interest; and (ii) regenerating a plant from the transformed cell;

Specification plant promoter The present invention relates to plant promoters.

In terms of quantity, the main structural components of plant cell walls are cellulose and hemicellulose. polysaccharides, including pectin polymers, but some specialized cell types ( For example, in vascular/epidermal cells), other components that match the functional role of the cell It supplements these.

Dicotyledonous plant cell walls also contain two types of structural proteins: phosphorus-rich glycoprotein (HGRP) and, more recently, glycine-rich protein. It was found to include members of the group Quality (GRP). HGR called extensin P has a characteristic S-P4 repeat structure, and the proline residue is hydroxylated. and then glycosylated.

Recently, many proline-rich proteins (PRPs) have been isolated from dicotyledonous plants. Ta. Many of these are known to be related to the cell wall, but their functions are is still not clear. In each cell type and each species, the P present PP seems to be different.

From the isolation of genomic and cDNA clones encoding PPP, this It was discovered that this is a group of proteins containing structurally distinct sequences. PPP The expression of some of the encoded genes is highly regulated at the messenger RNA level. pattern, caused by wounds, fungal infections, auxin, or during nodule formation It turned out to be induced. In recent years, two papers have shown that the expression of a specific PRP gene is It has now become clear that this phenomenon is correlated with specific developmental phenomena.

In contrast to the case of dicots, there is little data on PPPs from monocot species. It's not easy. Purification of extensin-like proteins from maize was reported. However, hydroxyproline residues were only present at low levels. Following this, The highly repetitive PRP cDNA sequence from maize was published and the mRNA Expression studies showed that the highest levels were present in root tips and coleoptiles. this The protein is a dicotyledonous plant, except that it contains a high proportion of threonine residues. It shares a significant level of similarity with PRP from species.

The present inventors have now discovered two types of proline-rich protein (PPP) genes. Promoter sequences for each were identified.

Therefore, the present invention provides the following nucleotide sequence: (a) Upstream of the PRP140 gene -1369 to -49, or (b) -2 upstream of the PPP378 gene 316 to -12, this sequence is modified one or more salts, as long as the sequence has the ability to act as a promoter. by substitution, insertion and/or deletion of groups and/or at both or one end. may be modified by elongation.

The present invention also provides a protein operably linked to a heterologous gene encoding a protein. A DNA fragment consisting of the promoter is provided.

Furthermore, it contains a heterologous gene encoding a protein under the control of said promoter. A vector is provided, whereby plant cells transformed with the vector are can express the gene. A suitable vector is one in which the promoter is ``It is directly connected to the end. The vector further integrates into the plant cell genome. Contains a region that allows for transfer and stable integration of the gene and promoter. You can stay there. This vector is usually a plasmid.

Plant cells can be transformed using this type of vector. Therefore, the original The protein is further operably linked to a heterologous gene encoding a protein. A plant cell carrying the promoter described above is provided. Transcription from such plant cells can regenerate sugenic plants. The resulting transgenic plant has its cells wherein said protein is operably linked to a heterologous gene encoding the protein; Owns a lomotor. Seeds can be collected from transgenic plants .

The present invention further provides a method for producing a target protein in a plant cell, and this method teeth: (i) transforming a plant cell with a vector according to the invention, provided that The target protein is the protein encoded by the gene under the control of the motor. and (ii) the transformed plant cell is capable of expressing the protein. Cultivate under conditions that It consists of

Furthermore, the present invention enables the production of transgenic plants capable of producing target proteins. (i) transfecting plant cells with a vector according to the invention; converting, provided that the gene encoded by the gene under the control of said promoter and (ii) the protein derived from the transformed cell is the protein of interest; It consists of regenerating the body.

The target protein is obtained from the transformed plant cells obtained by the first method or from the second method. It can be isolated from plants obtained by the method described above.

The promoter of the present invention is wheat PRP14 from base-1369 to base-49. 0 gene upstream sequence or wheat PR from base -2316 to base -12 Consists of the upstream sequence of the P378 gene, base I is the ATG translation initiation codon of PRP. This is A. The sequence of PRP140 promoter is shown in Figure 6, PRP378 promoter The arrangement of the data can be estimated from Figure 5.

