JP2004350692A - Mutant dna encoding alpha subunit of first isozyme of anthranilate synthase of rice plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA encoding α-subunit of a first isozyme of an anthranilate synthase (ASA) of a rice plant, or a mutant of such DNA. <P>SOLUTION: Disclosed are a DNA sequence encoding an α-subunit protein of the first isozyme of the anthranilate synthase (ASA) of a rice plant and a DNA encoding α-subunit protein of a second isozyme of the ASA. An example of the DNA sequence encoding the α-subunit protein of the first isozyme of ASA is a DNA having a base sequence represented by SEQ No. 1 in the sequence table. An example of the DNA sequence encoding the α-subunit protein of the second isozyme of ASA is a DNA having a base sequence represented by SEQ No. 10 in the sequence table. A DNA having a base sequence represented by SEQ No. 12 in the sequence table is also disclosed as a mutant derived from the DNA having a base sequence represented by SEQ No. 1 in the sequence table. A recombinant vector comprising incorporating a DNA fragment having the DNA sequence of the invention into the downstream of promoter is constituted, and when a plant cell which is introduced with the vector is cultured, a transformed plant having high tryptophane content can be regenerated. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel variant of a gene DNA encoding the α subunit of the first isozyme of two isozymes of rice anthranilate synthase (isoenzymes). Specifically, the present invention relates to two isozymes of anthranilate synthase involved in the biosynthesis of tryptophan in rice plants, that is, a protein which is the α subunit of the first isozyme of the first isozyme and the second isozyme. A novel variant of the DNA encoding

  That is, the present invention relates to a novel modified DNA encoding a novel protein having the activity of the α-subunit protein of the first isozyme of anthranilate synthase. The present invention also relates to a novel recombinant vector into which the novel modified DNA has been incorporated. The present invention further includes Escherichia coli transformed with the novel modified DNA, or plants and seeds transformed with the novel modified DNA.

  Furthermore, the present invention relates to a method for increasing the tryptophan content of a plant by using the novel modified DNA. Furthermore, the present invention relates to a method for selecting a transformed cell of a plant containing the novel modified DNA, and to a method for producing a transformed plant containing the novel modified DNA.

  Grain seeds such as rice, corn and wheat are important nutrient sources for humans and livestock. However, these seeds have a low content of tryptophan, one of the essential amino acids, and are inferior in nutritional value. There is a demand for a new variety of plants capable of producing cereal seeds having a high tryptophan content and excellent nutritional value.

  In the biosynthetic pathway of tryptophan in plants, chorismic acid biosynthesizes anthranilic acid, and the production of anthranilic acid involves the catalytic action of anthranilate synthase (hereinafter abbreviated as "ASA"). Thus, it is known that anthranilic acid is produced by this, and that tryptophan is produced from anthranilic acid through indole by a six-step enzymatic reaction (Non-Patent Document 1: Biochemistry Experimental Course, Vol. 11, p. 652) 65653 pages, published by Tokyo Chemical Dojin in 1976).

  It is known that a conventionally known anthranilate synthase of a plant is composed of a plurality of subunits. For example, the anthranilate synthase of Arabidopsis thaliana (Japanese name: Arabidopsis thaliana) consists of two types of isozymes, and both the first isozyme and the second isozyme are dimers of α subunit and β subunit, respectively. It has been known. Gene encoding the α subunit of the first isozyme of Arabidopsis anthranilate synthase (abbreviated to ASA1) (asa1) and gene encoding the α subunit of the second isozyme (abbreviated to ASA2) (asa2) And the nucleotide sequences of the DNAs of these genes have been elucidated (Non-patent Document 2: The Plant Cell, Vol. 4, pp. 721-733, 1992).

  On the other hand, first, the present inventors focused on the anthranilate synthase α subunit, which is suggested to have an important functional region for regulating tryptophan biosynthesis in rice, and regulated the biosynthesis of tryptophan and the plant hormone IAA. In 1996, we conducted a study to isolate a gene encoding an anthranilate synthase protein with the aim of gaining insight into the mechanism. As a summary of the report of this study, the present inventors extracted mRNA and genomic DNA from rice (Agriculture and Forestry No. 8) seedlings, prepared cDNA libraries and genomic DNA libraries, and used these libraries. Using a cDNA fragment of the Arabidopsis asa gene as a probe, genomic Southern analysis and screening of the library were performed, and as a result, it was reported that DNA that was considered to correspond to the asa gene of rice anthranilate synthase was obtained ( Non-Patent Document 3: “Breeding Science Journal”, vol. 46, separate volume 2, p. 28, 1996). In the summary of the research report published in this document, it was reported that a DNA fragment corresponding to the asa gene of rice anthranilate synthase was obtained, and the specific method used for obtaining the DNA fragment was described by the present invention. They reported that they were not disclosed and that the nucleotide sequence of the DNA fragment had not yet been determined. In addition, in the summary of the above research report, it was mentioned that there are two types of DNA in the DNA which is considered to correspond to the gene encoding rice anthranilate synthase (the above-mentioned “Breeding Journal”, vol. 46). Separate volume 2, page 28, 1996).

In addition, a report on introducing the DNA of the ASA gene of the α subunit of the first isozyme of Arabidopsis ASA and a DNA fragment obtained by modifying the DNA into a tobacco plant and expressing the function of the gene in tobacco has been reported. (Non-Patent Document 4: Massachusetts Institute of Technology, Cambridge, MA, 1993).
"Biochemistry Experiment Course", Vol. 11, pp. 652-653, (1976) "The Plant Cell," Volume 4, 721-733, (1992) The Journal of Breeding Science, Volume 46, Separate Volume 2, Page 28 (1996) "Massachusetts Institute of Technology, Cambridge, MA" (1993) However, as far as the present inventor knows, the analysis of the amino acid sequence of the protein which is the α subunit of rice ASA isozyme, and the analysis of rice ASA isozyme There is no known literature reporting a specific method by which a gene encoding the α subunit of Example 1 could be obtained. Further, a promoter sequence related to the expression of the gene is not known. Further, there is no known literature reporting the use of a gene encoding rice ASA.

  One object of the present invention is to obtain from a rice plant a gene related to rice ASA, in particular, a novel DNA encoding the α subunit of the first isozyme of rice ASA. Is to determine the base sequence.

  Another object of the present invention is to provide a novel DNA capable of encoding a novel protein having the activity of the α subunit of the first isozyme of rice ASA, but which is insensitive to feedback suppression by tryptophan. Still another object of the present invention is to transform useful plants such as corn, rice, soybean, wheat, barley, tomato, and potato with the novel DNA, and produce seeds having a high tryptophan content. It is to provide a new transformed variety of useful plants that can be used. Furthermore, another object of the present invention is to create a novel DNA fragment capable of encoding a protein having the activity of the α subunit of the first isozyme of rice ASA but insensitive to feedback suppression by tryptophan, Another object of the present invention is to provide a plant cell transformed with the novel DNA fragment and a method for efficiently producing a transformed plant. Other objects of the present invention will become clear from the following description.

  In order to achieve the above objects, the present inventors have conducted a series of various studies. First, a study was conducted to obtain genes encoding the α subunits of each of the two isozymes of ASA from rice plants. As a result of this research, total RNA was extracted from rice seedling tissue, for example, crushed green foliage by a method known in the art of genetic engineering, and total mRNA was isolated from the extracted total RNA by a conventional method. From the total mRNA, rice cDNA was successfully obtained using a commercially available cDNA synthesis kit. The obtained total cDNA was further prepared by ligating the ends of EcoRI-cut pieces of the λgt11 phage vector to a phage vector (commercially available from STRATAGEN) treated with calf intestine-derived alkaline phosphatase. Many trial and error results have shown that packaging the recombinant vector into lambda-phage allows construction of a replicable recombinant lambda-phage.

  When the recombinant lambda-phage is infected with Escherichia coli Y1088 and incubated, a large number of recombinant λ phages are obtained as many plaques (plaques), and a group of recombinant λ phages contained in the obtained many plaques Are various phages containing all the rice-derived cDNAs, which were found to be usable as rice cDNA libraries.

  On the other hand, the amino acid sequence of the protein which is the α subunit of the first isozyme of ASA of Arabidopsis described in Non-Patent Document 2: “The Plant Cell”, vol. 4, pp. 721-733 (1992), and Reference is made to the predicted nucleotide sequence of the gene encoding the protein, the amino acid sequence of the protein which is the α subunit of the second isozyme of Arabidopsis ASA, and the nucleotide sequence of the gene encoding the protein predicted from this. Meanwhile, the present inventors produced a first oligonucleotide consisting of 21 nucleotides and a second oligonucleotide consisting of 24 nucleotides, which were considered to be suitable as primers for the PCR method, by chemical synthesis.

  When a PCR amplification reaction is performed using a mixture of the first oligonucleotide, the second oligonucleotide, and a commercially available Arabidopsis cDNA library (used as a template), the first and second oligonucleotides are obtained. Oligonucleotides can serve as the required primers (complementary DNA) in the PCR method and replace a portion of the DNA sequence corresponding to the gene encoding the α subunit of each of the two isozymes of Arabidopsis ASA. It was confirmed that the constituent DNA fragments could be amplified. From the PCR amplification reaction solution, a partial amplification product of the gene encoding the α subunit of each of the first and second isozymes of Arabidopsis ASA was successfully recovered as probe DNA.

  By utilizing the above-obtained probe DNA thus obtained, a phage plaque-hybridization method was used to obtain a rice cDNA library (i.e., 300,000 plaques of the recombinant λ phage) obtained previously. As a result of many trial-and-error tests, plaques of recombinant λ phage containing a gene encoding the α subunit of the first isozyme of rice and a gene encoding the α subunit of the second isozyme of rice ASA 8 I was lucky enough to isolate the individual. After separately amplifying the recombinant λ phage of these eight plaques, each λ phage DNA was isolated by a conventional method.

  The recombinant phage DNA containing a DNA sequence found to correspond to the gene encoding the α subunit of each of the first and second isozymes of rice ASA obtained as described above was digested with the restriction enzyme EcoRI. Then, the DNA fragment obtained by digestion with the above was inserted into the EcoRI cleavage site of a commercially available known plasmid vector p Bluescript II SK (+) using a DNA ligation kit and ligated. Escherichia coli XL1-Blue MRF ′ could be transformed with the recombinant plasmid vector thus obtained. By incubating the obtained Escherichia coli transformant, many cells could be obtained. Plasmid DNA was collected from the obtained cells. By subjecting the plasmid DNA thus collected to DNA base sequence analysis, the plasmid DNA contained in this DNA fragment and corresponding to the α-subunit of each of the first and second isozymes of rice ASA was encoded. At this time, it was confirmed that the base sequence of the first DNA sequence of the two DNA sequences determined to have the base sequence described in SEQ ID NO: 1 in the sequence listing described below. In addition, it has now been confirmed that the base sequence of the second DNA sequence of the two DNA sequences has the base sequence described in SEQ ID NO: 10 in the sequence listing.

  Furthermore, to the knowledge of the present inventors, the DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing and the DNA having the nucleotide sequence of SEQ ID NO: 10 are not described in any document. It was identified as a novel DNA sequence.

  The protein encoded by the DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing is assumed to be a protein having the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, and this protein is the first isozyme of rice ASA It is recognized as a protein constituting the α subunit.

  Accordingly, the first invention provides a protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein the DNA encodes the α subunit of the first isozyme of rice anthranilate synthase.

  The above-mentioned DNA according to the first invention can be specifically a DNA having the base sequence of SEQ ID NO: 1 in the sequence listing.

  The DNA of the first invention is a DNA encoding a protein which is the α subunit of the first isozyme of rice ASA. As described above, the DNA of the first invention was obtained from a rice cDNA library by a genetic engineering technique when conducting the research of the present inventors. However, since the base sequence of the DNA has been elucidated in the present invention, it can be obtained also by chemical synthesis from nucleotides with reference to the base sequence of SEQ ID NO: 1 in the sequence listing. Alternatively, a synthetic nucleotide may be prepared and used as a probe with reference to the nucleotide sequence of SEQ ID NO: 1 or a polymerase chain may be used from a rice chromosome DNA library by a known method using the synthetic oligonucleotide as a primer. The DNA of the first invention can also be obtained by a reaction (PCR) method or hybridization.

  Furthermore, the protein encoded by the DNA having the nucleotide sequence of SEQ ID NO: 10 in the sequence listing was recognized to be a protein having the amino acid sequence of SEQ ID NO: 11 in the sequence listing, and this protein was the second protein of rice ASA. It is recognized as a protein constituting the α subunit of the isozyme.

  Accordingly, the second invention provides a protein having the amino acid sequence shown in SEQ ID NO: 11 in the Sequence Listing, which DNA encodes the α-subunit of the second isozyme of rice anthranilate synthase.

  The above-mentioned DNA according to the second invention can be specifically a DNA having the base sequence of SEQ ID NO: 10 in the sequence listing.

  The DNA of the second invention is a DNA encoding a protein that is the α subunit of the second isozyme of rice ASA. As described above, the DNA of the second invention was obtained from a rice cDNA library by genetic engineering when conducting the research of the present inventors. However, since the nucleotide sequence of the DNA has been elucidated in the present invention, it can also be obtained by chemical synthesis from nucleotides with reference to the nucleotide sequence of SEQ ID NO: 10 in the sequence listing. Further, a synthetic nucleotide is prepared and used as a probe with reference to the nucleotide sequence of SEQ ID NO: 10, or a polymerase chain is prepared from a rice chromosome DNA library by a known method using the synthetic oligonucleotide as a primer. The DNA of the second invention can also be obtained by a reaction (PCR) method or hybridization.

