CA2191624C - Dna fragment, recombinant vector containing the same and method for expressing foreign genes using the same - Google Patents

Abstract

A novel DNA which has a sequence different from those of publicly known DNAs capable of promoting the expression of an alien gene and can remarkably promote the expression of an alien gene. An isolated DNA fragment having a base sequence represented by SEQ ID NO:1 in the Sequence Listing; a nd an isolated DNA fragment represented by this base sequence wherein one or more nucleotides have been added, inserted, deleted or replaced and having the effect of promoting the expression of a gene located in the downstream thereof.

Description

2~.9~~H~
SPECIFICATION
DNA Fragment, Recombinant Vector.Containing the Same and Method for Expressing Foreign Genes Using the Same mRC'.HNICAL FIELD
The present invention relates to a novel DNA
fragment having function to promote expression of genes, a vector -containing the same and a method for expressing foreign genes using the same.
BACKGROUND ART
Promotion of expression of foreign genes is one of the most important techniques in applying genetic engineering processes to plants. One of the methods therefor is utilization of a DNA having a nucleotide _ sequence which promotes expression of a gene.
Known nucleotide sequences which promote expression of foreign genes include the intron of the catalase gene, of castor bean (Japanese Laid-open Patent Application (Kokai) No. 3-103182; Tanaka et al., Nucleic Acids Res.
18, 67'67-6770 (1990)). However, since there are wide varieties of plants to be manipulated and since promotion of expression of genes is required in each of the desired growth stages ox tissues of organs, it is desired that wide varieties of DNAs which promote expression of genes can be utilized.
pjSCT,nSURF OF THE INVENTION
Accordingly, an object of the present invention is-to provide a novel DNA, which can promote expression of foreign genes and which has a nucleotide sequence different from those of known DNAs that promote expression of foreign genes.
The present inventors intensively studied to discover introns of rice phospholipase D (hereinafter also referred to as "PLD") gene by comparing a rice cDNA
and a rice genomic DNA, and discovered that one of the introns has a function'to prominently promote expression of the gene downstream thereof, thereby completing the present invention.
That is, the present invention provides an isolated DNA fragment having a nucleotide sequence shown in SEQ ID
NO. 1 in Sequence Listing or having a nucleotide sequence which is the same as the nucleotide sequence shown in SEQ
ID N0. 1 in Sequence Listing except that one or a plurality of nucleotides are added, inserted, deleted or substituted, the latter nucleotide sequence having a function to promote expression of a gene downstream thereof. More specifically, the present invention provides for an isolated DNA fragment having a nucleotide sequence shown in SEQ ID NO. 1 in Sequence Listing or having a nucleotide sequence, which hybridizes with said nucleotide sequence shown in SEQ ID N0. 1 when the hybridization is carried out in a hybridization solution containing 0.5M
sodium phosphate buffer (pH7.2), 7% SDS, 1 mM EDTA and 100 ug/ml of salmon sperm DNA, at 65°C for 16 hours, and then washing the product twice with a washing solution containing 0.5xSSC and 0.1o SDS at 65°C for 20 minutes 2a each, the latter nucleotide sequence having a function to promote expression of a gene downstream thereof in a plant.
The present invention also provides a recombinant vector comprising the above-mentioned DNA fragment according to the present invention and foreign gene to be expressed, which is operably linked to the DNA fragment at a downstream region of the DNA fragment. The present invention further provides a method for expressing a foreign gene comprising introducing the recombinant vector according to the present invention into host cells and expressing the foreign gene.