This promoter was used to create a wheat DNA genomic library and to use PRP14. Screening a library of 0 or PRP378 genes, wheat PRP 140 or the upstream sequence of the PRP378 gene with an appropriate restriction enzyme and/or Obtained by digestion with exonuclease. Upstream of PRP140 gene There is an N5pBII restriction site at base -49 of the sequence and an Xbal restriction site at base -1369. There is a rank. Notl restriction site at base +72 of upstream sequence of PRP378 gene However, there is a former ndl11 restriction site at base -2316.

Upstream of wheat PRP140 or PRP378 gene consisting of promoter sequence Several plasmid vectors were created containing the sequence. These vectors contain p lPKH-12/2.3, pXNot/1.45, pBIXN/1.3 and p Includes HN/2.4. E. coli MC1022 carrying these vectors is National Collection of Aberdeen, UK on November 9, 1989 Industrial and Marine Bacteria (National Co. 11ection of Industrial and MarineBac teria) with accession numbers NCIMB 40223, NCIMO40224, NC Deposited as IMB40225 and NCIMB40226, respectively.

pBIXN/1.3 was deposited under the name plPXN/1.3.

PRP140 promoter is pXNot/1.45 with XbaI and N5pBII It can be excised from this plasmid by digestion with PRP378 The promoter was linearized with pHN/2.4 using Notl-BstXI, and the nuclease The plasmid populations were religated and the 3′ of the PRP378 gene A plasmid with a deletion from the end to base −12 was selected, and this plasmid was transformed into It can be excised by digestion with old ndlll.

The promoter sequence may contain one or more base substitutions, insertions and/or deletions. and/or by extension at both or one end. . However, the modified promoter sequence still functions as a promoter. There must be. Salt from base -769 of upstream sequence of wheat PRP140 gene Sequences up to group -49, and shorter sequences, produce proteins at satisfactory levels. It was found that this was not sufficient to induce the expression of On the other hand, wheat PRP140 gene The upstream sequence from base -961 to base -49 showed a satisfactory level of protein content. expresses the dark qualities. Generally, modified sequences and salts upstream of the wheat PRP140 gene Unmodified natural sequence from base -1369 to base -49 or wheat PPP378 residue There are few unmodified natural sequences from base -2316 to base -12 upstream of the gene. Both require 60% homology. homology is at least 75%, at least 85 %, or at least 95%.

Base-1369 of PRP140 gene or base-231 of PRP378 gene Longer promoter sequences extending upstream of 6 (e.g. to other restriction sites) may also be used. provided. The upstream extension is generally at base -1369 of the PRP140 gene or consists of the natural nucleotide sequence upstream of base -2316 of the PPP378 gene. . Longer sequences of this type were obtained from wheat DNA genomic libraries as described above. Obtainable.

Even in the case of the wheat PRP140 gene from base -961 to base -49, the upstream The sequence may be modified by one or more base substitutions, insertions and/or deletions. It may be modified by extension and/or extension at both or one end. In this case as well, A modified sequence such as must function as a promoter. longer qualification There is at least 60%, such as at least 75%, less separation between the sequence and the unmodified sequence. There may be at least 85% homology, or at least 95% homology.

Modified promoter sequences are obtained by introducing changes to the native promoter sequence. It will be done. This includes endonuclease restriction of the native sequence, insertion of linkers, Use of xonucleases and/or polymerases, site-directed mutagenesis This can be achieved using suitable techniques such as Therefore, shorter DNA sequences, e.g. Using exonucleases such as Exonuclease Ill and BAL 31 , by removing nucleotides from the 5' or 3' end of the native promoter sequence. can be obtained.

It is easy to check whether a modified sequence can function as a promoter. For example, a bacterial reporter gene encodes a protein such as β-gluironidase. Place this modified sequence upstream of the sequence. Next, transform tobacco leaf discs. Ru. The proteins expressed by the transformed cells are then assayed. β-gluk The case of lonidase is as described in Example 1.