  Hereinafter, a method for obtaining the DNA according to the first invention and the DNA according to the second invention from the stems and leaves of rice by a genetic engineering technique will be schematically described.

(1) Preparation of rice mRNA and construction of rice cDNA library Extracting total RNA from various tissues of rice (Oriza sativa), for example, foliage, roots, callus and other tissues, preferably green foliage, by a conventional method . After removing contaminants such as proteins from the extracted total RNA, rice (mRNA) can be obtained by further purifying poly (A) + RNA through a column packed with oligo dT cellulose.

  Next, a total cDNA of rice is synthesized from the total mRNA using a commercially available cDNA synthesis kit. The whole synthesized cDNA is ligated to a phage vector, for example, a λgt11 vector or a λZAPII vector. The obtained recombinant vector is integrated into lambda phage to obtain a large number of recombinant phages. By infecting Escherichia coli with these recombinant phages as a host and incubating, a large number of recombinant phages can be obtained as plaques. These series of operations can be performed using a commercially available cDNA cloning kit.

  Since a large number of recombinant phages obtained as lysed plaques of host Escherichia coli are composed of a wide variety of phages containing all the rice-derived cDNAs, they can be used as rice cDNA libraries.

(2) Construction of primers for PCR method Common storage between the known base sequence of the α-subunit gene of the first isozyme of Arabidopsis ASA and the known base sequence of the α-subunit gene of the second isozyme With reference to the base sequence (online database EMBL: M92353) and considering that the first isozyme of Arabidopsis ASA has a stronger expression power in plants, the present inventors (Oligonucleotides of SEQ ID NOs: 8 and 9 in the sequence listing described later) were prepared by chemical synthesis as primers (complementary DNA) for PCR.

(3) Preparation of probe DNA In a rice cDNA library obtained as a plaque consisting of a large number of recombinant phages, a DNA encoding the α subunit of the desired rice ASA is selectively collected. To prepare a probe DNA used for For the preparation of this probe DNA, a primer consisting of the above-mentioned synthetic oligonucleotide was used, and the α-subunit of the first and second isozymes of ASA of Arabidopsis was obtained from a cDNA library of Arabidopsis as a template by PCR. Amplify the DNA encoding a part of the gene.

  After the amplification reaction is repeated by the PCR method, an amplification product of a DNA fragment which is a part of the DNA sequence corresponding to the gene of the α subunit of Arabidopsis ASA is collected as a desired probe DNA from the amplification reaction solution. .

(4) Selection of DNA for α-subunit gene of rice ASA from rice cDNA library Screening of the above-described probe DNA from a large number of plaques of recombinant phage obtained earlier as a rice cDNA library Several plaques consisting of recombinant phage into which a DNA sequence corresponding to the gene of the α subunit of the target rice ASA has been integrated are selected by the phage plaque-hybridization method used in the present invention.

  The recombinant phage collected as plaques selected in this manner is a recombinant phage in which a DNA fragment containing the target DNA sequence corresponding to the DNA of the α subunit gene of rice ASA is integrated. belongs to.

  That is, specifically, the phage of the plaque selected by plaque hybridization as described above is collected, and the phage DNA is further recovered from the collected phage. By treating the collected phage DNA by the dideoxy method or the like, the nucleotide sequence of the rice-derived inserted DNA fragment present inside the phage DNA can be determined. When the amino acid sequence defined from the protein coding region (open reading frame) in the base sequence of the rice-inserted DNA sequence is compared with the known amino acid sequence of the protein that is the α subunit of Arabidopsis ASA, By determining the sex, it can be specified that the phage DNA recovered above is a DNA fragment containing a DNA sequence corresponding to the gene for the α subunit of rice ASA.

  Therefore, the inserted DNA fragment determined to contain the DNA sequence corresponding to the gene for the α subunit of rice ASA can be obtained by cutting out the phage DNA of the collected phage selected as described above with a restriction enzyme.

(5) Cloning of cDNA corresponding to α-subunit gene of rice ASA As described above, a DNA fragment containing the DNA sequence corresponding to the α-subunit gene of rice ASA was cut out from the phage and obtained. The obtained DNA is inserted into an EcoRI cleavage site of a plasmid vector p Bluescript II SK (+) and ligated to construct a recombinant plasmid vector. Escherichia coli XL1-Blue MRF ′ is transformed with the thus constructed recombinant plasmid vector. By culturing and growing the E. coli transformant thus obtained, the above-described recombinant plasmid carrying a DNA fragment containing a DNA sequence corresponding to the α subunit gene of rice ASA can be cloned. Therefore, a DNA fragment containing the DNA sequence corresponding to the gene for the α subunit of rice ASA can be cloned.

  In the present invention, a DNA sequence obtained by cloning in Escherichia coli ligated to the plasmid vector p Bluescript II SK (+) as described above and containing the DNA sequence corresponding to the gene for the α subunit of rice ASA is contained therein. As DNA fragments, two types of DNA fragments having different numbers of bases were obtained. The small-sized DNA fragment obtained here is tentatively referred to herein as a DNA fragment X, and the large-sized DNA fragment is tentatively referred to herein as a DNA fragment Y.

(6) Sequence analysis of cloned DNA
(i) Two types of DNA fragments containing the DNA sequence corresponding to the gene for the α subunit of rice ASA by the restriction enzyme EcoRI from the cloned recombinant plasmid described above, DNA fragment X and DNA fragment Y, Cut out separately. When each of the excised DNA fragment X and the DNA fragment Y is treated with a commercially available nucleotide sequencing kit, a DNA fragment X or a DNA fragment Y containing a DNA sequence corresponding to the gene for the α subunit of rice ASA is obtained. The entire nucleotide sequence can be determined. For the above DNA fragment X, the DNA sequence having the nucleotide sequence determined in this way and encoding the α subunit of the first isozyme of rice ASA is the nucleotide sequence described in SEQ ID NO: 1 in the sequence listing described below. And consists of 1734 bases. This DNA sequence is named the sequence OSASA-1 sequence. This DNA sequence OSASA-1 is an example of the DNA according to the first invention.

  In addition, a DNA sequence having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing obtained in Example 1 described below and corresponding to the α subunit gene of the first isozyme of rice ASA is contained therein. The DNA fragment X contained was inserted into an EcoRI cleavage site of a p Bluescript II SK (+) plasmid vector (manufactured by Stragene) and ligated. Escherichia coli XL1-Blue MRF ′ (Escherichia coli) XL1-Blue MRF ′ (OS-asa1) transformed by introducing the thus obtained recombinant plasmid vector (designated vector pOSASA-1) was used. ) And deposited with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Tsukuba, Ibaraki, Japan, on August 18, 1997, under the accession number FERM P-16388. Further, Escherichia coli OS-asa1 has been deposited with the aforementioned laboratory under the terms of the Budapest Treaty under accession number FERM BP-6453 since August 7, 1998.

  The DNA according to the first invention uses the nucleotide sequence of the DNA elucidated in the present invention as a guideline to obtain a large amount of a protein that is the α subunit of the first isozyme of rice ASA by chemical synthesis. It is useful in making it possible and can contribute to advance enzymological studies on the protein that is the α subunit of the first isozyme of rice ASA.

  (ii) Further, for the DNA fragment Y, the DNA sequence having the nucleotide sequence determined as described above and encoding the α subunit of the second isozyme of rice ASA is described in SEQ ID NO: 10 in the sequence listing. This DNA sequence was designated as OSASA-2 sequence and had 1821 bases. This DNA sequence OSASA-2 is an example of the DNA according to the second invention.

  In addition, the DNA sequence having the nucleotide sequence of SEQ ID NO: 10 obtained in Example 1 described below and corresponding to the α subunit gene of the second isozyme of rice ASA was internally stored. Was inserted and ligated into the EcoRI cleavage site of the pBluescript II SK (+) plasmid vector. The Escherichia coli XL1-Blue MRF '(Os-asa2) was transformed with the thus obtained recombinant plasmid vector (designated as vector pOSASA-2) and transformed into Escherichia coli XL1-Blue MRF'. Was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-16853 on June 18, 1998, and from August 7, 1998, under FERM BP-6454 under the terms of the Budapest Treaty. Deposited by number.

  On the other hand, in general, one or more amino acids in the amino acid sequence that constitutes a protein having a biological activity is deleted, and / or is replaced with another amino acid, and (or) It is widely recognized by those skilled in the art that the amino acid sequence formed by adding or removing one or more amino acids to the amino acid sequence may maintain the physiological activity of the protein having the original amino acid sequence. Have been. The DNA of the first invention can be a DNA encoding a protein having the activity of the α subunit of the first isozyme of rice ASA even after a part or a part of the base sequence is modified. It is.

  In other words, the DNA according to the first aspect of the present invention has a rice sequence even after one or more bases in its base sequence, for example, 1, 2 or 3 to 10 bases have been modified to another base. Can retain the ability to encode a protein having the activity of the α subunit of the first isozyme of ASA.

  Accordingly, a protein having a modified amino acid sequence formed by substituting one amino acid in the amino acid sequence shown in SEQ ID NO: 2 with another amino acid, wherein the α-sub of the first isozyme of anthranilate synthase It is possible to provide a DNA encoding a protein having unit activity.

  The DNA modified from the DNA according to the first invention encodes, for example, a protein having an amino acid sequence in which an amino acid at a specific site in a protein encoded by the DNA is replaced with another amino acid by site-directed mutagenesis. Thus, it is obtained by modifying the nucleotide sequence of the DNA of the first invention.

  Further, a cell having a DNA fragment containing the DNA of the first invention is subjected to a mutation treatment, and a stringent condition is applied to the DNA having the nucleotide sequence described in SEQ ID NO: 1 in the mutated cell, for example. The above-mentioned modified DNA can also be obtained by selecting a DNA that can hybridize below but has a nucleotide sequence partially different from the nucleotide sequence of SEQ ID NO: 1. The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid to the DNA of the first invention is formed and a non-specific hybrid is not formed. This condition is difficult to clearly specify by numerical values, but as an example, two nucleic acids having a high homology, DNAs having a high homology, for example, a DNA having a homology of 98% or more Are allowed to hybridize, but do not allow nucleic acids having lower homology to hybridize.

  In the third invention (the invention according to claim 1 of the claims), the protein is a protein having the amino acid sequence of SEQ ID NO: 13 in the sequence listing, and the α-form of the first isozyme of rice anthranilate synthase DNA is provided which encodes a protein having the activity of a subunit, but which is insensitive to feedback suppression by tryptophan.

  Specific examples of the above-mentioned DNA according to the third invention include those prepared by the method described in Example 2 described below, named as modified D sequences in Example 2, and described in SEQ ID NO: 12 in the sequence listing. The DNA sequence according to claim 2, which has a base sequence.

  The above-mentioned DNA having the nucleotide sequence of SEQ ID NO: 12 in the sequence listing and named as the modified D sequence has the activity of the α subunit of the first isozyme of rice ASA, but is not susceptible to feedback suppression by tryptophan. DNA encoding the above protein that is insensitive.

  In the present specification, simply referred to as “ASA α-subunit gene” constitutes a holoenzyme having anthranilate synthase activity in cooperation with the β-subunit constituting rice anthranilate synthase. Refers to the DNA encoding the α-subunit protein to be obtained. The term “α subunit” used herein means one or both α subunits of the first and second isozymes of rice ASA.

  A specific example of the third novel DNA according to the present invention is, as described above, a DNA sequence prepared by the method described in Example 2 described below and designated as a modified D sequence.

  The DNA sequence designated as the modified D sequence is a DNA having the base sequence described in SEQ ID NO: 12 in the sequence listing. This modified D sequence is the 967th, 968th, and 969th GAC sequence (codon encoding aspartic acid) in the DNA of the first invention having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. It corresponds to a DNA modified such that one base G (guanine) at position 967 is replaced with one A (alanine) and the sequence (codon encoding asparagine) is located at position 967 of the base sequence.

  The protein encoded by this modified D sequence has the amino acid sequence of SEQ ID NO: 13 in the sequence listing and has the activity of the α subunit of the first isozyme of rice ASA.

  Further, a protein encoded by the above-mentioned modified D sequence, which is a specific example of the DNA of the third present invention, is characterized in that ASA involved in the tryptophan biosynthetic pathway inhibits feedback inhibition of enzyme activity by tryptophan as a biosynthetic product. It is a novel protein whose enzyme activity has been modified so as not to be affected by it. The modified DNA encoding this novel protein is used in plant transformation for the purpose of increasing the tryptophan content of the plant, as described below.

  In general, as a method of modifying a base sequence in a partial region of a DNA sequence, (Kunkel method `` Methods in Enzymology '' Vol. 154, No. 367), and oligonucleotide-direct dual amber method, and other methods The method is known.

  In order to modify the first isozyme α subunit of ASA encoded by the DNA of the first invention so as not to receive feedback inhibition of enzyme activity by tryptophan, a part of the base sequence of the DNA of the first invention is modified. As a policy of modification, the present invention is described with reference to a report on a known ASA gene of a mutant Arabidopsis which is resistant to a tryptophan analog described in “Plant Physiology”, Vol. 110, pp. 51-59, 1996. The present inventors have found that a modified DNA prepared by replacing guanine (G) with adenine (A) at position 967 of the nucleotide sequence of DNA (OSASA-1 sequence) described in SEQ ID NO: 1 in the sequence listing. I got the anticipated idea that it would be effective for the purpose of the period.

  Based on this idea, the present inventors have conducted various studies, and as a result of many trials and errors, the DNA sequence corresponding to the gene of the α subunit of the first isozyme of rice ASA (that is, OSASA- A recombinant plasmid vector (hereinafter, referred to as pOSASA-1) obtained by inserting and ligating a DNA fragment containing (1 sequence) between the EcoRI cleavage sites of the plasmid vector p Bluescript II SK (+) using a ligation kit. And found that it was suitable as a starting material for the preparation of the novel modified DNA for the purpose of the third invention.