As experimentally confirmed in the Example described below, the DNA fragment according to the present -invention largely promotes expression of the gene downstream of the DNA fragment. Therefore, it_is expected that the presentinvention will largely contribute to expression of foreign-genes by genetic engineering processes.
RRTEF DESCRIPTION F THE DRAWINGS
Fig. 1 shows the important part of a genetic map of pBI221 into which the DNA fragment according to the present invention is inserted, which was prepared in the Example of the present invention.
BEST MODE FOR CARRING OUT THE INVENTION __ As mentioned above,-the DNA fragment according to the present invention has a nucleotide sequence shown in SEQ ID N0. I in the Sequence Listing. As will be described in detail in the Example below, introns located upstream of rice PLD gene were identified by comparing the nucleotide sequence of the cDNA of rice PLD gene and that of the rice genomic DNA. A fragment containing one of these intron sequences having a size of 173 by located at the 5'-flanking region was prepared by PCR and the DNA
fragment was inserted into an upstream site of a reporter gene of an expression vector containing the reporter gene..
By checking the expression activity of the reporter gene, it was confirmed that the DNA fragment has a function to promote expression of the gene downstream thereof. The ~1~1s~~
nucleotide sequence of the DNA fragment according to the present invention corresponds to 1661nt to 1843nt of the nucleotide sequence of the rice genomic PLD gene, which nucleotide sequence is shown in SEQ ID N0. 3 of the Sequence Listing.
The nucleotide sequence of the above-mentioned intron sequence having a size-of 173 bp, which is located upstream of the rice PLD gene, is shown in SEQ ID NO. 4 in the Sequence Listing. Needless to say, the sequence shown in SEQ ID N0. 4 also has a function to promote expression of the gene downstream thereof. The nucleotide sequence shown in SEQ ID NO. 4 corresponds to 1666nt to 1838nt of the-nucleotide sequence of the rice -genomic PLD gene, which is shown in SEQ ID NO. 3 in the Sequence Listing.
Since the DNA fragment according to the present invention is an intron existing upstream of the rice PLD
gene, and since its nucleotide sequence was determined according to the present invention, the DNA fragment may easily be prepared by PCR using the rice genomic DNA as a template. PCR-is a conventional technique widely used in the field of genetic engineering and a kit therefor is commercially available, so that those skilled in the art can easily perform the PCR. One concrete example thereof is described in detail in the Example below.
It is well-known in the art that there are cases wherein the physiological activity of a physiologically active DNA sequence is retained even if the nucleotide sequence of the DNA is modified to a small extent, that is, even if one or more nucleotides are added, inserted, deleted or substituted. Therefore, DNA fragments having the same nucleotide sequence as shown in SEQ ID NO. 1 except-that the DNA fragments have such modifications, which have the function to promote expression of the gene downstream thereof, are included within the scope of the present invention. That is, the DNA fragments having the same nucleotide sequence as shown in SEQ ID N0. 1 except that one or more nucleotides are added, deleted or substituted, which have the function to promote expression of the gene downstream thereof, are included within the scope of the present invention. Particularly, in the nucleotide sequence shown in SEQ ID N0. 1, the 5 nucleotides at the 5'-end and the 6 nucleotides at the 3'-end are the nucleotides in the exon regions, so that it is thought that the nucleotide sequences which do not have these regions also have the function to promote gene expression. Thus, these DNA fragments are within the scope of the present invention.
Modification of DNA which brings about addition, deletion or substitution of the amino acid sequence encoded thereby can be attained by the site-specific mutagenesis which is well-known in the art (e. g., Nucleic Acid Research, Vol. 10, No. 20, p6487-6500, 1982). In the present specification, "one or a plurality of 6 2~9~62~
nucleotides" means the number of nucleotides which can be added, deleted or substituted by the site: specific mutagenesis.
Site-specific mutagenesis may be carried out by, for example, using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA except that the desired mutation as follows. That is, using the above-mentioned synthetic oligonucleotide.as a primer,-a complementary chain is produced by a phage, and host bacterial cells are transformed with the obtained double-stranded DNA. The culture of the transformed bacterial _ cells is plated on agar and plaques are formed from a single cell containing the phage. Theoretically, 50$ of -the new colonies contain the phage having a single-stranded chain carrying the mutation and remaining 50~ of -the colonies contain the phage having the original sequence. The obtained plaques are then subjected to hybridization with a kinase-treated synthetic probe at a temperature at which the probe is hybridized with the DNA
having exactly the same sequence as the DNA having the desired mutation but not with the original DNA sequence.
that is not completely complementary with the probe.
Then the plaques in which the hybridization was observed are picked up, cultured and the DNA is collected.
In addition to the above-mentioned site-specific ._-mutagenesis, the methods for substituting, deleting or adding one or more amino-acids without losing the 2191~~~
function include a method in which the gene is treated _..
with a mutagen and a method in which the gene is selectively cleaved, a selected nucleotide is removed, added or substituted and then the gene is ligated.
The DNA fragment according to the present invention has a function to promote expression of the gene downstream thereof. Therefore, by inserting the DNA
fragment according to the present invention into the transcriptional region of a desired foreign gene to be expressed, preferably into the 5'-end region of the transcriptional region, expression of the foreign gene is promoted. The method for expressing a foreign gene has already been established in the field of genetic engineering. That is, by inserting the desired foreign gene into a cloning site of an expression vector, introducing the resulting vector into host cells. and expressing it, the foreign gene may be expressed.
According to the method of the present invention, the DNA
fragmeht-according to the present invention is inserted at a site upstream of the foreign genein a manner such that the DNA fragment is operably linked to the foreign _ gene, and the foreign gene is expressed. The term that the DNA fragment according to the present invention is "operably linked" to the foreign gene meansthat expression of the foreign gene is delectably increased by inserting the DNA fragment according to the present invention when compared with the case wherein the DNA

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fragment according to the present invention is not _.
inserted. The DNA according to the present invention may be inserted.into the site immediately upstream of the foreign gene. Alternatively, another sequence may be located between the DNA according to the present invention and the foreign gene. Although the size of this intervening sequence is not restricted, it usually has a size of 0 - 1000- bp. A promoter sequence is located upstream of the DNA fragment according to the present invention. The DNA fragment according to. the present invention may be inserted into the site ._ immediately downstream of the promoter, or another sequence may be located between the promoter and the DNA
according to the present invention. Although the size of this intervening sequence is not restricted, it is usually 0 - 1000 bp. In summary, all recombinant vectors with which the expression of the- foreign gene is detectably increased by inserting the DNA fragment according to the present invention when compared with the case wherein the DNA fragment is not inserted, are within the scope of the present invention.
Since the nucleotide sequence of the cloning site of an expression vector is known, the DNA fragment according to the present invention may easily be inserted into the vector:.
Wide varieties of such an expression vectorare well-known in the art and are commercially available.