A promoter is operably linked to a heterologous gene that encodes a protein. . The heterologous gene may encode any protein desired to be expressed. “Unusual "Species" means that the gene is operably linked to its promoter in nature. means not tied. Therefore, the promoter of this gene is PRP1 does not encode PRP140 when derived from the upstream sequence of the 40 gene; PRP3 if the promoter is derived from the upstream sequence of the PRP378 gene. Do not code 7B. A protein is encoded within a heterologous gene sequence at its N-terminus. The transport peptide sequence may also include a transit peptide sequence.

Promoters are commonly used to control the expression of genes in plant tissues.

Proteins whose expression is controlled by promoters are co-regulated by herbicide resistance genes. Proteins that are encoded or that biologically control pests and pathogens It can be. This protein thus confers resistance to leaf-eating insects. can be an insecticidal protein such as B. thuringiensis toxin. .

Other uses of this promoter include viral coat proteins that protect against viral infection. production of high-value proteins such as pharmaceuticals, and changing the taste and nutritional value of grass. This includes the production of protein that can be used to grow.

The promoter sequence is linked to the heterologous gene directly or via a linker. linker - The sequence may contain introns. Excluding the length of intronic sequences, The car may consist of up to 45 bases, e.g. up to 30 bases, or up to 15 bases. can do. The linker sequence is a transport amino acid sequence (e.g. chloroplast or mitochondrial). Transport molecules that can direct proteins to predetermined subcellular regions, such as Doria, It may also contain an array encoding a column. In addition, the linker sequence can be used to control the expression level may contain sequences with enhancer properties.

DNA fragments and vectors in which the promoter is operably linked to a heterologous gene It is possible to create a tar. Even if these fragments and vectors are single-stranded, , may be double-stranded. using such fragments directly or using vectors of this kind. can be used to transform plant cells. The vector is under the control of a promoter. A foreign gene is integrated. This vector is a plant cell transformed with the vector. further comprises regulatory elements capable of expressing the gene in the protein. This kind of tone In addition to the promoter, the node elements include translation initiation and/or termination sequences. can be given.

Vectors generally contain chimeric genes and linked regulatory control elements in the plant cell genome. It also includes areas that enable transmission and stable integration.

Therefore, vectors generally contain transcriptional regulatory sequences and/or the coding sequence of the gene. If not present at the 3" end of the column, a stop futon is provided. Thus, the DNA The fragments also contain terminator sequences and other components that allow expression of the gene in plant cells. The array is included.

Can increase or decrease expression levels in certain parts of the plant or under certain conditions En/)cer or pond elements may also be incorporated into the DNA fragment and/or vector. can be done. Furthermore, the vector ζ usually confers resistance to transformed plant cells. It has an antibiotic resistance gene and can be grown by growing in an appropriate medium containing antibiotics. , transformed cells, tissues and plants can be selected.

Transformed cells are selected by growth in an appropriate medium. Thus, for example, plant cells A plant cell that contains a heterologous gene under the control of a promoter in its genome. A physical structure is obtained. Therefore, this gene can be expressed in plant cells. after that , heterologous genes and promoters integrated into the cell, e.g. into the plant cell genome. As a result, a plant that can express the gene can be regenerated. Re The grown plants can be propagated, for example, seeds can be collected.

A preferred method for transforming plant cells involves producing a protein operably linked to a heterologous gene. Agrobacterium tumefaciens (A grobacterium tumefaciens). . Therefore, the following elements: (a) express a gene when integrated into the genome of a plant cell; A heterologous gene under the control of said promoter and other regulatory elements that can : (b) at least one DNA delimiting the DNA to be integrated into the plant genome; array: and (C) a DNA sequence that can transfer this DNA to the plant genome; A printed plasmid vector is used.

Generally, the DNA integrated into the plant cell genome is the T-DNA of the Ti-plasmid. Bounded by a border array. When there is only one border array , preferably it is a right-most border array. This DNA is converted into a plant cell genome. The DNA sequences that can be transferred to the TI-plasmid are generally virulence (v ir) area.