  For such a purpose, the present inventors studied to prepare a novel modified DNA from the above-mentioned starting material, the recombinant plasmid vector pOSASA-1, according to the PCR method. As primers that can be appropriately used for the PCR method, four types of primers, namely, a primer OSASN1 consisting of an oligonucleotide having a base sequence described in SEQ ID NO: 16 in the sequence listing below, and a base described in SEQ ID NO: 17 A primer OSASN2 consisting of an oligonucleotide having a sequence, a primer OSASC1 consisting of an oligonucleotide having a base sequence of SEQ ID NO: 18, and a primer OSASC2 consisting of an oligonucleotide having a base sequence of SEQ ID NO: 19, obtained by chemical synthesis. Produced.

  By using the above-mentioned recombinant plasmid vector pOSASA-1 and the above-mentioned primers composed of the four kinds of synthetic oligonucleotides, the method described in Example 2 described below is carried out. The DNA of SEQ ID NO: 12, which is a specific example of the DNA, that is, the modified “D sequence” was successfully obtained.

  Next, a procedure which can be suitably used to prepare a DNA fragment containing the above-mentioned modified D sequence (claim 2) which is a specific example of the DNA according to the third invention, and which is exemplified in Example 2 described below, The method of altering the resulting DNA is briefly described here.

(1) Cloning of the DNA of the first invention The DNA of the first invention consisting of 1734 bases described in SEQ ID NO: 1 in the sequence listing, that is, a DNA fragment containing the OSASA-1 sequence, Using the ligation kit, the above-mentioned recombinant plasmid vector pOSASA-1 is obtained by inserting and ligating between the EcoRI cleavage sites of the vector p Bluescript II SK (+). This vector pOSASA-1 is introduced into Escherichia coli XL1-Blue MRF ', and the transformed Escherichia coli thus obtained is propagated. A large amount of the recombinant plasmid vector pOSASA-1 is obtained from the grown Escherichia coli cells by extraction in a conventional manner. By this operation, the DNA sequence according to the first invention, that is, the OSASA-1 sequence is cloned.

(2) Construction of CR primer
Using a DNA synthesizer (Model-391, manufactured by Applied Biosystems), four types of oligonucleotides having the following nucleotide sequences are synthesized as primers. That is, as described below, a primer OSASN1 comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 in the sequence listing, a primer OSASN2 comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 17, and SEQ ID NO: 18 A primer OSASC1 consisting of an oligonucleotide having the base sequence of SEQ ID NO: 19 and a primer OSASC2 consisting of an oligonucleotide having the base sequence of SEQ ID NO: 19 are produced by chemical synthesis.

(i) primer OSASAN1 (primer having a base sequence described in SEQ ID NO: 16 in the sequence listing and described below):
5'-GAGTCAGTTGACGAAGCGTATGAGG-3 '
(ii) primer OSASAN2 (primer having a base sequence described in SEQ ID NO: 17 in the sequence listing and described below):
5'-GTACATTTGCTAACCCCTTTGAGG-3 '
(iii) primer OSASAC1 (primer having a base sequence described in SEQ ID NO: 18 in the sequence listing and described below):
5'-CAAAGGGGTTAGCAAATGTACGC-3 '
(iv) Primer OSASAC2 (primer having a base sequence described in SEQ ID NO: 19 in the sequence listing and described below):
5'-GTTCAACGTTCATCAGTTTCTCCACC-3 '
(3) Propagation and Recovery of Required DNA Fragments by PCR In order to obtain the required DNA fragments by proliferating, the first step of the PCR method involves two reactions, namely the following (A) reaction and (B) reaction. I do.

  The (A) reaction was carried out using the above-mentioned recombinant plasmid vector pOSASA-1 used as a template, the above-mentioned primer OSASAN1 (synthetic oligonucleotide of SEQ ID NO: 16) used as a primer, and the primer OSASAC1 (synthetic oligonucleotide of SEQ ID NO: 18). A nucleotide, and a GTT at the 8th to 10th position from the 5'-end is a primer capable of inducing a modified portion AAC of the modified D sequence) and a normal reaction solution for PCR (Tris-HCl, MgCl2, KCl, 4 types) Containing deoxynucleotide triphosphate (dNTP) and LaTaq DNA polymerase), followed by performing a growth reaction.

  By the reaction (A), a DNA fragment having a base sequence in a certain region of the modified D sequence (referred to as DNA fragment-A) is generated by a proliferation reaction.

  (B) The reaction was performed using the recombinant plasmid vector pOSASA-1 used as a template, the primer OSASAC2 (synthetic oligonucleotide of SEQ ID NO: 19) used as a primer, and the primer OSASAN2 (synthetic oligonucleotide of SEQ ID NO: 17). A nucleotide, a 12th to 14th AAC from the 5′-end is a primer capable of inducing a modified AAC of the modified D sequence) and (A) to the same normal reaction solution for the PCR method used in the reaction, and then Performing a proliferative reaction.

  By the reaction (B), a DNA fragment containing a base sequence in a certain region of the modified D sequence (referred to as DNA fragment-B) is produced by a proliferation reaction. The above-mentioned proliferation reaction by the PCR method can be performed using a commercially available PCR reaction device.

  After completion of the growth reaction, the growth reaction solution of the above reaction (A) was fractionated by low-melting point agarose electrophoresis, and a band containing a 268 bp (base pair) DNA fragment-A as a growth product was removed from the agarose gel. Cut out. Similarly, the proliferation reaction solution of the above-mentioned reaction (B) is fractionated by low-melting point agarose electrophoresis, and a band containing a DNA fragment-B of 336 bp (base pair) is cut out from the agarose gel.

  Next, the two gel slices are purified using a DNA purification kit, for example, Genclean II (manufactured by Funakoshi), thereby recovering a purified DNA fragment-A and a purified DNA fragment-B.

  Further, as a second step of the PCR method, a DNA sequence having a nucleotide sequence obtained by substituting guanine at position 967 of the nucleotide sequence described in SEQ ID NO: 1 with adenine (that is, the nucleotide sequence of SEQ ID NO: 12 A 583 bp (base pair) DNA fragment (C fragment) corresponding to a partial region of the modified D sequence fragment according to the third aspect of the present invention.

  For this purpose, a purified product of the DNA fragment-A (sequence length 268 bp), which is a by-product of the reaction (A), used as a template, and a DNA fragment-, a by-product of the reaction (B), Add purified product of B (sequence length 336 bp) to a normal growth reaction solution for PCR (containing Tris-HCl, MgCl2, KCl, four dNTPs, and La Taq DNA polymerase). Do. After completion of the reaction, the reaction solution is fractionated by low-melting point agarose electrophoresis, and a band containing a desired DNA fragment having a sequence length of 583 bp (referred to as DNA fragment-C) is cut out from the agarose gel.

  The gel slice is purified using a DNA purification kit, for example, Genclean II kit (Funakoshi) to obtain a purified DNA fragment-C. This DNA fragment-C has a nucleotide sequence corresponding to the partial region of the DNA fragment of the modified D sequence according to the third invention, and has a structure containing a cleavage site for restriction enzymes AflII and BglII therein. .

  Further, this DNA fragment-C contains a substitution site of the target nucleotide sequence by digestion with restriction enzymes AflII and BglII, and a restriction enzyme AflII cleavage site at the 5′-terminal and a restriction site at the 3′-terminal. A 288 bp DNA fragment-α having a BglII enzyme cleavage site can be obtained.

(4) Cloning of DNA fragment containing modified D sequence Next, using the DNA fragment-C obtained in the above section (3), a DNA fragment containing the desired modified D sequence is obtained.

  For that purpose, first, the above DNA fragment-C is treated with a restriction enzyme AflII and a restriction enzyme BglII. Thus, a DNA fragment having a part of the modified D sequence having an AflII cleavage site on the 5′-side and a BglII cleavage site on the 3′-side is cut out from the DNA fragment-C. Thus, a sample (i) of a DNA fragment containing the DNA sequence corresponding to the target modified D sequence is obtained.

  Next, by treating the plasmid vector pOSASA-1 containing the DNA described in SEQ ID NO: 1 of the sequence listing (that is, the OSASA-1 sequence) with the restriction enzymes AflII and BglII, a BglII cleavage site was placed on the 5′-side. Having an AflII cleavage site on the 3′-side, and a partial region from the 1st base to the 933th base and the 1220th base to the 1734th base of the DNA described in SEQ ID NO: 1. A plasmid fragment (ii) containing

  The thus obtained AflII-BglII-plasmid fragment (ii) is mixed with a DNA fragment sample (i) containing a DNA sequence corresponding to the modified D sequence, and then subjected to a ligation reaction using a DNA ligation kit. And a recombinant plasmid containing the modified D sequence of SEQ ID NO: 12 (hereinafter, referred to as pBluescript-DNA-D plasmid) can be prepared.

  This recombinant plasmid, i.e., the pBluescript-DNA-D plasmid was introduced into Escherichia coli XL1-Blue MRF 'for transformation, and the transformed Escherichia coli thus obtained (referred to as Escherichia coli XL1-Blue MRF' / pBluescript-DNA-D). : Deposit No. FERM BP-6451) is cultured and grown in a liquid medium. Escherichia coli cells grown in large quantities contain the recombinant plasmid described above, ie, the Coppii of the pBluescript-DNA-D plasmid. In this way, the modified D sequence can be cloned. From the Escherichia coli culture cells, a plasmid containing the modified D sequence is extracted and taken by a conventional method.

(5) Recovery of DNA fragment of modified D sequence The plasmid containing the modified D sequence obtained in (4) above is digested by treating with a restriction enzyme EcoRI. Thus, a digestion reaction containing a DNA fragment containing a modified D sequence having the base sequence ATG at the 5′-side end adjacent to the EcoRI cleavage site and having an extension having the EcoRI cleavage site also at the 3′-side end A liquid can be obtained.

  The digestion reaction solution is fractionated by low melting point agarose electrophoresis, and the band containing the DNA fragment is cut out from the agarose gel. The excised agarose gel piece is dissolved in a TE buffer, and the resulting solution is extracted with phenol, whereby the DNA fragment is recovered in a phenol extract. The phenol extract containing the DNA fragment is mixed with a 3M aqueous sodium acetate solution and ethanol, and the mixture is left at 20 ° C. for about 6 hours, and further centrifuged at a low temperature to precipitate the DNA fragment. When this is dried under reduced pressure, a DNA fragment containing the desired modified D sequence therein is obtained as a powder. The powder of the DNA fragment containing the modified D sequence is soluble in water.

  In the above description, a DNA fragment containing a modified D sequence, which is a specific example of the DNA according to the third invention, was produced by a method utilizing genetic engineering techniques. However, it can also be produced by a conventionally known polynucleotide chemical synthesis method with reference to the nucleotide sequence of SEQ ID NO: 12 in the sequence listing.

  The method of implementing the third invention has been described for the case where the guanine at position 967 in the nucleotide sequence of the DNA of SEQ ID NO: 1 is modified by adenine according to the first invention. However, if the DNA described in SEQ ID NO: 1 according to the first invention is used as a template and used as a primer in combination with several types of synthetic oligonucleotides having appropriately devised base sequences, the SEQ ID NO: 1 according to the first invention is used. It is possible to produce another modified DNA by replacing a base at a different position from the 967th position of one DNA with another base.

  Furthermore, the present inventors have proceeded with further research. As a result, the plant is transformed by introducing a foreign gene into the plant, and by utilizing a conventionally known biotechnology technique for expressing the foreign gene in the obtained transgenic plant, the first is achieved. Neither the novel DNA encoding the α subunit of the first isozyme of rice ASA according to the invention nor the novel modified DNA created from the DNA encoding the α subunit of the first isozyme of rice ASA according to the third invention, It has been found that they can be introduced into a plant after being integrated into a recombinant vector and can be expressed in the plant.

  Therefore, in the fourth invention (the invention of claim 3 of the claims), the present invention has the activity of the α subunit of the first isozyme of rice anthranilate synthase but is insensitive to feedback suppression by tryptophan. A plant cell transformed with a recombinant vector carrying a DNA having the nucleotide sequence of SEQ ID NO: 12 (ie, DNA having a modified D sequence), which is a DNA according to the third invention encoding a certain protein. And a transgenic plant, characterized in that the introduced DNA can be expressed.

  In addition, it has been found that when the transformed plant according to the fourth aspect of the present invention is a plant that can produce seeds when cultivated, the plant can be cultivated under ordinary conditions to harvest the seeds of the plant.

  Therefore, in the fifth invention, (the invention of claim 4 of the present invention) which is obtained by introducing a recombinant vector carrying the modified DNA according to the third invention into a plant cell and the DNA is expressed Provided is a seed of a transformed plant, which is obtained by cultivating a transformed plant that is possible, and further, being a seed collected from a fruited plant by the cultivation.

  Furthermore, according to the sixth invention (the invention of claim 5 of the claims), a DNA fragment having the base sequence of SEQ ID NO: 12 in the sequence listing and carrying a DNA sequence designated as a modified D sequence is An integrated recombinant vector is provided which is capable of expressing the DNA sequence in a host cell.

  According to the seventh invention (the invention of claim 6 of the claims), a DNA fragment having the base sequence of SEQ ID NO: 12 and carrying a DNA sequence designated as D sequence is incorporated. Escherichia coli transformed with the recombinant vector, which is capable of expressing the DNA sequence in a host cell, is provided as a novel microorganism.

  An example of the transformed Escherichia coli according to the seventh invention may be the aforementioned Escherichia coli XL1-Blue MRF '/ p Bluescript-DNA-D deposited under the accession number FERM BP-6451 under the terms of the Budapest Treaty.