9 2I916?4 These expression vectors contain at least a replication origin for replication in the host cells, a promoter, a cloning site giving a restriction site for inserting the foreign gene, and a selection marker such as drug resistance, and usually contain a terminator which stably terminates transcription, and an SD sequence when the host is a bacterium. In the method of the present invention, any of these known expression vectors may be employed.
Example The present invention will now be described in more detail by way of examples thereof.- However, the present invention is not restricted to the examples.
1. pmr~~~~ation of PZD of Rice Bran _ For purification, a reference--(Takano et al., Journal of Japan Food-Industry Association, 34, 8-13 (1987) was referred. The enzyme activity was measured-by employing phosphatidylcholine as a substrate and quantifying the choline generated by the enzyme reaction (Imamura et al., J. Biochem. 83, 677-680 (1978)). It should be noted, however, the enzyme reaction was stopped by heat treatment at 95°G for-5 minutes.
That is, to 100 g of bran of rice (Oryza sativa), variety "KOSHIHIKARI", one liter of hexane was added and the mixture was stirred for a whole day and night, thereby defatting the rice bran. To the resultant, 10 g of Polycral AT (trademark, polyvinylpyrrolidone, commercially available from GAF Chemical) and 500 ml of mM Tris-HC1 buffer (pH7) containing 1 mM CaCI2 and 5 mM 2-mercaptoethanol were added, and the resulting mixture was stirred for 1 hour to extract the enzyme.
5 The extract was filtered through an 8-layered gauze and the filtrate was centrifuged at 15,000 x g for 20 minutes, followed by recovering the middle layer asa crude extract. The crude extract was treated with ammonium sulfate (65~ satuxation) and the generated precipitates 10 were collected by centrifugation (15,000 x g, 20 minutes), followed by dialyzing the precipitates after dissolution against the above-mentioned buffer. After the dialysis, precipitates were eliminated by filtration to obtain ammonium sulfate fraction.
The ammonium sulfate fraction was- applied to a column (2.0 x IO cm) of DEAE-Cellulose (commercially available from Whattman) equilibrated with buffer A (10 mM Tris-HCl, pH 7, 1 mM CaClz, I mM 2-mercaptoethanol).
After washing the column with about 100 ml of buffer A
containing 50 mM NaCl, elution was carried out with 120 m1 of buffer A having a linear gradient of NaCl concentration from 50 mM to 350 mM. P2~D was eluted at a NaCl concentration of about 0.2 M. The fraction having PLD activity was collected as an eluted solution_(DEAE-cellulose).
To the eluted solution (DEAE-cellulose), 3 M
ammonium sulfate was added in an amount attainingthe 1l final concentration of ammonium sulfate of 1 M, and the resulting mixture was applied to a Phenyl Sepharose column (commercially available from Pharmacia, 2.6 x 10 cm) equilibrated with buffer A containing 1 M ammonium sulfate. Elution was performed using 240 ml of buffer A
having a linear gradient of ammonium sulfate concentration from 1.0 M to 0 M. PLD was eluted at a concentration of ammonium sulfate of about 0.1 M. The fraction having the activity was recovered and dialyzed against buffer A to obtain an eluted solution (Phenyl Sepharose).
The eluted solution (Phenyl Sepharose) was applied to Mono Q column (anion-exchange column commercially available from Pharmacia, 16 x 10 cm) equilibrated with buffer A, and elution was performed using 150 ml of buffer A having a gradient of NaCl concentration from 50 mM to 350 mM. PLD was eluted at NaCl concentration from 210 mM to 235 mM. The fraction having PLD activity was recovered and dialyzed against buffer A to obtain an eluted solution (Mono Q*lst).
The eluted solution (Mono Q 1st) was concentrated to 0.5 ml by ultrafiltration and applied to Superose 6 column (commercially available from Pharmacia, 1.0 x 30 cm equilibrated with buffer A containing 0.1 M NaCl and elution was performed using the same buffer. The molecular weight of PLD was estimated to be 78 kDa. The fraction having PLD activity was recovered as an eluted * Trademarks 219.16~~
i2 solution (Superose 6).
To the eluted solution (Superose 6), 2.5 ml of 40°s -Carrier Ampholite (commercially available from Pharmacia, pH4.0-6.0) and distilled water were added to attain a final volume of 50 ml and isoelectric electrophoresis was carried out using Rotofore (commercially available from -Biorad). Electrophoresis was performed at 2°C with a constant power of 12W for 4 hours. PLD activity was observed at about pH 4.9. The fraction having PLD
activity was collected and dialyzed against buffer A to -obtain an isoelectric electrophoresis fraction.
The isoelectric electrophoresis fraction was applied to Mono Q column (commercially available from Pharmacia, 0.5 x 5 cm) and eluted with NaCl having a linear gradient of concentration of 50 mM to 350 mM. PLD was eluted at _ NaCl concentrations of about 210 mM and about 235 mM.
The two fractions having PLD activity were recovered as eluted solutions (Mono Q 2nd-I, II).
Purities of the eluted solutions (Mono Q 2nd-I, II) were checked by SDS-polyacrylamide electrophoresis (Laemmli (1970)) using 7.5$ acrylamide. After the electrophoresis, the gel was stained with Coomassie brilliant blue R-250. With either eluted solution, a main band was observed at a position corresponding to a molecular weight of 82 kDa. With the eluted solution (Mono Q 2nd-II), only a single band was observed.
By the purification described above, the purification magnificati-ons of the eluted solutions (Mono Q 2nd-I, II) were 380 times and 760 times, respectively, with respect to the crude extract.
Properties of the enzymes contained in the two fractions were determined. The results are shown in Table 1. The buffer solutions used for the measurement of the optimum pH were sodium acetate (pH 4-6), MES-NaOH
(pH 5.5-7.0) and Tris-HC1 (pH 7-9), all of which have a concentration of 100 mM in all of the buffer solutions.
The pH stability means the pH range in which decrease in the enzyme activity is not observed after leaving the enzyme at the respective pH at 25°C for-30 minutes. The temperature stability was measured by measuring the remaining activity after leaving the enzyme to stand at 4°C, 25°C, 37°C or 50°C for 30 minutes. The substrate specificity was measured at a substrate concentration of 5 mM and expressed in terms of the relative activity taking the enzyme activity to phosphatidylcholine as 100.