Thus, the heterologous gene and its transcriptional and translational regulatory elements (including the promoter) - Can be inserted between the T-DNA border sequences of the plasmid. this plus The medium may be a disabled Ti-plasmid in which the oncogenic gene has been deleted. deer However, the heterologous gene and its transcriptional and translational regulatory elements (including promoters) A binary vector (binaryv) in trans with a Ti-plasmid containing the ir region ector) is inserted between the T-DNA borders. A binary vector like this is the following element: (a) Before a gene can be expressed when integrated into the genome of a plant cell a heterologous gene under the control of a promoter and other regulatory elements; and (b) at least one DNA delimiting the DNA to be integrated into the plant genome; array; including.

Therefore, a hybrid plasmid vector or a Ti-plasmid vector with a vir region Agrobacterium tumefashen containing a binary vector in sumid and trans can be used to transform plant cells. like a stem or leaf disc inoculate tissue explants with this bacterium or plant protoplasts for regeneration. This bacterium is co-cultivated. Plant protoplasts also encode heterologous genes. DNA fragment (with promoter and appropriate other transcriptional and translational regulatory elements present) or Alternatively, transformation may be performed by direct introduction of a vector containing such a fragment. straight For contact introduction, electroporation, polyethylene glycol, microinjection This can be achieved using traction or particle bombardment.

Plant cells from monocots or dicots can be transformed according to the present invention. Wear. Monocot species include barley, wheat, corn, and rice. . Dicotyledonous plant species include tobacco, tomato, sunflower, petunia, cotton, and sugar beet. yam, potatoes, lettuce, melons, soybeans, rapeseed, poplars, etc. Ru.

Growing tissue cultures of transformed plant cells results in differentiated and fully transformed cells. The plant body is regenerated. The transformed plant cells are grown in a suitable medium, preferably selectable. Culture in growth medium. Plants can also be regenerated from callus. write to give functional to foreign genes integrated into cells, e.g. into their genomes. A transgenic plant can be obtained that carries a promoter linked to . As a result, this gene can be expressed intracellularly.

Seeds can be collected from the regenerated plants for future use.

The following examples illustrate the invention. In the attached drawings: Figure 1 shows the restriction map of the insert DNA of phage 3cX2; Figure 2 shows the restriction map of the insert DNA of phage 3cX2; Restriction map of the insert DNA of E3.2 is shown; Figure 3 shows the sequence of the PRP378 gene. Figure 4 shows the sequence of the PRP140 gene: Figure 5 shows the sequence of the PPP378 gene. Figure 6 shows the upstream regulatory sequences of the PRP140 gene. Figure 7 shows the construction of pBII (N/2.4; Figure 8 shows the construction of pBIXN/1.3. (7) Construction; Figure 9 shows pBID-234, -400, -540, -76 Construction of 9 and -961N, 10, plPKH-6, -12 and -50 Figure 11 shows the construction of plPH-6/2.3; Figure 12 shows the construction of plP S-6/762 and plP P-6/615; Figure 1 3 is plPD-6/1839.1510.1289, +023 and 816 Show construction; Figure 14 shows the sequence of the fusion junction of the BIN 19-based construct; , shows the sequence of the fusion junction of the puc19-based construct; Showing the results of Gus test for 40 promoter deletion types; Figure 17 shows the results of the Gus test for the PRP378 deletion type; The nucleotide sequence of the cDNA clone (WPRPI) is shown.

The strategy adopted to isolate the wheat PRP promoter was to first isolate the wheat DN Create a genomic library of A, and then use this library for the PRP gene. The purpose was to screen using the cDNA probe of

Triticum aestivum cv, Chinese Spring High molecular weight DNA was isolated from shoots grown in the dark (Lazarus et al. al,, Plant Mol.

Biol, 5.8-24.1985). Establishing conditions for partial digestion by 5au3A did.