  When a plant is transformed by using the DNA according to the first invention or the DNA according to the third invention as a foreign gene, the types of plants that can be transformed are diverse, and foreign plants must be used for plant transformation. The method of introducing the DNA of the present invention as a gene into a plant can be any of various conventionally known biotechnology techniques performed for the purpose.

A method which can be suitably carried out to introduce the DNA of the first invention or the DNA of the third invention as a foreign gene into a rice plant and is exemplified in Example 3 to be described later is summarized below.

(a) Preparation of a recombinant vector for introducing a foreign gene Conventionally known corn ubiquitin promoter, 1st intron and NOS terminator, and a phosphinothricin resistance gene, and an ampicillin resistance gene that is effective only in microorganisms When treated with the restriction enzymes BamHI and SacI in a buffer, the known plasmid vector pUBA (Plant Molecular Biology, Vol. 18, No. 4, pp. 675-689, 1992) containing the 1st intron downstream of the ubiquitin promoter. , A vector fragment having a size of about 4.8 Kb, which is cleaved at a BamHI cleavage site downstream of and a SacI cleavage site upstream of a NOS terminator, can be obtained.

  The aqueous solution of the vector DNA fragment is mixed with the aqueous solution of the DNA fragment containing the DNA of the present invention, and the mixture is treated with a DNA ligation kit to perform a ligation reaction. According to this, a recombinant vector having a DNA fragment containing the DNA of the present invention inserted and ligated between the ubiquitin promoter and the NOS terminator region of the vector DNA fragment can be obtained.

  The selected recombinant vector is introduced into E. coli JM109 to obtain transformed E. coli.

  The resulting transformed Escherichia coli is inoculated and cultured in a medium containing the antibiotic ampicillin to obtain several ampicillin-resistant colonies of Escherichia coli. These colonies are further separately grown in a medium containing ampicillin.

  The plasmid is recovered from each ampicillin-resistant E. coli grown in each colony. The recovered plasmids include various plasmids having different inserted DNA directions. Plasmids recovered for each colony were digested with appropriate restriction enzymes and digested with various DNA fragments cut out and subjected to agarose gel electrophoresis to analyze the size and base sequence of those DNA fragments. An appropriate plasmid (about 6.5 Kb in size) in which the DNA of the present invention is inserted and ligated in the normal direction downstream of the ubiquitin promoter of the recombinant plasmid can be selected.

  A plasmid incorporating the DNA of SEQ ID NO: 1 of the sequence listing of the first invention is referred to as a vector pUBdW1, and a plasmid incorporating the DNA of SEQ ID NO: 10 of the sequence listing of the second invention is referred to as a vector pUBdW2, Further, a plasmid in which the DNA of the modified D sequence of SEQ ID NO: 12 in the sequence listing of the third invention is integrated is referred to as a vector pUBdD.

  In addition, the known plasmid vector plG121-Hm (Plant Cell Phisiol, Vol. 31, pp. 805-813, 1990) containing the hygromycin resistance gene as a recombinant vector for gene transfer in the Agrobacterium method was restricted in the buffer. When treated with the enzymes PmeI and SacI, a vector fragment having a size of about 9.8 Kb can be obtained.

  Further, the above plasmid vector pUBdW1, pUBdW2 or pUBdD is treated with SphI and SacI in a buffer, and the SphI cleavage site is blunted to thereby remove the ubiquitin promoter, a vector fragment having the DNA of the present invention connected downstream of the 1st intron. Obtainable.

  Using each of these vector fragments to which the DNA of the present invention is ligated, the DNA of the present invention is inserted in a normal orientation by performing the ligation reaction, the production of transformed E. coli, and the collection of the plasmid in the same manner as described above. A linked recombinant vector for gene transfer by the Agrobacterium method can be obtained.

  A plasmid in which the DNA of SEQ ID NO: 1 in the sequence listing of the first invention is incorporated, referred to as a vector pUb-OSASAW1, and a plasmid in which the DNA of SEQ ID NO: 10 in the later-described sequence listing of the second invention is incorporated. The plasmid in which the DNA of the fragment of the modified D sequence of SEQ ID NO: 12 in the sequence listing of the third invention is incorporated is referred to as a vector pUb-OSASA1D.

(b) Preparation of rice callus Rice husks are removed from the mature rice seeds, and the obtained rice seeds with hulls are sterilized with an ethanol solution, then sterilized with a dilute aqueous solution of sodium hypochlorite, and further washed with sterilized water.

  The rice seed with the hulls is placed on a callus formation medium composed of MS medium supplemented with sucrose, 2,4-PA as a plant hormone, and agar. When cultured at 28 ° C. for 15 to 18 hours per day for 15 to 18 hours under sunlight of 1500 to 2500 lux for 40 to 50 days, rice calli are formed. The callus is cut from the endosperm portion of the seed to obtain a callus.

(c) Introduction of a foreign gene into rice callus cells The recombinant vector prepared as described in the preceding section (a), into which the DNA of the present invention has been normally inserted and ligated (that is, the aforementioned vector pUb-OSASAW1 In order to introduce pUb-OSASAW2 or pUb-OSASA1D) into callus cells by the known Agrobacterium method, first, it is incorporated into Agrobacterium tumefaciens which is a host. Integration is performed by a known electroporation method (plant tissue culture, Vol. 10, No. 2, pp. 194-196, 1993).

  Callus cells obtained as shown in the above (b) and Agrobacterium bacteria thus obtained were prepared by a known method (Cell Engineering Separate Volume, Experimental Protocol for Model Plants, pp. 93-98, 1996). The co-culture is performed according to the method described in (1) published by Shujunsha, and the DNA of the present invention can be introduced into rice callus cells. Then, a plant cell which is resistant to hygromycin and transformed with the DNA of the present invention is obtained.

(d) Re-selection of transformed plant cells From the hygromycin-resistant transformed plant cells obtained as described above, only transformed plant cells containing the DNA of the present invention in a sufficiently effective amount as a foreign gene are used. Reselect.

  For this purpose, the former transformed cells were reconstituted by adding sucrose, 2,4-PA, gellite, and 5MT (ie, 5-methyltryptophan), which is an analog of tryptophan, to N6 medium. Implant on selection medium.

  The transplanted cells are cultured for 16 hours per day at 25-28 ° C. for 25-30 days while irradiating with 2000 lux light.

  Transformed plant cells containing the DNA of the present invention in a sufficiently effective amount as a foreign gene are resistant to 5MT and can grow even if 5MT acting as a cell growth inhibitor is present in the medium. 5MT-resistant cultured plant cells that can grow in the above-mentioned medium supplemented with 5MT are selected.

(e) Regeneration of Plants from Reselected 5MT-Resistant Transformed Plant Cells The 5HT-resistant cultured plant cells reselected as described above are converted into MS medium for plant tissue culture as sucrose and plant hormone. Of benzyladenine, naphthaleneacetic acid, and gellite.

  The transplanted cells are cultured at 25-28 ° C. for 25-30 days while irradiating with 2000 lux light for 16 hours per day. In this manner, shoots and roots can be regenerated from the cultured transformed plant cells by differentiation.

  After the regenerated buds and the sprouts containing the roots grow to a length of 10 to 30 mm, the sprouts are transplanted to a medium for acclimation obtained by adding sucrose and gellite to an MS medium. Furthermore, the plants are grown at 25-28 ° C. for 18-20 days while irradiating light of 2000 lux for 16 hours per day.

  Thus, the transformed plant can be regenerated. When the plant thus obtained is transplanted to soil in a greenhouse and cultivated under normal conditions, it grows up to normal length and can cultivate rice seeds after cultivation for 3 to 6 months.

(f) Confirmation of introduced foreign gene Green leaves are collected from the transformed rice plant regenerated as described above. The harvested leaves are frozen with liquid nitrogen and then crushed. DNA is extracted from the crushed leaves according to the method of J. Sambrook et al. (Molecular Cloning, 2nd edition, described in Cold Spring Habor Laboratory Press, 1989).

  On the other hand, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 14 in the sequence listing described below and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 are chemically synthesized and used as primers.

  Using the DNA extracted as described above from the regenerated rice plant as a template and using the above-mentioned two synthetic oligonucleotides as primers, the DNA is amplified by a known PCR method. The obtained amplification reaction solution is fractionated by agarose electrophoresis according to a conventional method, and contains DNA fragments corresponding to the introduced foreign gene DNA among various DNA fractions of DNA extracted from regenerated rice plants. Take a gel band.

  When the base sequence of the DNA fragment contained in this band is analyzed by a known Southern method, it can be confirmed whether or not it corresponds to the DNA of the present invention.

  Extraction and measurement of the content of tryptophan in a plant can be performed by a known method (Hopkins-Cole method, Biochemistry Experiment Course Vol. 11, published by Tokyo Chemical Dojinsha), which is described in Examples below. It can also be performed by such a method using HPLC. The extraction of tryptophan and the measurement of the content can be appropriately changed according to the target plant species, the target plant site, and the growth stage of the target plant.

  Next, a method for selecting a plant cell transformed by introducing the DNA of the present invention and a method for producing a transformed plant will be described.

(a) Preparation of recombinant vector for selection For production of transgenic plants, as a recombinant vector used in a method of direct introduction into plant cells, a conventionally known cauliflower mosaic virus 35S promoter and NOS When a known plasmid vector pBI221 (manufactured by Clontech) containing a terminator and an ampicillin resistance gene expressed only in a microorganism is treated with restriction enzymes XbaI and SacI in a buffer, an XbaI cleavage site downstream of the 35S promoter is obtained. And a vector fragment of about 3.8 Kb in size cut at the SacI cleavage site upstream of the NOS terminator can be obtained.

  The aqueous solution of the vector DNA fragment is mixed with an aqueous solution of a DNA fragment containing the DNA (modified D sequence) of the third invention, and the mixture is treated with a DNA ligation kit to perform a ligation reaction. According to this, it is possible to obtain a recombinant vector in which a DNA fragment containing the DNA of the third invention (modified D sequence) is inserted and ligated between the 35S promoter and the NOS terminator region of the vector DNA fragment.

  The selected recombinant vector is introduced into E. coli JM109 to obtain transformed E. coli.

  The resulting transformed Escherichia coli is inoculated and cultured in a medium containing the antibiotic ampicillin to obtain several ampicillin-resistant colonies of Escherichia coli. These colonies are further separately grown in a medium containing ampicillin.

  The plasmid is recovered from each ampicillin-resistant E. coli grown in each colony. The recovered plasmids include various plasmids having different inserted DNA directions. Plasmids recovered from each colony were digested with appropriate restriction enzymes and digested with various DNA fragments and subjected to agarose gel electrophoresis to analyze the size and base sequence of these DNA fragments. An appropriate plasmid (about 5.6 Kb in size) can be selected in which the DNA of the third invention (modified D sequence) is inserted in the normal direction downstream of the 35S promoter of the recombinant plasmid and ligated. Also, for the production of transformed plants, the above-mentioned vector pUBdD as a recombinant vector used in a method of direct introduction into plant cells, or the above-mentioned vector pUb-OSASA1D as a recombinant vector used in an Agrobacterium method It can be used as a recombinant vector for selection.

(b) Preparation of rice callus Rice husks are removed from the mature rice seeds, and the obtained rice seeds with hulls are sterilized with an ethanol solution, then sterilized with a dilute aqueous solution of sodium hypochlorite, and further washed with sterilized water.

  The rice seed with the hulls is placed on a callus formation medium composed of MS medium supplemented with sucrose, 2,4-PA as a plant hormone, and agar. When cultured at 28 ° C. for 15 to 18 hours per day for 15 to 18 hours under sunlight of 1500 to 2500 lux for 40 to 50 days, rice calli are formed. The callus is cut from the endosperm portion of the seed to obtain a callus.

(c) Introduction of the recombinant vector for selection into rice callus cells The DNA of the present invention, prepared as described in (a) above, was inserted and ligated into a known recombinant vector pUBdD, which is known in the art. The callus cells are introduced by a direct introduction method using whiskers. In addition, the recombinant vector pUb-OSASA1D for selection is introduced into callus cells by a known Agrobacterium method (Cell Engineering Separate Volume, Experimental Protocol for Model Plants, pp. 93-98, 1996, published by Shujunsha). .

(d) Selection of transformed plant cells Callus cells containing the recombinant vector for selection obtained as described above, sucrose in N6 medium, 2,4-PA, gellite and 10 mg of a tryptophan analog as a selection drug. / L to 200 mg / L to 200 mg / L, preferably 30 mg / L to 50 mg / L. Further, the cells are cultured at 25 to 28 ° C. for 20 to 60 days, preferably 25 to 30 days, while irradiating them in the dark or at 2,000 lux of light for 16 hours per day.

  Thus, plant cells which are resistant to the tryptophan analog and are transformed with the recombinant vector for selection are selected.

(e) Selection of transformed plants From the transformed plant cells resistant to tryptophan analogs obtained as described above, in order to obtain transformed plants, the former transformed cells were converted into MS medium as sucrose and plant hormone. A benzyladenine, naphthalene acetic acid, gellite and a tryptophan analog compound as a selection agent are added to a selective differentiation medium for plant regeneration by adding 10 mg / L to 200 mg / L, preferably 30 mg / L to 50 mg / L.

  The transplanted cells are cultured at 25-28 ° C. for 25-30 days while irradiating with 2000 lux light for 16 hours per day. In this manner, shoots and roots can be regenerated from the cultured transformed plant cells by differentiation.

  After the regenerated buds and the sprouts containing the roots grow to a length of 10 to 30 mm, the sprouts are transplanted to a medium for acclimation obtained by adding sucrose and gellite to an MS medium. Furthermore, the plants are grown at 25-28 ° C. for 18-20 days while irradiating light of 2000 lux for 16 hours per day. Thus, the transformed plant can be regenerated.