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Table 1 Mono Q 2nd-I Mono Q 2nd-II

Km Value 0.29 mM 0.29 mM

Optimum pH 6 6 pH Stability 7-8 7-8 Temperature Stability 4-37C 4-37C

Ca2+ Dependency not less not less than than 20 mM 20 mM

Substrate Specificity Phosphatidylcholine 100 100 Lysophosphatidylcholine 13 12 Sphingomyelin 6 4 2. Prnnf that Purified Protein is PLD _ Each of the eluted solutions (Mono Q 2nd-I, II) was-subjected to SDS-polyacrylamide gel electrophoresis in the same manner as in the purity test, and the obtained patterns were transferred to PVDF membranes (commercially available from Millipore), followed by staining the membranes. The band of the protein having the molecular weight of 82 kDa was cut out and the amino acid sequence of the N-terminal region of the protein was determined by a protein sequencer (commercially available from Shimazu Seisakusho, PSQ-1). For both proteins, amino acid sequence up to 10 residues from the N-terminal could be -determined, and the determined sequences were identical.
The sequence was as follows.
Val Gly Lys Gly Ala Thr Lys Val Tyr Ser Although the relationship between the proteins l having the molecular weight of 82 kDa contained in the two fractions having the enzyme activity is not clear, it is thought that they have high homology in their amino acid sequences, so that it was judged that there would be no problem even if a mixture of the fractions is used as an antigen for preparing an antibody.
A mixture of the eluted solutions (Mono Q 2nd-I, II) was subjected to SDS-polyacrylamide gel electrophoresis using 7.50 acrylamide, and the gel was stained with Coomassie brilliant blue R-250. The band of the protein having the molecular weight of 82 kDa was cut. out and recovered by electroelution (25 mM Tris, 192 mM glycine, 0.0250 SDS, 100V, 10 hours). Then SDS was removed by electrodialysis (15 mM ammonium bicarbonate, 200 V, 5 hours) and the resultant was lyophilized. For the electroelution and electrodialysis, BIOTRAP*(commercially available from Schleicher & Schuell) was used.
The protein having the molecular weight of 82 kDa highly purified by the above-described method was administered to a rabbit in an amount of 50 )gig per time at 7 days' intervals. Immunological titration test was performed for the sera before the immunization and after the third immunization. To the PLD solution containing 8.6 x 10 3 units of PLD, were added 0 - 50 ~1. of the serum before the immunization or after the third immunization, 50 u1 of 250 mM Tris-HC1 (pH~.O), 5 u1 of 50 mM CaCl2, 50 u1 of 0.2% Triton X-100 (trademark) and * Trademark 21~16~~

water to a total volume of 250 u1, and the mixture was left to stand at room temperature for 2_5 hours. To the resultant, 200 u1 of Protein A Sepharose (commercially available from Pharmacia) was added and the resulting mixture was gently shaken at room temperature for 2 hours.
The mixture was then centrifuged (500 x g, 5 minutes) and the enzyme activity in the supernatantwas measured.
Taking the measured enzyme activity in the case where the serum was not added as 100$, the enzyme activities in - cases where 20 p1 and 50 u1 of the serum before _ immunization were added were-95$ and 88$, respectively, and the enzyme activities in cases where 20 u1 and 50 u1 of the serum after the third immunization were added were .
75$ arid 30$, respectively. These results prove that the protein having the molecular weight of 82- kDa is PLD.
3. Determination of Aminn Acid sequence of Internal Regions The PLD protein was fragmentated in a gel (Cleveland et al., J. Biol. Chem., 252, 1I02(I977)). The cut out gel containing the PLD protein was inserted into a stacking gel well on a 15$ acrylamide gel prepared for separation of peptides, and Staphylococcus aureus V8 protease (commercially available from Wako Pure Chemical Industries, Ltd) in an amount of 1/10 volume of the PLD
protein was overlaid, followed by starting electrophoresis. The electrophoresis was stopped at the time point at which the bromophenol blue reached the 219~62~
center:of the stacking gel and then restarted 30 minutes-later.. After the electrophoresis, the pattern was transferred to a PVDF membrane and the membrane was stained. Clear bands were observed at the positions corresponding to molecular weights of 20, I4, 13, 11 and kDa. Each of the bands of the peptide fragments having molecular weights of 20, 14 and 13 kDa were cut out and their amino acid sequences were determined by a -protein sequencer. The determined sequences are as IO follows.
kDa Asn Tyr Phe His Gly Ser Asp Val Asn ? Val Leu ? Pro Arg Asn Pro Asp Asp (Asp) ?
?