A 18-25 kb DNA fragment was purified by size fractionation using a sucrose density gradient. Manufactured. Lambda Sharon 35 (Loenen and Blattner, Prepared by Gene gradient purification. Vector using T4 DNA ligase DNA (1.5 μg) and wheat DNA (3.5 μg) at high concentration (500 ng) /u I) Te ligation, then i using a commercially available extract (Stratagene) Packaged in vitro.

Approximately 2.6 x 106 recombinants were plated on host E. coli and plated The DNA lift from the tube was transferred to a nitrocellulose filter. Wheat P RP cDNA WPRPI (reference example) was randomly plated with P-dATP. was used to search for library lifts. hybrida The positive plaques that appear are purified to homogeneity through several rounds of screening, and then DNA from these positive phages was purified for further analysis.

2. Characterization of the PPP gene Control was obtained from the insert DNA of positive phages 3cX2 (Figure 1) and E3.2 (Figure 2). A restriction map was created and the position and direction of the PPP gene was determined by hybridization experiments. It has been decided. DNA fragments containing these regions were injected into 1 μl of Stratagene. plasmid, which is a pUcI9 derivative containing the polylinker region of the escript plasmid. It was subcloned into the lasmid vector pUBsl (Raines et al. l,, Nucl, Ac1ds, Res, 16.7931-7942゜1 988). Exonuclease II! Digestion and double-stranded dideoxynucleotide sequences Analysis yielded overlapping sequences from these clones.

Data were collected and analyzed using the 5taden package. The range of the determined array The boundaries are shown in Figures 1 and 2.

Comparing the DNA sequences of the PRP378 gene and the cDNA probe used for its isolation A comparison reveals that several bases differ, indicating that the isolated gene copy This suggested that it was an allele of the cloned mRNA. PRP] 40 genes ( Figure 4) is clearly a new type of PRP.

3. Upstream sequence of PRP The regulatory sequences upstream of the PRP378 and PRP140 genes were assembled into the following constructs. 5 and 6, respectively, including all those used therein. The subsequent claw Restriction enzyme sites used in the coding method are underlined. These arrays are , allowing the correct expression of downstream protein-coding sequences under their control. It was proposed that it would contain all the necessary promoters and other regulatory elements.

We tested this proposal and tested PRP378 and PRP14 in the context of plant transformation. A series of constructs were generated to evaluate the utility of the 0 promoter. Each In this case, part of the PRP378 or PRP140 upstream region is Bacterial reporter genes β-glucuronidase and tubalin synthase terminus placed in front of the tar array.

In the case of the PRP378 promoter, this It was a high copy number pUc19-based plasmid suitable for transient expression. P In the case of the RP140 promoter, this is the It was an old N19-based vector suitable for transformation. In both cases, PPP Promoter-controlled expression is possible from transformed plants or protoplasts. Determination of β-glucuronidase (Gus) activity using fluorogenic substrates in extracts of It was possible to detect it by elementary test.

(a) plPPKGT and pBIPKGTplPPKGT are pRAJ275 (Jefferson, Plant, Mo1. Biol.

EcoR1/Satl of Reporter 5.387-405.1987) The insert was blunt-ended using the Klenow fragment of E. coli DNA polymerase I. and pUcPolyTer (pLIcI9 as a 5stl/EcoR1 fragment) Klenow blunt version of tubalin synthase terminator fragment cloned in It is linked to the terminated Asp718 site. The corresponding old N19 based program The lasmid is pBIPKGT. plPPKGT and pBIPKGT are mutually exclusive. A 2.4 kb Hindll-Notl fragment of the PRP378 gene obtained from (Fig. 1) was subcloned into the plasmid vector pLIBsl to create a plasmid. pHN72.4 was prepared. pi (N/2.4 is linear with Notl, this part The position was repaired using the Klenow fragment of E. coli DNA polymerase I. . This insert was excised by old nd111 digestion and Hindlll-3mal digestion. pB+101.3 (Jefferson, Plant, Mo1. Bi ol, Reporter 5.387-405゜1987) and pBI HN/2.4 was produced. The fusion junction was examined by nucleotide sequence analysis and its The sequence and deduced amino acid sequence are shown in FIG.