  The DNA according to the third aspect of the present invention can be used as a foreign gene not only for the rice plant described above but also for other types of plants to transform the plant.

  Further, the DNA of the third invention can be used not only for increasing and selecting the tryptophan content of the rice plant described above, but also for other types of plants.

  Accordingly, a method for introducing the DNA of the third invention into a general plant, a method for increasing the tryptophan content, and a method for selecting transformed cells and transformed plants will now be described in general terms.

  Plants into which the DNA of the third invention can be introduced are not particularly limited, and include, for example, rice, corn, wheat, barley, and other monocotyledonous plants, as well as tobacco, soybean, cotton, tomato, Chinese cabbage, Examples include dicotyledonous plants such as cucumber and lettuce. First, it is convenient to prepare cultured cells from these plants and introduce the DNA of the third invention as a foreign gene into the obtained cultured cells.

  The production of the cultured cells used for introducing the DNA of the present invention may be any explant derived from a plant, for example, scutellum, growth point, pollen, anther, leaf blade, stem, petiole, and root-derived explants. Cultures can be used.

  The explants described above, a medium for callus formation, such as MS medium (Murashige et al., Physiologia Plantarum, 1962, Vol. 15, pp. 473-497), or R2 medium (Ojima et al., `` Plant and Cell Physiology, 1973, Vol. 14, pages 1113-1121, or N6 medium (Chu et al., 1978, `` In Proc.Symp.Plant Tissue Culture, Science Press Peking, '' pages 43-50, etc. In a medium for plant tissue culture containing an inorganic salt component and vitamins as components, as a plant hormone, for example, 2,4-PA (2,4-dichlorophenoxyacetic acid) 0.1 to 5 mg / liter, and as a carbon source, For example, it is convenient to introduce the DNA of the present invention into cultured cells obtained by placing the cells on a medium containing 10 to 60 g / liter of sucrose and 1 to 5 g / liter of gellite and culturing them. is there.

  Plant cells to which the DNA of the third invention is introduced include, for example, dedifferentiated cultured cells such as callus and suspension cells or somatic embryos, cultured cells such as shoot primordia, or plant tissues such as leaves, roots, Preference is given to callus cells and suspension cells made from cells such as stems, embryos, growth points and the like.

  When the explant is placed on a callus-forming medium to prepare cultured cells for introducing the DNA of the third invention, the culture period until the cultured cells for introducing the DNA of the present invention is obtained is particularly limited. It is not done. However, since it is necessary to regenerate the transformed plant, it is possible to regenerate the plant from the cultured cells, that is, to obtain the plant within the period in which the plant has the ability to regenerate the plant. It is important to use cultured cells that have been used.

  The cultured cells for introducing the DNA of the third invention may be suspension cells cultured in a liquid medium as long as the cells have a plant regeneration ability.

  In order to introduce the DNA of the third invention into plant cells, it is necessary to first prepare a recombinant vector in which the DNA of the third invention has been incorporated into an expression vector. In the recombinant vector, the DNA of the present invention is arranged downstream of the expression promoter, and the terminator is further arranged downstream of the DNA of the third invention so that the DNA of the present invention introduced into the plant is expressed in the plant. It must be a recombinant vector adjusted to The recombinant vector used for this purpose can be any of various vectors used for normal plant transformation, depending on the type of the method for introducing the plant. For example, when using a direct DNA transfer method to plant cells by an electroporation method, a particle gun method, a whisker method, or the like, a plasmid vector that can be propagated in Escherichia coli such as a pUC system or a pBR322 system should be used. When the Agrobacterium method is used, a plasmid vector such as a plan system is preferably used.

  The promoter arranged upstream of the DNA of the third invention in the recombinant vector is, for example, CaMV35S derived from cauliflower mosaic virus “The EMBO Journal”, Vol. 6, pp. 3901-3907, 1987, or No. 6-315381, corn ubiquitin promoter [Japanese Unexamined Patent Publication No. 2-79983], and phaseolin promoter "Plant Cell, Volume 1, pages 839 to 853, 1989". The terminator arranged downstream of the DNA of the present invention is, for example, a terminator derived from a cauliflower mosaic virus or a terminator derived from a nopaline synthase gene [The EMBO J. Vol. 6, pp. 3901-3907, 1987]. However, any promoter or terminator that functions in a plant body may be used.

  In order to efficiently select a plant cell transformed by introducing the DNA of the present invention, the above-mentioned recombinant vector to be used should be introduced into a plant cell together with a plasmid vector containing an appropriate selection marker gene. Is preferred. The selectable marker gene used for this purpose is the hygromycin phosphotransferase gene, which is resistant to the antibiotic hygromycin, or the neomycin phosphotransferase gene, which is resistant to kanamycin or gentamicin, or the herbicide phosphinothricin Acetyltransferase gene [The EMBO Journal, Vol. 6, pp. 2513-2518, 1987, or JP-A-2-171188].

  Methods for introducing the DNA of the present invention into plants as a foreign gene include the Agrobacterium method [Bio / technology, Vol. 6, pp. 915-922, 1988], and the electroporation method [Plant cell Rep., No. 10]. Vol. 106-110, 1991], particle gun method [Theor. Appl. Genet., Vol. 79, pages 337-341, 1990], whisker method, but especially these It is not limited to.

  Next, in order to efficiently reselect a transformed cell containing the recombinant vector containing the DNA of the present invention in a sufficiently effective amount, the cells were cultured on a medium supplemented with a tryptophan analog which is a cell growth inhibitor. I do.

  As the tryptophan analog, for example, 5-methyltryptophan (5MT) can be added to the medium for reselection at a concentration of 10 mg / l to 1000 mg / l, preferably 20 mg / l to 100 mg / l.

  Next, a plant body is regenerated from a transformed plant cell containing a recombinant vector into which the DNA of the present invention reselected as described above has been incorporated as a foreign gene and a vector for a selection marker. Regeneration of the plant can be performed in a known manner. For example, the plant can be regenerated by placing the transformed plant cells re-selected as described above on a known plant regeneration medium and culturing them.

  The transformed cells placed in the medium for plant regeneration are cultured at a temperature of 15 to 30 ° C., preferably 20 to 28 ° C., for 500 to 2000 lux, preferably 800 to 1000 lux while irradiating with light. The culture is performed for up to 60 days, preferably 30 to 40 days.

  As a result, a plant transformed with the recombinant vector carrying the foreign gene comprising the DNA of the present invention can be regenerated from individual plant cells.

  The plant regenerated from the transformed cells is then cultured in a conditioned medium. After that, when the acclimated regenerated plants are grown under normal cultivation conditions in a greenhouse, the plants can mature and set fruit after 3 to 6 months of cultivation to obtain seeds.

  The presence of the introduced foreign gene in the transformed and cultivated transformed plant was determined by a known PCR method and Southern method (Southern, "J. Mol. Biol.," Vol. 98, No. 503- 517, 1975) can be confirmed by analyzing the nucleotide sequence of DNA in a plant.

  In this case, extraction of DNA from the transformed plant can be performed according to a known method of J. Sambrook et al. (“Molecular Cloning”, 2nd edition, Cold Spring Habor Laboratory Press, 1989).

  When the foreign gene consisting of the DNA of the present invention present in the regenerated plant is analyzed by the PCR method, the DNA extracted from the regenerated plant as described above is used as the template, and the modified DNA of the present invention is used as a template. Using a synthesized oligonucleotide having a base sequence appropriately selected in accordance with the above base sequence as a primer, these are mixed and added to a reaction solution for PCR to carry out an amplification reaction. In this amplification reaction, if the denaturation, annealing, and extension reactions of DNA are repeated several tens of times, an amplification product of a DNA fragment containing the DNA sequence of the present invention can be obtained.

  When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, various amplified DNA fragments are fractionated. A piece of a band agarose gel containing a DNA fragment recognized to contain a DNA sequence corresponding to the DNA of the present invention, which is an introduced foreign gene, is cut out. When the base sequence of the DNA sequence of the DNA fragment contained in the cut gel piece is analyzed by the Southern method, it can be confirmed whether or not the DNA sequence corresponds to the DNA of the present invention.

  Tryptophan analogs can be used as selection agents that can be used for the selection of transformed cells and the selection of transformed plants of the present invention.Specifically, 5-methyltryptophan (5MT), 4-methyltryptophan (4MT) ), 6-methyltryptophan (6MT), 7-methyltryptophan (7MT), 6-methylanthranilic acid (6MA), 5-methylanthranilic acid (5MA), 3-methylanthranilic acid (3MA), 5-fluoroanthranilic acid Biosynthetic intermediates of tryptophan analogs such as (5FA) and 6-fluoroanthranilic acid (6FA) can also be used.

  As described above, the novel modified DNA according to the third aspect of the present invention can be used as a foreign gene for transforming a plant or a plant cell having an enhanced tryptophan content by introducing it into a plant or a plant cell.

  Therefore, in the eighth invention (the invention of claim 7), a protein which is insensitive to feedback suppression by tryptophan and has the activity of the α subunit of the first isozyme of anthranilate synthase is used. A DNA encoding the DNA according to the third invention, which is a recombinant vector carrying a DNA having the base sequence of SEQ ID NO: 12 (DNA named as modified D sequence), wherein the DNA is a plant Introducing a recombinant vector that can be expressed in a plant cell callus, thereby obtaining a plant cell callus transformed with the DNA, and further regenerating the callus into a plant. Provided is a method for increasing the tryptophan content of

  In the ninth invention (the invention of claim 8), a protein which is insensitive to feedback suppression by tryptophan and has an activity of the α subunit of the first isozyme of anthranilate synthase is used. A DNA according to the third invention, which is a recombinant vector carrying a DNA having a base sequence of SEQ ID NO: 12 in the sequence listing (modified D sequence) and an antibiotic resistance gene. A recombinant vector that can be expressed in a plant is introduced into a plant cell, thereby imparting the plant cell with resistance to a tryptophan analog that inhibits the growth of the plant cell, and further expressing the resistance to the tryptophan analog. The present invention provides a method for selecting transformed plant cells, the method comprising selecting for cells that have been transformed.

  Furthermore, in the tenth invention (the invention of claim 9), the protein which is insensitive to feedback suppression by tryptophan and has the activity of the α subunit of the first isozyme of anthranilate synthase A DNA having the nucleotide sequence of SEQ ID NO: 12 in the Sequence Listing (modified D sequence) and a recombinant vector carrying an antibiotic resistance gene, A recombinant vector whose DNA can be expressed in a plant is introduced into a plant cell, thereby imparting the plant cell with resistance to a tryptophan analog that inhibits the growth of the plant cell, and expressing the resistance to the tryptophan analog. And regenerating a plant from the transformed cells thus selected. It increased creating a transformed plant of the tryptophan content is provided.

  Furthermore, the present inventors have separately prepared a gene DNA encoding the α subunit of the first isozyme of rice ASA or a promoter DNA useful for the expression of the modified DNA of the third invention. Conducted a study. As a result of the study, it contains a gene DNA of the α subunit of the first isozyme of rice ASA or a promoter DNA sequence effective to express the DNA of the third invention by the procedure described below. The DNA fragment was successfully obtained. An operation procedure for obtaining the DNA fragment is briefly described below (for details of the procedure, see Example 5 described later).

(a) Preparation of genomic DNA of rice Genomic DNA is extracted from tissues such as foliage, roots and callus of rice (Oriza sativa), preferably foliage or callus by a conventional method. Contaminants such as proteins are removed from the extracted genomic DNA, and a purified genomic DNA is obtained by ultracentrifugation.

(b) Preparation of rice genomic DNA fragment The genomic DNA (purified product) obtained above is partially digested with a restriction enzyme EcoRI. The resulting partially digested DNA is electrophoresed on an agarose gel. The DNA fragments fractionated by electrophoresis are transferred to Nylon membrane Hybond N. The transcribed DNA is fixed on a nylon membrane by a denaturation treatment.

  Each fraction of the DNA immobilized on the nylon membrane in this manner is subjected to a hybridization reaction with a labeled probe DNA labeled with DIG prepared from Arabidopsis in Example 1 (5) described later. A DNA fraction that emits a signal can be assayed at a DNA size of about 6 kb on the nylon membrane. The DNA fraction in the agarose gel corresponding to the DNA fraction that generated this signal is partially digested with the restriction enzyme RcoRI in the agarose, and then cut out from the gel. The excised DNA is purified, and the obtained DNA fragment (purified product) is dissolved in a TE buffer, whereby a fractionated genomic DNA is obtained.

(c) Construction of Rice Genomic DNA Library The obtained genomic DNA is ligated to a phage vector, and the obtained recombinant vector is packaged in λ phage. When the thus obtained recombinant λ phage is infected with Escherichia coli and grown, a large amount of the above-mentioned recombinant λ phage is obtained. This large amount of recombinant λ phage is used as a fractionated genomic DNA library of rice.

(d) Selection of promoter gene from rice genomic DNA library Using recombinant phage which is the rice genomic DNA library constructed above, and DIG label produced from Arabidopsis in (5) of Example 1 By using the labeled probe DNA thus obtained, a recombinant phage containing a DNA sequence corresponding to the promoter gene for the rice ASA gene can be screened from the genomic DNA library by plaque hybridization. As a result of this screening, three phage plaques in which it was determined that the promoter gene of the ASA gene had been integrated were successfully isolated from 100,000 phage plaques of the library.

  These three plaques are separately digested with the restriction enzyme RcoRI to obtain a digestion solution containing an EcoRI-DNA fragment of DNA.

(e) Cloning of genomic DNA The EcoRI-DNA fragment obtained above is ligated to the EcoRI cleavage site of plasmid vector p Bluecsript II SK (+). The obtained recombinant plasmid vector is introduced into E. coli and cloned. Isolate the recombinant plasmid vector clone from E. coli.