I1e 14 kDa Thr ? Asn Val Gln Leu Phe Arg Ser I1e Asp 15 G1y Gly Ala Ala Phe G1y Phe Pro Asp Thr Pro Glu Glu Ala Ala Lys ? G1y Leu Va1 Ser Gly 13 kDa Ile Ala Met GIy Gly Tyr Gln Phe Tyr His Leu Ala Thr Arg Gln Pro Ala Arg Gly Gln Ile His-Gly Phe Arg Met Ala Leu ? Tyr Glu His Leu ...-20 Gly Met Leu ? Asp Val Phe (In the sequences, "?" means ino residue which the acid am could not be d, nd ino residue in identifie a the acid am parentheses means that the amino acid residue could not be identified confidentially.
4. Preparation of cDNA Library of Rice Immature Seeds Total RNAs were extracted from immature seeds obtained after 5 days from flowering by the SDS-phenol method, and prepared by the lithium chloride precipitation. Poly(A)rRNA was prepared using Oligotex-dT30 (commercially available from Takara Shuao) according to the instructions provided by the manufacturer. For the cDNA cloning, cDNA synthesis System Plus (commercially available from Amersham) and cDNA Cloning System ~.gtl0 (commercially available from Amersham) were used. However, a,2APII*vector (commercially available from Stratagene) was used as the cloning vector and XLl-Blue was used as the host cells.
5. Preparation of Probes Oligonucleotides corresponding to the amino acid sequences of PLD were synthesized by a DNA synthesizer (commercially available from Applied Biosystems). The sequences thereof as well as the corresponding amino acid sequences are as follows.
20KF 5' AAYTAYTTYCAYGG 3' 20KR1 5' RTCRTCRTCNGGRTT 3' (In these sequences, "R" represents a purine base A or G;
"Y" represents a pyrimidine base T or G; and N represents G, A, T or C. ) The 20KF is a mixture of 32 kinds of oligonucleotides containing the DNA sequences encoding the amino acid sequence of Asn Tyr Phe His Gly found in a peptide having a molecular weight of 20 kDa, and the 20KR1 is a mixture of 128 kinds of * Trademarks oligonucleotides containing complementary chains of the DNA sequences encoding the amino acid sequence of Asn Pro Asp Asp(Asp) found in the same peptide.
The cDNA synthesis was carried out using 10 ng of Poly(A)+RNA, 0.3 ug of random hexamer (N6), 10 LI of an RNase inhibitor (RNA Guard, commercially available from Pharmacia), 1 mM each of dATP, dCTP, dGTP and dTTP, 1 x PCR buffer (commercially available from Takara Shuzo), 50 mM of magnesium chloride and 100 U of a reverse transcriptase (M-MuLV RTase, commercially available from BRL) in a total volume of 10 u1. The reaction was carried out at 37°C for 30 minutes and the reaction mixture was then heated at 95°C for 5 minutes, followed by retaining the reaction mixture in ice.
Polymerase chain reaction (PCR) was performed using the above-described cDNA as a template and 20KF and 20KR1 as primers. The reaction was performed using 10 u1 of the cDNA synthesis reaction mixture, a mixture of 50 pmol each of the primers, 200 uM each of dATP, dCTP, dGTP and dTTP, 1 x PCR buffer (commercially available from TAKARA
SHUZO), and 2.5 U of AmpliTaq DNA polymerase (commercially available from TAKARA SHUZO) in a total volume of 50 u1. A cycle of temperature conditions of 94°C for 1 minute, 40°C for 1 minute and 72°C for 2.5 minutes was repeated 30 times in a DNA Thermocycler * Trademark (commercially available from Perkin Elmer Cetus).
The PCR product was separated on 2o agarose gel. A
small number of fragments were detected by the ethidium bromide staining method. One of them had a size of 94 by 5 as expected.
The PCR fragment was cut out from the gel and subcloned into pUCl9 plasmid. The DNA sequence of the subcloned PCR fragment was determined by the dideoxy method using T7 sequencing kit (commercially available 10 from Pharmacia). Between the two primers, a DNA sequence encoding the expected amino acid sequence was observed.
The nucleotide sequence of the DNA between the primers and the amino acid sequence encoded thereby are as follows.

Ser Asp Val Asn Cys Val Leu Cys Pro Ar_g Isotope 32P (commercially available from Amersham) was incorporated into the oligonucleotide using a DNA 5'-end labelling kit MEGALABEL*(commercially available from 20 Takara Shuzo) to obtain a radioactive oligonucleotide probe.