The 1.45 kb Xbal-Notl fragment (Fig. 2) of the PRP140 gene was Subcloned into Sumid vector pUBs 1 to create pXNot/1.45. Manufactured.

Digest with XbaI and N5pBII to excise the 1.3kb fragment (Figure 8), Insert into bal-3mal digested pBIPKGT to create pBIXN/1.3. Ta. The sequence of the fusion junction is shown in FIG.

Construction of mido pBID-234, -400, -540, -769 and -961N (Figure 9) Plasmid pXNot/1.45 (Figure 8) was erased by Xbal-Pstl. It became linear. Incubate with exonuclease II+ for various times. Sequences of different lengths were deleted from the Xbal end. Nuclease Sl and Escherichia coli D Close the plasmid population by treating with the Klenow fragment of NA polymerase. Religated as circular DNA. Using nucleotide sequence analysis to represent this population. The degree of deletion from the Xba l end of the superficial portion was examined.

23 5° of the translation initiation codon of PRP140, based on information from sequence analysis. 4. Deletion plasmids up to 400.540.769 and 961 nucleotides was selected for evaluation of promoter activity (Fig. 6). Hind! II and N5 Five restriction fragments were excised by digestion with pBII, and 1 (indlll-5ma p[11D-234, -400, -540, - 769 and 1961N were produced. In all five constructs, the fusion junction The sequence of pBIXN/1.3 is identical to that of pBIXN/1.3 (Figure 14).

Plasmid pHN/2.4 (Fig. 7) was digested with Notl-BstXl to make it linear. did. Notl ends were removed by incubation with exonuclease III for various times. Sequences of different lengths were deleted from. Nuclease St and Escherichia coli DNA polymer The plasmid population was transformed into closed circular DNA by treatment with the Klenow fragment of and reconnected. Using nucleotide sequence analysis, N Ot! The degree of deletion from the end was examined.

Based on information from sequence analysis, downstream from the Hindl11 site, PRP378 Approximately 2.3 kb extending to 6.12 and 50 nucleotides 5' of the translation initiation codon. Promoting plasmids pHD-6, -12, -50/2.3 with long inserts selected for the evaluation of motor activity (Fig. 5).

Each of the three plasmids was linearized by partial 5stl digestion, and the overhanging parts were The ends were made blunt with Mung Bean nuclease. three inserts Digest and cut out the old ndlll, digest the old ndlll-3mal, plPPKGT plPK+4-6, -12 and -50/2.3 were created by inserting into .

The sequence of the fusion junction is shown in FIG.

(ii) Construction of plPH-6/2.3 (Figure 11) This plasmid is plPKH -6/2.3, but pB1201.1 (Jefferson, 1 987) with the full-length PPP378 promoter inserted, and the “Kozak” co-promoter It has a different connection part (FIG. 15) in which there is no initiating ATG.

Plasmid plPH-6/2.3 (Figure 11) was digested with Pstl or partially 5st Digested to make it into a linear shape, and the protruding part was made into a Mung Bean nucleus. The ends were made blunt using Aase. Carcinogens with GUS gene and NOS terminator nucleotides 762 and 615 of the PPP378 promoter in the set. The respective inserts were excised by EcoRI digestion, and EcoRl/Smal digested p Inserted into UcI9. In both constructs, the sequence of the fusion junction is plPHJ/ It is the same as that of 2.3 (Fig. 15).

Plasmid pHN/2.4 (Figure 7) was digested with old ndlll-Apal and linearized. And so. Hind by incubating with exonuclease Il+ for various times. Sequences of different lengths were deleted from the lll end. Nuclease St and E. coli DN The plasmid population was closed by treatment with the Klenow fragment of A polymerase. It was religated as a similar DNA. Representatives of this population using nucleotide sequence analysis The extent of the deletion from the former ndllll end of the target portion was examined.