(f) Sequence analysis of cloned plasmid DNA The clone of the recombinant plasmid vector obtained as described above is digested with the restriction enzymes RcoRI and BamHI. The EccoRI-BamHI fragment thus obtained is subjected to nucleotide sequence analysis.

  From the genomic DNA clone obtained as described above, a DNA fragment containing a promoter region DNA sequence for expression of the α subunit gene of the first isozyme of rice ASA (DNA fragment Z and here Tentatively referred to as ") with reference to the base sequence of the DNA fragment.

  The entire nucleotide sequence of the DNA fragment Z containing the promoter DNA thus obtained was determined using a conventional sequencing kit, and the determined overall nucleotide sequence was represented by SEQ ID NO: 3 in the sequence listing. Was confirmed. The promoter DNA contained inside this DNA fragment was described in SEQ ID NO: 7 of the sequence listing with reference to the base sequence known about the DNA of the promoter region of the gene for the α subunit of the first isozyme of abidopsis. It was confirmed to have a base sequence.

  All or part of the nucleotide sequence of the DNA fragment Z has been confirmed to have promoter activity from the test of Example 5 described later.

  A DNA fragment Z containing a promoter DNA having the nucleotide sequence of SEQ ID NO: 7 in the sequence listing was cloned into a pBluescript II SK (+) plasmid vector, and Escherichia coli XL1-Blue transfected with the thus obtained recombinant vector was introduced. MRF 'has been named Escherichia coli (Os-asa # 7) and has been deposited with the National Institute of Bioscience and Human Technology on August 18, 1997 under the accession number FERM P-16387. Since August 7, 1998, it has been deposited under the terms of the Budapest Treaty under the accession number FERM BP-6452.

(g) Assay of promoter activity As contained in the DNA fragment Z isolated from the rice genomic DNA clone obtained above, the promoter region of the gene for the α subunit of the first isozyme of ASA, In order to confirm the function as a promoter, an assay for its activity was performed. That is, a recombinant plasmid vector was prepared by inserting the DNA fragment Z into a restriction site of a commercially available pBI101 plasmid vector (manufactured by Clontech) carrying a β-glucuronidase (GUS) gene as a reporter gene. did. This recombinant plasmid vector is introduced into a plant cell, for example, a cultured rice cell, by a conventional method, and it can be confirmed by a commercially available kit for measuring GUS activity that the promoter region is effective for expression of GUS activity.

  The DNA of the gene for the α subunit of the isozyme of ASA according to the first invention and the DNA sequence of the promoter thereof are isolated from the cDNA library or the genomic DNA library as described above when completing the present invention. It is something. According to the present invention, the base sequences of the DNAs of these promoters have been elucidated, and the promoters can also be obtained by chemical synthesis based on the base sequences shown in the sequence listing below. In addition, it can also be obtained by PCR from a rice cDNA library or a chromosomal DNA library by a known method using a synthetic oligonucleotide primer prepared based on the above nucleotide sequence.

  Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

  Unless otherwise specified, the procedure of the experimental operation performed in the following Examples was in accordance with the method described in “Molecular Cloning”, 2nd edition (J. Sambrook et al., Cold Spring Habor Laboratory press, 1989). Was.

Example 1
In this example, the gene encoding the α subunit of the first isozyme (ASA1) of rice anthranilate synthase (ASA) and the gene encoding the α subunit of the second isozyme (ASA2) are isolated. The method is illustrated.

(1) Preparation of rice mRNA Rice (cultivar: Nipponbare) seeds were sown. Thereafter, 2 g of foliage of the young plant on the seventh day of cultivation was frozen with liquid nitrogen. The frozen foliage was ground in a mortar. From the pulverized product, a known AGPC method (a method using Acid Guanidinium thiocyanate-phenol-chloroform) (Experimental Medicine, Vol. 9, No. 15 (November issue), pp. 99-102, 1991) , About 2 mg of the total RNA was extracted. Next, mRNA was isolated from the total RNA obtained above using the mRNA Purification Kit (Pharmacia Biotech). Thus, about 30 μg of rice total mRNA was obtained.

(2) Construction of rice cDNA library From the whole rice mRNA obtained in the above (1), a whole rice cDNA was obtained using a DNA synthesis kit (TimeSaver cDNA Synthesis Kit manufactured by Pharmacia Biotech).

  This total cDNA was ligated to a phage vector, which was a λgt11 phage vector (Lambda gtll / EcoRI / CIAP-Treated Vector Kit (manufactured by STRATGENE)) in which EcoRI-cut ends were treated with calf intestinal alkaline phosphatase. The recombinant vector thus obtained was packaged into lambda phage using an in vitro packaging kit (Gigapack II Gold Packaging Extract).

  Escherichia coli Y1088 was infected with the recombinant lambda phage packaged with the recombinant vector and propagated. A large number of the recombinant lambda phages were obtained as lysed plaques of the grown E. coli. The recombinant lambda phage in the plaques consisted of various phages containing all the cDNAs derived from rice, and these various phages were used as a rice cDNA library.

(3) Construction of primers for PCR method For cloning cDNA fragments encoding the α subunits of the first isozyme (ASA1) and the second isozyme (ASA2) of rice anthranilate synthase (ASA) A DNA probe to be used is prepared for the PCR method. For this purpose, first, a primer was designed. For this purpose, first, referring to the known nucleotide sequence and known amino acid sequence of the gene encoding the α-subunit of each of the first and second isozymes of anthranilate synthase of Arabidopsis (Japanese name: Arabidopsis thaliana) Thus, two types of oligonucleotides having the base sequences shown below were prepared by chemical synthesis as primer No. 1 and primer No. 2.

Primer No. 1 (oligonucleotide having the base sequence shown in SEQ ID NO: 8 in the sequence listing)):
5'-CATATGTCTTCCTCTATGAAC-3 '
Primer No. 2 (oligonucleotide having the base sequence of SEQ ID NO: 9):
5'-GGATCCTCATTTTTTCACAAATGC-3 '
The above two types of oligonucleotides were prepared by synthesizing oligonucleotides using a DNA synthesizer (Model 391, manufactured by Applied Biosystems) and purifying the synthesized product by ion-exchange HPLC. .

(4) Preparation of probe DNA Next, using 10 p.M of each of the two types of synthetic oligonucleotides constructed as described above as a first primer and a second primer, and a commercially available cDNA library of Arabidopsis (STRATAGENE) was used as a template. The first and second primers and the Arabidopsis cDNA library were subjected to an amplification reaction solution for PCR (10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.001% gelatin, pH 8.3; 4 types , And a DNA polymerase (Takara Ex Taq, 2.5 units) (50 μl), and a DNA amplification reaction was performed. The amplification reaction solution used here was prepared with a PCR kit (PCR Amplification Kit (Takara Shuzo Co., Ltd.)).

  The above amplification reaction of DNA by the PCR method is performed by using a PCR reaction device (manufactured by PERKIN ELMER, DNA, Thermal Cycler 480) for denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 1 minute, and extension (extention ) At 72 ° C. for 2 minutes was repeated 35 times.

  As described above, an amplification product of a DNA fragment constituting the entire DNA sequence (full length) corresponding to the gene encoding the α subunit of each of the two isozymes of Arabidopsis ASA is generated. The amplification products of these DNA fragments are collected as probe DNA. The probe DNA was used for cloning a rice cDNA library as follows.

(5) Selection of the DNA of the gene encoding the α subunit of the two rice ASA isozymes (ASA1 and ASA2) from the rice cDNA library, the rice cDNA library constructed in (2) above From the recombinant lambda phage, the DNA sequence corresponding to the gene encoding the α subunit of the two isozymes of rice ASA was integrated by using the probe DNA prepared from the Arabidopsis DNA library in (4) above. Lambda phage is collected by screening by the plaque hybridization method.

  For the purpose of this screening, plaques of the recombinant lambda phage, a rice cDNA library obtained in (2) above, were formed on 1.5% agar medium in a plurality of Petri dishes. These phage plaques were transferred to a plurality of nylon membranes (manufactured by Amersham) High Bond N. The phage DNA contained in the plaque of the phage transferred to the nylon membrane in this manner was treated with an alkaline denaturing solution (1.5 M NaCl, 2.0 M NaOH) and a neutralizing solution (1.0 M Tris-HCl pH5, 2.0 M NaCl), respectively. After that, the cells were fixed on a nylon membrane by UV irradiation.

  Next, the probe DNA obtained from the Arabidopsis cDNA library in (4) above was labeled with digoxigenin (DIG) to obtain a labeled probe DNA. The labeled probe was plaque-hybridized to a nylon membrane on which the phage DNA (ie, rice cDNA library) was immobilized. The labeling of the probe DNA was performed using a DIG-ELISA DNA Labeling & Detection Kit (Boehringer Mannheim).

  In the case of this plaque hybridization, the nylon membrane on which the phage DNA was immobilized was immersed in a hybridization solution (500 mM Na-Pi buffer, pH 7.2, 7% SDS, 1 mM EDTA), and further at 65 ° C. for 10 minutes. An immersion treatment was performed. Next, the above-described DIG-labeled labeled probe DNA (10 ng / ml) was added, and the mixture was incubated at 65 ° C. for 15 hours, whereby the plaque hybridization reaction was performed.

  After the completion of the reaction, the nylon membrane was washed three times with a washing solution (40 mM Na-Pi buffer, pH 7.2, 1% SDS) for 20 minutes each. Thereafter, using the DIG-ELISA Labeling & Detection Kit described above, the target recombinant phage containing DNA encoding the α subunit of rice ASA was detected. Eight recombinant phage plaques that hybridize and give a strong signal on X-ray film, i.e. eight phage plaques that seem to have integrated genes encoding rice ASA, were replaced with 300,000 phage plaques From this, it was possible to detect on the nylon membrane. Then, the above-mentioned eight recombinant phage plaques incorporating the rice ASA gene were isolated and selected.

  From the collected phage of the eight collected plaques, λ DNA was isolated separately for every eight phage plaques using a λ DNA isolation kit (Lambda DNA Purification Kit (manufactured by STRATAGENE)).

  At this time, the above-mentioned λDNA was isolated as follows. That is, 50 μl of DNaseI (20 mg / ml) and 200 μl of RNaseA (2 mg / ml) were added to 5 ml of each culture solution in which each phage of each of the eight plaques was separately and massively grown, and left at room temperature for 15 minutes. did. The resulting phage growth solution was centrifuged at 15000 rpm at 4 ° C. for 10 minutes. 25 ml of 80% DEAE-cellulose was added to the obtained supernatant and incubated at room temperature for 10 minutes. The mixed solution thus incubated was centrifuged, and 2 ml of 0.5 M EDTA and 770 μl of Pronase (50 mg / ml) were added to the obtained supernatant, and the mixture was allowed to stand at 37 ° C. for 15 minutes. Further, 1.5 ml of a 5% CTAB solution (1% CTAB (Cetyltrimethyl-ammonium bromide), 50 mM Tris-HCl, pH 8.0, 10 mM EDTA) was added. The resulting mixture was treated at 65 ° C. for 3 minutes and then left on ice for 5 minutes. To each of the reaction solutions thus obtained, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added, and the mixture was allowed to stand at -20 ° C for about 6 hours. The phage DNA precipitated from was separately dried and dissolved and stored in 5 ml of water, respectively.

  Eight kinds of phage DNAs isolated by the above operation were obtained. 5 μl of each of these DNAs was separately digested with 10 units of the restriction enzyme EcoRI in H buffer, and the obtained digestion reactions were separately analyzed.

  Thus, the inserted DNA fragment in the phage DNA, which was determined to contain the DNA sequence corresponding to the gene encoding the α subunit of the two isozymes of rice ASA, was obtained from the collected phage selected as described above. The phage DNA was obtained by digesting the phage DNA with the restriction enzyme EcoRI and excising it as described above.

(6) Cloning of cDNA containing a DNA sequence corresponding to the gene encoding each α subunit of two rice ASA isozymes (ASA1 and ASA2) As described above, α of two rice ASA isozymes As the DNA fragments determined to contain the DNA sequence corresponding to the gene encoding the subunit, there are eight DNA fragments obtained by excision from each of the eight types of recombinant phages. A recombinant plasmid vector was constructed by inserting and ligating each of such DNA fragments to the EcoRI cleavage site of plasmid vector p Bluescript II SK (+) using a DNA ligation kit. Escherichia coli XL1-Blue MRF ′ was transformed by introducing the thus constructed recombinant plasmid vector.

  The operation of introducing the constructed recombinant plasmid vector into Escherichia coli XL1-Blue MRF 'for the purpose of transformation as described above was performed in detail as follows. That is, 10 μg of the recombinant phage DNA of the collected phage obtained above was first digested with 10 units of the restriction enzyme EcoRI in H buffer to obtain a digestion reaction solution. On the other hand, 10 μg of the plasmid vector p Bluescript II SK (+) was similarly digested with EcoRI to obtain a digestion reaction solution. 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were separately added to each DNA digestion reaction solution obtained as described above, and the mixture was left at -20 ° C for about 6 hours. The obtained DNA solutions were centrifuged, and the DNA precipitated from each solution was dried and dissolved in 5 μl of water. The aqueous solution of phage-derived DNA thus obtained and 5 μl of each of the plasmid-derived DNA aqueous solutions were mixed, and the resulting mixture was treated with a DNA Ligation kit (manufactured by Takara Shuzo Co., Ltd.). Connected. One tenth volume of 3M sodium acetate and two volumes of ethanol were added to the obtained DNA ligation reaction solution, and the mixture was allowed to stand at -20 ° C for about 6 hours. The obtained mixture was centrifuged, and the precipitated DNA was dried. Thus, the ligated vector DNA was collected. This ligated vector DNA was dissolved in 10 μl of water.