6. Screening of PLD Gene-containing Clones Using the radioactive oligonucleotide as a probe, a cDNA library was screened. Hybridization solution contained 0.5 M sodium phosphate buffer (pH 7.2), 7o SDS, 1 mM EDTA and 100 ug/ml of salmon sperm DNA, and hybridization was performed after adding the probe to the * Trademark hybridization solution at 45°C for 16 hours. The washing solution contained 0.3 M NaCl and 30 mM sodium citrate, and washing was performed twice at 45°C for 20 minutes each. Positive plaques were isolated and subcloned in vivo into pBluescript* plasmid (commercially available from Stratagene) in accordance with the instructions provided by the manufacturer of a,2APII cloning vector.
The nucleotide sequence was determined by the dideoxy method. As a result, a region encoding the internal amino acid sequence determined in the "Section 3" existed.

7. Determination of Nucleotide Sequence of 5'-end Re,~n Since a clone containing the full length of cDNA
could not be isolated, a DNA fragment having the 5'-end region was prepared by RACE method (Edwards et al., Nucleic Acids Res., 19, 5227-5232 (1991)). 5'-AmpliFINDER RACE Kit (commercially available from Clonetech) was used in accordance with the manual attached to the product. An oligoDNA was synthesized based on the nucleotide sequence of the cDNA determined in "Section 6", and PCR was performed using the mRNA
prepared by the method described in "Section 4" as a template. The PCR product was subcloned into a PCRII
vector (commercially available from Invitrogen) and the nucleotide sequence was determined by the dideoxy method.
The thus determined nucleotide sequence of the cDNA of rice PLD as well as the deduced amino acid sequence * Trademarks encoded thereby is shown in SEQ ID N0. 2 in the Sequence Listing. It is thought that translation is initiated from the 182nd nucleotide shown in SEQ ID NO. 2 since a termination codon exists at 36 bases upstream thereof.

8. Isolation of PLD Genomic Clone Corresponding to PLD
cDNA and Identification of Promoter Re Qion To isolate a genomic DNA clone having tree regulatory sequence of the PLD gene corresponding to the PLD cDNA
determined in "section 6", which was cloned into pBluescript plasmid, a genomic library of rice, variety "KOSHIHIKARI" was prepared. This was carried out by partially digesting DNAs from live leaves of KOSHIHIKARI
with Mbo I, purifying a fraction having a size of 16 - 20 kb by sucrose gradient centrifugation, and using lambda DASH II (commercially available from Stratagene) and GigapackII*Gold (commercially available from Stratagene).
The genomic library was screened with the PLD cDNA clone as a probe. The screening was carried out in the same manner as in "Section 6" except that hybridization was performed at 65°C for 16 hours, the washing solution contained 0.5 x SSC and 0.1o SDS, and that the washing was performed twice at 65°C for 20 minutes each. The nucleotide sequence of the hybridized genomic clone was determined by the dideoxy method. As a result, a region homologous to the cDNA sequence determined in "Section 6"
existed.
The transcription initiation site was determined by * Trademarks ~19162~
z3 the method described in "Section 7". In the vicinity of the transcription initiation site, a "TATA" consensus sequence box was observed. The ATG translation -initiation site was determined based on the determined DNA sequence as the most upstream ATG codon in the translation open reading frame of the clone and as the ATG codon which is first accessible in the mRNA
synthesized in rice.
The DNA sequence of a-part of the genomic clone hybridized with the cDNA clone is shown in SEQ ID N0. 3.
In the genomic DNA sequence, an open reading frame starting from the ATG translation initiation codon, which overlaps with the corresponding cDNA sequence has been identified. The promoter region exists upstream of the ATG translation initiation codon and starts from the sits immediately upstream thereof.

9, rrlPnt;f;ration of Introns and Analysis of Functions thereof on Expression of Genes From comparison between the cDNA (SEQ ID N0. 2) and the genomic DNA (SEQ ID N0. 3), it was proved that 3 introns exist in PLD gene. Among these, the intron having a size of 173 by located at the 5'-flanking region of the mRNA (i.e., the nucleotide sequence between 1666nt and 1838nt of the nucleotide sequence shown in SEQ ID N0.
3, the sequence being shown in SEQ ID N0. 4) was tested _.
for its influence on expression of a gene in plant cells.
Primers of l5mer each of which contains 5 bases of.exon , 2~9I6~~

region (5'-ACCCGGTAAGCCCAG-3', 3'-CCCCCGCGTCCATCC-5') were synthesized and PCR was carried out using the genomic clone as a template according to the method described in the Section of "5. Preparation of Probes". The PCR product was subcloned into PCRII
vector and a fragment was cut out with Eco RI. The _ fragment was blunted and inserted into the Sma I
site of a plasmid pBI221-(commercially available from Toyobo) (see Fig. 1). The obtained recombinant plasmid was introduced into rice cultured cells (Baba et al., Plant Cell Physiol. 27, 463-471 (1986)) in accordance with the reported method (Shimamoto et.al., Nature, 338, 274-276 (1989)) and (3-glucuronidase (GUS) activity was measured. As shown in Table 2, by introducing the intron, the GUS
activity was increased. Further, as shown in Table 3, increase in the GUS activity was also observed in the case where the intron was inserted in the reverse direction. The direction of the intron was determined based on the sizes of the fragments cut out with Bg1 II and Bam HI, utilizing the Bg1 II
site existing in the intron sequence and the Bam HI
site existing in pBI221.