18 5° of the translation initiation codon of PRP378, based on information from sequence analysis. Plasmid pD-1 with an insert extending from 39 to 816 nucleotides 839, -1510, -1289, -1023 and -816N to Kpnl-3 digested. The excised fragment was partially digested with EcoRl with 5stll pIP. H-6/2.3 (PRP37 with GUS gene and NOS terminator 8 promoter) and then EcoR1-Kpn I-digested puc+9 was ligated. Again, in all constructs, the fusion link The sequence of the knot is identical to that of pIPH-6/2.3 (Figure 15).

(1984), the construct was transformed from E. coli MC1022 to Agrobacterium tumefaciens. Moved to Shens LBA 4404. Nicotiana tabacum Describes the leaf disc of the Samsun variant of a. tabacum. As per Horsch et al., 5science 223.49 6-498°1984) transformed and containing 100 μg/ml kanamycin Selection was made on germination medium. Generally, plants containing each construct will regenerate 5-15 plants, with lateral The activity of the PRP promoter in each transformant was determined by β-glucurin in leaf extracts. This was investigated by measuring the nidase activity. Prepare tissue extracts and follow the instructions as described. (Jefferson, 1987) reaction product 4-methylumbellife Ron's fluorescence was analyzed. The reaction mixture is typically incubated at 37°C for 4 hours and then Aliquots were taken at hourly intervals. Determine the protein concentration of each extract and (using the Bio-Rad kit).

The results of the assay for the expression of the PRP140 promoter-inducible construct are nmol 4 - Methylumbelliferone/n+in/mg protein, shown in Figure 16. It is. 1369 to 9 due to the variation normally observed between individual transformed plants. Excision of the PRP140 promoter up to 61 nucleotides in length significantly reduced activity. It is difficult to ascertain whether it will lead downwards or not. However, 769 and 5 The 40 nucleotide form has virtually no activity; the 400 and 234 nucleotide forms It is clear that the leotide form is virtually inactive.

pUc19-based constructs were constructed using Zea mays according to the method of Chen et al. Ca of protoplast of cell suspension (cell line Ba Tan Ba1) -Temporary expression by PEG-mediated transformation was assayed (Chen.

D, F, Ta5sie, A, Dale, P, J, Goldsb rough, A, P,, Liang, W,.

Bevan, M.W., Flavell, R.B., and Xia. Z, A. (1988), In: Pulte.

K1. , Dons, Ji, M., , Huizing, IL.J., , Kool, A.J. ,,Korneef.

M, and Krens, F, A, (eds.) Progress in Pla nt Protoplast Research (Advances in plant protoplast research) ), Kluwer Academic Publishers, Dordre cht) o Generally, for each construct, 50 μg of DNA and l Three replicate experiments were performed using rotoplasts. As a control, protoplasts Equal amounts of garlic sperm DNA and cauliflower mosaic virus (CaMV) 3 Plasmid CG20 carrying an 850 bp copy of the 5S promoter Converted.

The lysate of transformed protoplasts was prepared using the same method as for transformed plants. (Jerson, 1987). tested in the same experiment The expression activity per 1 x 10' protoplasts between the constructs was compared.

Assay results for the PPP378 promoter-inducible construct are nmol 4-methyl Umbelliferone/min/l x 10’ expressed in protoplasts, Table 1 and Fig. 17 is shown.

Table 1. Gus assay/PRP378 promoter 3' deletion type CG20 (CaM V35S) 3.59±2.96 (100%) Ning sperm DNA 0.03 08 (0,858)p IPKH-6/2.3 9.65±0.63 (26 9) -1211,92±4.22 (332) -504,7320.66 (1 32) The 2.3 kbp type of PPP378 promoter is a maize protoplast. It is more prominent than the 850 bp type of CaMV35S promoter in Yes (Table 1). Furthermore, it is susceptible to relatively short deletions from the 3′ end. It is. Therefore, plPKH-12/2.3 (starting “ATG” of GUS gene (Fig. 15) in which the promoter was placed under almost natural conditions (construct) was the best level of expression, but move the promoter further or closer to the ATG (FIG. 15), the activity decreases.