  10 μl of the aqueous solution of the ligated vector DNA thus obtained (10 ng of DNA) and 100 μl of commercially available Escherichia coli XL1-Blue MRF ′ competent cells (manufactured by STRATAGENE) were placed in a 1.5 ml tube, and placed on ice for 30 minutes, 42 minutes. Incubated for 30 seconds at 0 C and again for 2 minutes in ice. Thereafter, 900 μl of SOC liquid medium (containing 2% Bacto-tryptone, 0.5% Bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO4, 10 mM MgCl2, 20 mM Glucose) was added to the incubated mixture. The E. coli was shake-cultured at 37 ° C. for 1 hour.

100 μl of the obtained culture solution of E. coli, ampicillin at a concentration of 50 mg / l, X-gal (5-Bromo-4-Chloro-3-Indolyl-bD-Galactoside) at a concentration of 20 mg / l, IPTG
(Isopropyl-bD-thiogalactopyranoside) is added at a concentration of 20 mg / l in LB agar medium (1% Bacto-Tryptone, 0.5% Bacto-Yeast extract, 0.5% NaCl, 0.1% Glucose, pH 7.5, Agar 1.5% Contained). After culturing Escherichia coli at 37 ° C for 16 hours, Escherichia coli colonies that are colored white are transformed with the recombinant plasmid vector DNA to which the DNA fragment of the recombinant phage is linked from E. coli colonies that are not colored white. It was isolated as transformed E. coli.

  Ten colonies of the transformed E. coli that are white and have ampicillin resistance isolated in this way are grown in a liquid medium containing 50 mg / l of ampicillin. The plasmid DNA was separated and purified using a QIA filter Plasmid Midi Kit (manufactured by QIAGEN). By this purification, 50 μg (50 μl) of the recombinant plasmid DNA was obtained from the E. coli transformed with the ampicillin-resistant colony obtained above.

  Cloning was performed as described above from each of the recombinant phages (total of 8 types) isolated above, and 8 types of recombinant plasmid DNAs were obtained. The following operations were performed to analyze the base sequences of these eight DNAs.

(7) Sequence analysis of cloned DNA By treating each of the eight cloned recombinant plasmid DNAs with a commercially available nucleotide sequencing kit, the entire nucleotide sequence of those DNA fragments can be determined. Was. Then, the nucleotide sequence of the DNA corresponding to the gene encoding the α subunit of rice ASA1 contained in the inside of those DNA fragments, and the nucleotide sequence of the DNA corresponding to the gene encoding the α subunit of rice ASA2 Could be determined.

  In the determination of the nucleotide sequence of the above DNA, denaturation treatment was performed using a nucleotide sequence determination kit (Autoread Sequencing Kit (Pharmacia Biotech)), followed by automatic DNA sequencer ALF DNA Sequencer II (Pharmacia). The nucleotide sequence of the DNA fragment was determined by the above-mentioned method.

  As a result of determining the nucleotide sequence of the above DNA fragment, it was found that the DNA sequence encoding the α subunit of rice ASA had two different nucleotide sequences. Therefore, rice ASA is composed of two isozymes (isoenzymes), and the DNA encoding the α subunit of the first isozyme (ASA1) encodes the α subunit of the second isozyme (ASA2). It was found that the DNA had a base sequence different from that of the DNA.

  Then, the DNA corresponding to the gene encoding the α subunit of rice ASA1 was determined as described above, and has now been recognized as having the nucleotide sequence described in SEQ ID NO: 1 in the sequence listing below. .

  In addition, the DNA corresponding to the gene encoding the α subunit of rice ASA2 was determined as described above, and has now been recognized as having the nucleotide sequence described in SEQ ID NO: 10 in the sequence listing below. .

  The base sequence of the DNA of the gene encoding the α subunit of rice ASA1 consists of 1734 bases in a single open reading frame shown in SEQ ID NO: 1 in the sequence listing. The nucleotide sequence of the DNA of the gene encoding the α subunit of rice ASA1 according to the present invention shown in SEQ ID NO: 1 in the sequence listing encodes a protein consisting of 577 amino acid residues described in SEQ ID NO: 2. are doing. Comparing the amino acid sequence of SEQ ID NO: 2 with the known amino acid sequence of the protein of the α subunit of Arabidopsis ASA1 (The Plant Cell, Volume 4, pp. 721 to 733, 1992), 68% homology was found. Although it is observed, in other regions, it is clearly different from the α subunit of ASA1 of Arabidopsis, and it is a rice-specific sequence.

  The base sequence of the DNA of the gene encoding the α subunit of rice ASA2 consists of 1821 bases in a single open reading frame represented by SEQ ID NO: 10 in the sequence listing. Further, the nucleotide sequence of the DNA of the gene encoding the α subunit of rice ASA2 shown in SEQ ID NO: 10 encodes a protein consisting of 606 amino acid residues described in SEQ ID NO: 11. Comparing this amino acid sequence of SEQ ID NO: 11 with the known amino acid sequence of the protein of the α subunit of Arabidopsis ASA2 (The Plant Cell, Vol. 4, pp. 721 to 733, 1992), 72% homology was found. Although it is observed, in other regions, it is distinctly different from the α subunit of Arabidopsis ASA2, and is a rice-specific sequence.

(8) Deposition of Escherichia coli transformant into which the rice ASAα subunit gene has been introduced By the procedure described in (6) above, eight kinds of recombinant plasmid DNAs were finally obtained. The nucleotide sequence of the entire DNA fragment of each of these eight kinds of recombinant plasmid DNA fragments was determined by the nucleotide sequence determination kit in the preceding section (7).

  (i) Next, a DNA sequence consisting of 1734 bases described in SEQ ID NO: 1 in the sequence listing (named as DNA sequence OSASA-W1) (i.e., DNA encoding α subunit of rice ASA1) Was selected from the above eight types of recombinant plasmid DNA fragments. The selected recombinant plasmid DNA fragment was digested with a restriction enzyme EcoRI. From the digestion reaction solution, a DNA fragment determined to contain the DNA sequence OSASA-W1 (designated as DNA sequence OSASA-1) was fractionated by low melting point agarose electrophoresis and collected. The DNA sequence OSASA-1 thus collected was ligated to the EcoRI cleavage site of the plasmid vector p Bluescript II Sk (+) using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), and the recombinant plasmid vector pOSASA- Built one.

  The plasmid vector pOSASA-1 thus constructed was introduced into Escherichia coli XL1-Blue MRF ′ by the method described in the above section (6). The Escherichia coli strain transformed by introduction of the plasmid vector pOSASA-1 was named Escherichia coli XLl-Blue MRF '(Os-asa1). Escherichia coli XLl-Blue MRF '(Os-asa1) deposited with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Tsukuba, Ibaraki Prefecture, Japan on August 18, 1997 under the accession number of FERM P-16388 Have been. Furthermore, since August 7, 1998, it has been deposited with the aforementioned laboratory under the terms of the Budapest Treaty under the accession number FERM BP-6453.

  (ii) a DNA sequence consisting of 1821 bases described in SEQ ID NO: 10 of the sequence listing (named as DNA sequence OSASA-W2) (that is, DNA encoding α subunit of rice ASA2) Recombinant plasmid DNA fragments determined to be contained therein were selected from the above eight types of recombinant plasmid DNA fragments. The selected recombinant plasmid DNA fragment was digested with a restriction enzyme EcoRI. From the digestion reaction solution, a DNA fragment determined to contain the DNA sequence OSASA-2 (named DNA sequence OSASA-2) was collected by low-melting point agarose electrophoresis. The DNA sequence OSASA-W2 thus collected was ligated to the EcoRI cleavage site of the plasmid vector p Bluescript II Sk (+) using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), and the recombinant plasmid vector pOSASA- 2 built.

  The plasmid vector pOSASA-2 thus constructed was introduced into Escherichia coli XL1-Blue MRF ′ by the method described in the above section (6). The Escherichia coli strain transformed by introducing the plasmid vector pOSASA-2 was named Escherichia coli XLl-Blue MRF '(Os-asa2). Escherichia coli XLl-Blue MRF '(Os-asa2) was deposited at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Tsukuba, Ibaraki Prefecture, Japan, on June 18, 1998 under the accession number of FERM P-16853. Have been. Furthermore, since August 7, 1998, it has been deposited with the aforementioned laboratory under the terms of the Budapest Treaty under the accession number FERM BP-6454.

Example 2
In this example, a DNA sequence designated as a modified D sequence obtained by the third invention (the invention of claim 1) (that is, a base sequence consisting of 1734 bases described in SEQ ID NO: 12 in the sequence listing) was prepared. Production of a DNA fragment containing the DNA sequence

(1) Construction of synthetic oligonucleotides as primers for PCR
Four kinds of primers were synthesized using a DNA synthesizer (Model-391, manufactured by Applied Biosystems). That is, primer OSASN1, which is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 in the sequence listing, primer OSASN2 having the nucleotide sequence of SEQ ID NO: 17, and primer OSASC1 having the nucleotide sequence of SEQ ID NO: 18 And a primer OSASC2 having the nucleotide sequence of SEQ ID NO: 19 were constructed by chemical synthesis.

(2) Preparation of template for PCR method In section (8) of Example 1, a DNA sequence corresponding to the gene for the α subunit of rice ASA1, that is, a DNA fragment determined to contain the DNA sequence OSASA-W1 As a result, the DNA fragment OSASA-1 obtained by cutting out the recombinant phage with the restriction enzyme EcoRI was ligated to the EcoRI cleavage site of the plasmid vector p Bluescript II SK (+) using a DNA ligation kit, and the recombinant plasmid vector pOSASA-1). This plasmid vector pOSASA-1 was used as a template in the following PCR method.

(3) Amplification and recovery of required DNA fragments by PCR
(i) First Step of PCR Method In order to obtain required DNA fragments by an amplification reaction by the PCR method, the following (A) reaction and (B) reaction were performed as the first step of the PCR method.

  That is, in the reaction (A), 5 μl of the recombinant plasmid vector pOSASA-1 used as a template, 1 μM of the primer OSASAN1, and 1 μM of the primer OSASAC1 were mixed with an amplification reaction solution (10 mM Tris-HCl). (pH 8.3), 1 mM MgCl2, 50 mM KCl, a mixture of 0.2 mM each of four nucleotide dNTPs, and 2.5 units of LA Taq DNA polymerase) (100 μl), and an amplification reaction was performed. The DNA by-product obtained by the reaction (A) is referred to as DNA fragment-A.

  In the reaction (B), 5 μl of the plasmid vector pOSASA-1 used as a template, 1 μM of the primer OSASAC2, and 1 μM of the primer OSASAN2 were added to an amplification reaction solution having the same composition as that used in the reaction (A). Then, an amplification reaction was performed. The amplification product of DNA obtained by this (B) reaction is referred to as DNA fragment-B.

  In the amplification reaction, denaturation was performed at 94 ° C for 30 seconds, annealing was performed at 55 ° C for 30 seconds, and extension was performed at 72 ° C in a PCR reaction apparatus (Promram Temp Control System PC-700 (manufactured by Astec)). The reaction was performed by repeating three reaction operations for 20 minutes each 20 times.

  After the completion of the amplification reaction, the amplification reaction solution of the reaction (A) was fractionated by low-melting point agarose electrophoresis, and a band containing a 268 bp DNA fragment-A as an amplified DNA product was cut out from the agarose gel. Further, the amplification reaction solution of the reaction (B) was subjected to low-melting-point agarose electrophoresis to fractionate, and a band containing a 336 bp DNA fragment-B as an amplified DNA product was cut out from the agarose gel.

  From these two gel sections, a purified DNA fragment-A and a purified DNA fragment-B were obtained by using GENECLEAN II Kit (Funakoshi), respectively.

(ii) The second step of the PCR method Next, as the second step of the PCR method, 1 μl of the purified DNA fragment-A and the DNA fragment-B amplified in the above-mentioned reactions (A) and (B) were used. 1 μl of the purified product in 100 μl of an amplification reaction solution (containing a mixture of 10 mM Tris-HCl (pH 8.3), 1 mM MgCl2, 50 mM KCl, 0.2 mM each of dNTP and 0.2 unit of LA Taq DNA polymerase). In addition, an amplification reaction was performed. In the amplification reaction in this case, denaturation was performed at 94 ° C for 5 minutes, annealing was performed at 65 ° C for 10 minutes and 55 ° C for 10 minutes, and extension was performed once at 72 ° C for 1 minute.

  After the completion of the amplification reaction, the amplification reaction solution was fractionated by low-melting point agarose electrophoresis, and a band containing the expected DNA fragment-C of 583 bp as an amplified DNA product was cut out from the gel. The DNA fragment-C was separated from this gel slice using GENECLEAN II Kit (Funakoshi) and a purified DNA fragment-C was obtained.

(4) Next, using the DNA fragment-C obtained above, an operation of obtaining a DNA fragment containing the modified D sequence of interest according to the second invention in a sufficient amount by cloning is performed.

  (i) First, 10 μg of the DNA fragment-C obtained above was digested with 10 units of the restriction enzymes Afl II and 10 units of Bgl II in H buffer (manufactured by Takara Shuzo Co., Ltd.). The DNA fragment obtained by this digestion is called DNA fragment-α. On the other hand, 10 μg of the above plasmid vector pOSASA-1 was digested with 10 units of Afl II and 10 units of Bgl II in H buffer (manufactured by Takara Shuzo Co., Ltd.) to obtain a shortened plasmid. To each of the digestion reaction solution containing the DNA fragment-α and the digestion reaction solution containing the above-mentioned shortened plasmid, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were separately added, and the mixture was added at −20 ° C. for about 6 hours. Incubated for hours. Thereafter, the incubated solutions were centrifuged, and the DNA precipitated from each solution was dried and dissolved in 5 μl of water.