2191~2~
Table 2 Plasmid GUS Activity pBI221 10.4 pBI22I + intron 105.7 (pmol MU/min./mg protein) Table 3 Plasmid GUS Activity pBI221 8.8 pBI22I + intron 79-4 pBI221 + intron (reverse direction) 54.2 (pmol MU/min./mg protein) 2I9~~~~

SEQUENCE LISTING
SEQ ID NO.1 SEQUENCE LENGTH: 183 SEQUENCE TYPE: Nucleic Acid MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE
ORGANISM: Oryza sativa SEQUENCE DESCRIPTION

SEQ ID NO.: 2 SEQUENCE LENGTH: 3040 SEQUENCE TYPE: Nucleic Acid __ MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Oryza sativa SEQUENCE DESCRIPTION

Met Ala Gln Met Leu Leu His Gly Thr Leu His Ala Thr Ile Phe Glu 1 5 i0 15 Ala Ala Ser Leu Ser Asn Pro His Arg Ala Ser Gly Ser Ala Pro Lys PheIle Arg LysPhe Val GluGly Ile GluAsp Thr Val GlyVal Gly LysGly Ala ThrLys Val TyrSer Thr IleAsp Leu Glu LysAla Arg ValGly Arg ThrArg Met IleThr Asn GluPro Ile Asn ProArg Trp TyrGlu Ser PheHis Ile TyrCys Ala HisMet Ala Ser AsnVal Ile PheThr Val LysIle Asp AsnPro Ile GlyAla Thr Asn IleGly Arg AlaTyr Leu ProVal Gln GluLeu Leu AsnGly Glu Glu IleAsp Arg TrpLeu Asp IleCys Asp AsnAsn Arg GluSer Val Gly GluSer Lys IleHis Val LysLeu Gln TyrPhe Asp ValSer Lys Asp ArgAsn Trp AlaArg Gly ValArg Ser ThrLys Tyr ProGly Val Pro TyrThr Phe za ~~9~ ~~~

Phe Ser Gln Arg GlnGly Cys LysVal Thr LeuTyr Gln Asp AlaHis Val Pro Asp Asn PheIle Pro LysIle Pro LeuAla Asp Gly LysAsn Tyr Glu Pro His ArgCys Trp GluAsp Ile PheAsp Ala Ile SerAsn Ala Gln His Leu IleTyr Ile ThrGly Trp SerVal Tyr Thr GluIle Thr Leu Val Arg AspSer Asn ArgPro Lys ProGly Gly Asp ValThr Leu Gly Glu Leu LeuLys Lys LysAla Ser GluGly Val Arg ValLeu Met Leu Val Trp AspAsp Arg ThrSer Val GlyLeu Leu Lys ArgAsp Gly Leu Met Ala ThrHis Asp GluGlu Thr GluAsn Tyr Phe HisGly Ser Asp Val Asn CysVal Leu CysPro Arg AsnPro Asp Asp SerGly i Ser IleVal Gln AspLeu Ser Ile SerThr Met PheThr His HisGln Lys IleVal Val ValAsp His Glu LeuPro Asn GlnGly Ser GlnGln Arg ArgIle Val SerPhe Val Gly GlyLeu Asp LeuCys Asp GlyArg Tyr AspThr Gln TyrHis Ser Leu PheArg Thr LeuAsp Ser ThrHis His AspAsp Phe HisGln Pro Asn PheAla Thr AlaSer Ile LysLys Gly GlyPro Arg GluPro Trp His AspIle His SerArg Leu GluGly Pro IleAla Trp AspVal Leu Tyr AsnPhe Glu GlnArg Trp ArgLys Gln GlyGly Lys AspLeu Leu Leu GlnLeu Arg AspLeu Ser AspThr Ile IlePro Pro SerPro Val Met PhePro Glu AspArg Glu ThrTrp Asn ValGln Leu PheArg Ser Ile AspGly Gly AlaAla Phe GlyPhe 219~62~

ProAsp Thr ProGlu Glu AlaAla Lys AlaGly Leu Val SerGly Lys AspGln Ile IleAsp Arg SerIle Gln AspAla Tyr Ile HisAla Ile ArgArg Ala LysAsn Phe IleTyr Ile GluAsn Gln Tyr PheLeu Gly SerSer Tyr AlaTrp Lys ProGlu Gly IleLys Pro Glu AspIle Gly AlaLeu His LeuIle Pro LysGlu Leu AlaLeu Lys Val ValSer Lys IleGlu Ala GlyGlu Arg PheThr Val TyrVal Val Val ProMet Trp ProGlu Gly ValPro Glu SerGly Ser ValGln Ala Ile LeuAsp Trp GlnArg Arg ThrMet Glu MetMet Tyr ThrAsp Ile Thr GluAla Leu GlnAla Lys GlyIle Glu AlaAsn Pro LysAsp Tyr Leu ThrPhe Phe 2I916~4 Cys Leu Gly Asn Arg Glu Val Lys Gln Ala Gly Glu Tyr Gln Pro Glu Glu Gln Pro Glu Ala Asp Thr Asp Tyr Ser Arg Ala Gln Glu Ala Arg Arg Phe Met Ile Tyr Val His Thr Lys Met Met Ile Val Asp Asp Glu TyrIle IleIle Gly Ser AlaAsn Ile AsnGln Arg SerMet Asp Gly AlaArg AspSer Glu Ile AlaMet Gly GlyTyr Gln ProTyr His Leu AlaThr ArgGln Pro Ala ArgGly Gln IleHis Gly PheArg Met Ala LeuTrp TyrGlu His Leu GlyMet Leu AspAsp Val PheGln Arg Pro GluSer LeuGlu Cys Val GlnLys Val AsnArg Ile AlaGlu Lys Tyr TrpAsp MetTyr Ser Ser AspAsp Leu GlnGln Asp LeuPro Gly His 21916~~