Figure 17 shows the effect of a deletion from the 5'' end of the PPP378 promoter.8 The activity of the 00 bp deletion type is less than 60% of the full-length type, and the activity is dependent on the degree of deletion. Both continue to decline. In this experiment, the full-length version of the PRP378 promoter was Even if they do not have the same level of activity against MV 35S as those shown in Table 1, The fusion junction is different, and the starting AT of the β-glucuronidase gene G does not have the “KOZak” common sequence (FIG. 15).

Reference example A cDNA called WPRPI encoding wheat PPP was isolated, and its sequence was was decided. WPRPI clone was detected, but wheat cDNA library Lee was induced against chloroplast fructose-1,5-bisphosphatase. Screened using antibodies. Why does this antibody intersect with the WPRPI clone? I don't know if there will be a different reaction. The first cDNA isolated was full-length (1.3 kb ) was not. Complete sequences can be obtained by rescanning the cDNA library using short clones. It was determined from the longer cDNA obtained by cleaning.

Complete nucleotide sequence of wheat cDNA clone (WPRPI) and Its deduced amino acid sequence is shown in FIG. This 1548 bp cDNA is 1 137 base pairs of complete coding sequence and 101 bp of 5° non-coding sequence and polyadenylene It contains 310 bp of 3° non-cod sequence ending in a nylated tail.

Example 2 The localization of Gus expression in the tissues of the transformed tobacco of Example 1.4 was investigated. inspection Determination was performed as described in Example 1.5, but tissues other than leaves were also assayed. results It is shown in Table 2 below. Values shown are pmol/n+in/μg protein. mosquito Numbers in parentheses represent the ranking of tissue expression levels within individual transformed plants. all groups Significant expression levels are seen in the tissue. The expression level of petiole and stem is CaMV 3 Comparable to 53.

Claims (14)

[Claims] 1. The following nucleotide sequence: (a) −1369 to −49 upstream of the PRP140 gene, or (b) P It is a promoter with -2316 to -12 upstream of the RP378 gene. Therefore, the sequence is modified as long as the modified sequence has the ability to act as a promoter. by substitution, insertion and/or deletion of one or more bases, and / or the above-mentioned promoter may be modified by extension at one or both ends. Tar. 2. The upper part of the PRP140 gene, which may be modified as described in claim 1. The promoter according to claim 1, having the sequence from -961 to -49 of the sequence. 3. Claim 1 wherein the protein is operably linked to a heterologous gene encoding a protein. or a DNA fragment containing the promoter described in 2. 4. A variant encoding a protein under the control of the promoter according to claim 1 or 2. A vector containing a seed gene, which can be used in plant cells transformed with the vector. The above vector, in which the gene can be expressed. 5. 5. The promoter of claim 4, wherein the promoter is fused directly to the 5' end of the gene. vector. 6. The gene and promoter are transferred to the plant cell genome and assembled in a stable state. The vector according to claim 4 or 5, further comprising a region to be inserted. 7. 7. The vector according to any one of claims 4-6, which is a plasmid. 8. A plant cell transformed with a vector according to any one of claims 4-7. 9. Claim 1 wherein the protein is operably linked to a heterologous gene encoding a protein. or a plant cell carrying the promoter described in 2. 10. A transgenic plant regenerated from the plant cell according to claim 8 or 9. . 11. A claim operably linked to a heterologous gene encoding a protein A transgenic plant having the promoter according to 1 or 2 in its cells. 12. Seeds obtained from the transgenic plant according to claim 10 or 11. 13. A method for producing a target protein in a plant cell, comprising: (i) Claims Transforming plant cells with the vector described in any one of 4-7, provided that The protein encoded by the gene under the control of the promoter is the target protein. is protein; and (ii) culturing the transformed plant cells under conditions that allow expression of the protein; to nourish; A method consisting of 14. This is a method for creating transgenic plants that can produce the target protein. So: (i) transforming a plant cell with the vector according to any one of claims 4-7; However, the protein encoded by the gene under the control of said promoter the protein of interest is a protein of interest; and (ii) regenerating a plant from the transformed cell;