Leu Leu Ser Tyr Pro Ile Gly Val Ala Ser Asp Gly Val Val Thr Glu Leu Pro Gly Met Glu Tyr Phe Pro Asp Thr Arg Ala Arg Val Leu Gly Ala Lys Ser Asp Tyr Met Pro Pro Ile Leu Thr Ser SEQ ID NO.: 3 SEQUENCE LENGTH: 2799 SEQUENCE TYPE: Nucleic Acid MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE
ORGANISM: Oryza sativa SEQUENCE DESCRIPTION

Met Ala Gln Met Leu Leu His Gly Thr Leu His Ala Thr Ile Phe Glu Ala Ala Ser Leu Ser Asn Pro His Arg Ala Ser Gly Ser Ala Pro Lys Phe Ile Arg Lys Phe Val Glu Gly Ile Glu Asp Thr ValGly ValGly Lys GlyAla Thr LysVal Tyr Ser ThrIle Asp Leu GluLys AlaArg Val GlyArg Thr ArgMet Ile Thr AsnGlu Pro Ile AsnPro ArgTrp Tyr GluSer Phe HisIle Tyr Cys AlaHis Met Ala SerAsn ValIle Phe ThrVal Lys IleAsp Asn Pro IleGly Ala Thr Asn Ile Gly Arg Ala Tyr Leu Pro Val Gln Glu Leu Leu Asn Gly Glu Glu Ile Asp Arg SEQ ID NO.: 4 SEQUENCE LENGTH: 173 SEQUENCE TYPE: Nucleic Acid MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE
ORGANISM: Oryza sativa SEQUENCE DESCRIPTION

Claims (12)

1. An isolated DNA fragment having a nucleotide sequence shown in SEQ ID NO. 1 in Sequence Listing or having a nucleotide sequence which hybridizes with said nucleotide sequence shown in SEQ ID NO. 1 when the hybridization is carried out in a hybridization solution containing 0.5M
sodium phosphate buffer (pH7.2), 7% SDS, 1 mM EDTA and 100 µg/ml of salmon sperm DNA, at 65°C for 16 hours, and then washing the product twice with a washing solution containing 0.5×SSC and 0.1% SDS at 65°C for 20 minutes each, the latter nucleotide sequence having a function to promote expression of a gene downstream thereof in a plant.

2. The DNA fragment according to claim 1, which has a nucleotide sequence shown in SEQ ID NO. 1 in Sequence Listing.

3. An isolated DNA fragment having a nucleotide sequence shown in SEQ ID NO.4 in Sequence Listing or having a nucleotide sequence which hybridizes with said nucleotide sequence shown in SEQ ID NO. 4 when the hybridization solution containing 0.5M sodium phosphate buffer (pH7.2), 7% SDS, 1mM EDTA and 100 µg/ml of salmon sperm DNA, at 65°C
for 16 hours, and then washing the product twice with a washing solution containing 0.5×SSC and 0.1% SDS at 65°C
for 20 minutes each, the latter nucleotide sequence having a function to promote expression of a gene downstream thereof in a plant.

4. The DNA fragment according to claim 3, which has a nucleotide sequence shown in SEQ ID NO. 4 in Sequence Listing.

5. A recombinant vector comprising said DNA fragment according to claim 1 and a foreign gene to be expressed, which is operably linked to said DNA fragment at a downstream region of said DNA fragment.

6. The recombinant vector according to claim 5, wherein said DNA fragment has a nucleotide sequence shown in SEQ
ID NO. 1 in Sequence Listing.

7. A recombinant vector comprising said DNA fragment according to claim 3 and a foreign gene to be expressed, which is operably linked to said DNA fragment at a downstream region of said DNA fragment.

8. The recombinant vector according to claim 7, wherein said DNA fragment has a nucleotide sequence in SEQ ID No.4 in Sequence Listing.

9. A method for expressing a foreign gene comprising introducing said recombinant vector according to claim 5, into host plant cells and expressing said foreign gene.

10. The method according to claim 9, wherein said DNA
fragment has a nucleotide sequence shown in SEQ ID No.1 in Sequence Listing.

11. A method for expressing a foreign gene comprising introducing said recombinant vector according to claim 7, into host plant cells and expressing said foreign gene.

12. The method according to claim 11, wherein said DNA
fragment has a nucleotide sequence shown in SEQ ID No.4 in Sequence Listing.