  5 μl of the aqueous solution of DNA fragment-α thus obtained was mixed with 5 μl of the aqueous solution of the shortened plasmid DNA. Next, each DNA contained in the obtained mixture (10 μl) was subjected to a ligation reaction using a DNA Ligation kit (Takara Shuzo Co., Ltd.). 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to the ligation reaction solution, and the mixture was incubated at -20 ° C for about 6 hours. The incubated reaction solution was centrifuged, and the precipitated DNA ligation was dried and dissolved in 5 μl of water to make an aqueous solution.

  The DNA ligated product contained in this aqueous solution was a circular two-piece product formed by ligating the DNA fragment -α and the shortened plasmid obtained by Afl II and Bgl II digestion of the plasmid vector pOSASA-1 to each other. This is a strand recombinant plasmid (which is the pBluescript-DNA-D plasmid described above), and the modified D sequence was contained in the inserted DNA region in the plasmid DNA.

  (ii) Escherichia coli XL1-Blue MRF ′ was transformed by introducing the pBluescript-DNA-D plasmid into Escherichia coli XL1-Blue MRF ′.

  The Escherichia coli thus transformed was named Escherichia coli XLl-Blue MRF '/ p Bluescript-DNA-D, and was sent to the aforementioned Institute of Biotechnology and Industrial Technology on August 7, 1998 or less under the terms of the Budapest Treaty. Was deposited under the accession number of FERM BP-6451. The transformed E. coli cells were then cultured in liquid medium.

  The plasmid was further extracted from the cultured E. coli cells. Thus, the recombinant plasmid pBluescript-DNA-D containing the modified D sequence could be cloned.

(5) Recovery of DNA Fragment Containing Modified D Sequence 10 μl of the resulting plasmid DNA, plasmid p Bluescript-DNA-D, was digested with 10 units of EcoRI in H buffer (Takara Shuzo Co., Ltd.). . The digestion reaction solution was fractionated by low melting point agarose electrophoresis. A DNA fragment (referred to as DNA fragment-β) containing the DNA sequence OSASA-W1 sequence (1734 bp in size) was excised from agarose.

  An equal amount of TE buffer (containing 10 mM Tris-HCl, containing 1 mM EDTA, pH 8) was added to the agarose section containing the DNA fragment-β, and the agarose was dissolved by heating at 68 ° C. for 20 minutes. Agarose was removed by extracting the resulting solution twice with saturated phenol. To the phenol extract containing the obtained DNA, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added, and the mixture was allowed to stand at -20 ° C for about 6 hours and incubated. The incubated solution was centrifuged at 15000 rpm at 4 ° C. for 10 minutes. The obtained DNA precipitate was dried under reduced pressure, and the obtained DNA powder was dissolved in 10 μl of water.

  The DNA powder consisted of a DNA fragment-β containing the desired modified D sequence.

  The DNA fragment-β obtained in the above (5) of Example 2 was used in the same manner as described in (7) of Example 1, and an automatic DNA sequencer using a nucleotide sequence determination kit, ALF DNA Sequencer II. DNA fragment-β was confirmed to be a DNA fragment containing a modified D sequence having a base sequence of 1734 bases shown in SEQ ID NO: 12 in the sequence listing.

Example 3
In this example, the DNA sequence shown in SEQ ID NO: 1 of the sequence listing according to the first invention as a foreign gene, or the modified DNA sequence having the nucleotide sequence shown in SEQ ID NO: 12 according to the third invention as a foreign gene, as a rice plant A method for transforming a rice plant, which is introduced by the Agrobacterium method, is exemplified. Also, it was demonstrated that the tryptophan content of the transformed plant was increased in this example.

(1) Construction of recombinant vector pUBdD for introduction of foreign gene into rice plant
(i) Corn ubiquitin promoter, 1st intron, NOS terminator, 5.6 kb-containing plasmid vector pUBA DNA (10 μg) containing an ampicillin resistance gene, K buffer (manufactured by Takara Shuzo Co., Ltd.) Digested with 10 units of the restriction enzyme BamHI. DNA was precipitated from the digestion reaction, centrifuged and dried. The dried DNA was dissolved in 10 μl of sterile water, and then blunted with a DNA branding kit (Takara Shuzo). The DNA having blunt-ended ends was precipitated, centrifuged, and dried. The dried product was dissolved in 10 μl of sterilized water, and then digested with 10 units of the restriction enzyme SacI in L buffer (Takara Shuzo). DNA was precipitated from the digestion reaction, centrifuged and dried. In this way, a vector fragment of about 4.8 kb carrying the ubiquitin promoter, the 1st intron, the NOS terminator and the ampicillin resistance gene was obtained (see the left side of FIG. 1 in the attached drawings).

  (ii) Still further, a DNA (10 μg) of a recombinant plasmid pBluescript-DNA-D (1.7 kb in size) containing a modified D sequence therein, which is the DNA of the third invention, is added to an H buffer (Takara Shuzo ( Was digested with 10 units of the restriction enzyme EcoRV, DNA was precipitated from the digestion reaction solution, centrifuged, and dried. The dried DNA was dissolved in 10 μl of sterilized water, and digested with 10 units of the restriction enzyme SacI in L buffer (Takara Shuzo). The DNA was dried from the digestion reaction solution in the same manner as above to obtain a pBluescript-DNA-D vector fragment (see the right side of FIG. 1 in the attached drawings).

  (iii) The vector fragment having a size of about 4.8 kb obtained in (i) and the vector fragment obtained in (ii) were dissolved in 5 μl of sterilized water. Each 5 μl of the aqueous solution of the vector fragment was mixed. The resulting mixture was treated with a DNA ligation kit (Takara Shuzo Co., Ltd.) to perform a DNA ligation reaction. Thus, a circular recombinant vector (6.5 kb in size) was prepared by ligating the above-mentioned cleavage vector fragment of pBluescript-DNA-D to the cleavage vector fragment of the plasmid vector pUBA. This recombinant vector is called pUBdD (see the lower part of FIG. 1). The vector pUBdD has a 1st intron region downstream of the ubiquitin promoter region, and the modified D sequence of the third invention is inserted and linked between the 1st intron region and the NOS terminator region, and has an ampicillin resistance gene. It had a containing structure and a size of 6.5 kb.

(2) Construction of recombinant vector pUBdD-W1 for introduction of foreign gene into rice plant
(I) from the corn ubiquitin promoter, 1st intron, NOS terminator, and a DNA of a known plasmid vector pUBA (10 μg) having a size of 5.6 kb containing an ampicillin resistance gene, (i) and (ii) In the same manner as described above, a vector fragment of about 4.8 kb having the ubiquitin promoter, the 1st intron, the NOS terminator and the ampicillin resistance gene was obtained (see the left part of FIG. 1).

  (ii) Separately, a DNA (10 μg) of a recombinant plasmid pOSASA-W1 (4.7 kb in size) containing the DNA sequence OSASA-W1 of SEQ ID NO: 1 according to the first invention is added to an H buffer (Takara Shuzo Co., Ltd. )), The DNA was digested with 10 units of the restriction enzyme EcoRV, DNA was precipitated from the digestion reaction solution, centrifuged, and dried. The dried product was dissolved in 10 μl of sterilized water, and digested with 10 units of the restriction enzyme SacI in L buffer (Takara Shuzo). DNA was dried from the digestion reaction solution in the same manner as described above.

  (iii) The vector fragment having a size of about 4.8 kb obtained in the above (i) and the vector fragment obtained in the above (ii) were dissolved in 5 μl of sterilized water. Each 5 μl of the aqueous solution of the vector fragment was mixed. The resulting mixture was treated with a DNA ligation kit (Takara Shuzo Co., Ltd.) to perform a DNA ligation reaction. Thus, a circular recombinant vector was prepared by ligating the pOSA-W1 cleavage vector fragment to the pUBA cleavage vector fragment. This recombinant vector is called pUBdD-W1. The vector pUBdD-W1 has a 1st intron region downstream of the ubiquitin promoter region, and the sequence OSASA-W1 is inserted and linked between the 1st intron region and the NOS terminator region, and contains an ampicillin resistance gene. Having a structure.

(3) Preparation of recombinant vector pUb-OSASA1D or pUb-OSASAW1 for introduction of foreign gene into rice plant
(i) Digest the DNA (10 μg) of the plasmid vector pUBdD-W1 obtained in (2) (iii) above with 10 units of the restriction enzyme SphI in H buffer (manufactured by Takara Shuzo Co., Ltd.) and digest it. DNA was precipitated from the reaction, centrifuged and dried. The obtained vector fragment was dissolved in 10 μl of sterilized water (see the right of FIG. 2 in the attached drawings). 10 μl of the aqueous solution of the vector fragment was treated with a DNA branding kit (manufactured by Takara Shuzo Co., Ltd.) to perform a blunting reaction at the restriction enzyme cleavage site. DNA was precipitated from the reaction solution, centrifuged, dried, and then dissolved in 10 μl of sterilized water. The DNA (10 μg) of this vector fragment was digested with 10 units of the restriction enzyme SacI in L buffer (manufactured by Takara Shuzo Co., Ltd.), and the DNA was precipitated from the digestion reaction solution, centrifuged, and dried. In this way, a vector fragment having the ubiquitin promoter and the third modified D sequence of SEQ ID NO: 12 according to the present invention was obtained (see the third figure on the right in FIG. 2 of the attached drawings).

  (ii) The known plasmid vector plG121-Hm DNA (10 μg) having a size of 15 kb containing the hygromycin resistance gene was digested with 10 units of the restriction enzyme SacI in an L buffer (manufactured by Takara Shuzo Co., Ltd.) and digested. DNA was precipitated from the reaction, centrifuged and dried. The dried product was dissolved in 10 μl of sterilized water, and then digested with 10 units of the restriction enzyme PmeI in NEB4 buffer (New England Biolab). DNA was dried from the digestion reaction solution in the same manner as in the above (1) (i). In this manner, a vector fragment having a hygromycin resistance gene and having a size of about 9.8 kb was obtained (see left in FIG. 2).

  (iii) The vector fragment having a size of about 9.8 kb obtained in the above (ii) and the modified D sequence-containing vector fragment obtained in the above (i) were each dissolved in 5 μl of sterilized water. After mixing 5 μl of the aqueous solution of the vector fragment, the mixture was treated with a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) to perform a DNA ligation reaction. Thus, a circular recombinant vector was prepared by linking the modified D sequence fragment to the PmeI-SacI digested vector fragment of the plasmid vector plG121-Hm. This recombinant vector is called pUb-OSASA1D (see below in FIG. 2). The vector pUb-OSASA1D has a 1st intron region downstream of the ubiquitin promoter region, and a modified D sequence is inserted and ligated between the 1st intron region and the NOS terminator region, and is resistant to hygromycin expressed in plants. It had a structure containing the gene and the kanamycin resistance gene, and had a size of 11.5 kb.

  Furthermore, instead of the modified D sequence fragment, a cyclic recombinant vector obtained by ligating the OSASAW1 fragment to the vector fragment of about 9.8 kb obtained in the above (ii) in the same manner as described above is used. Created. This recombinant vector is called pUb-OSASAW1 (containing the DNA of SEQ ID NO: 1).

(4) Preparation of Agrobacterium bacteria
Agrobacterium (Agrobacterium tumefaciens LBA4404 (purchased from Clontech)) was inoculated into 300 ml of a liquid medium of LB medium (Bactotryptone 10 g / l, Bacto yeast extract 5 g / l, NaCl 5 μl pH7), and transferred at 30 ° C. for 16 hours. After culturing with shaking for a period of time, the culture solution was cooled at 4 ° C. for 10 minutes, and then a bacterial precipitate was obtained by centrifugation. The bacterial precipitate was washed three times with ice-cooled 10% glycerol and centrifuged three times, and then the precipitate was dissolved in 10 ml of 10 glycerol.

  Transfer 40 μl of the mixture to an Eppendorf tube, and put 5 μl of a lysate (40 ng) of the recombinant vector pUb-OSASA1D (containing the DNA of SEQ ID NO: 12) or the vector pUb-OSASAW1 (containing the DNA of SEQ ID NO: 1) in the tube. Was added and mixed well. After placing them on ice for 3 minutes, transfer them to a cuvette for electroporation (0.2 cm between electrodes), and use an electroporation device (Gene Pulser (manufactured by Bio-Rad)) at a voltage of 12.5 kV / cm and a capacitor capacity. Electroporation was performed under the conditions of 25 μF and a resistance value of 600Ω. 0.8 ml of an SOC medium (GIBCO BRL) was added to the cuvette, the suspension was transferred to a 2 ml test tube, and cultured with shaking at 30 ° C. for 1 hour. This culture solution was spread on an agar medium containing 50 mg / l of kanamycin in LB medium, and cultured at 30 ° C for 36 hours to obtain growing colonies as transformed Agrobacterium into which the recombinant vector was introduced. Was.

  Agrobacterium bacteria transformed with the recombinant vector pUb-OSASA1D are referred to as pUb-OSASA1D / LBA4404. Agrobacterium bacteria transformed with the vector pUb-OSASAW1 are referred to as pUb-OSASAW1 / LBA4404.

(5) Preparation of rice callus Rice hulls were threshed from mature rice (cultivar: Nipponbare) seeds. The seeds with the outer skin obtained were immersed in a 70% ethanol solution for 60 seconds and then in a sodium hypochlorite solution containing about 1% of available chlorine for 6 minutes to sterilize the seeds. The seeds were further washed with sterile water.

  The rice seeds sterilized as described above were placed on a medium obtained by adding 30 g / l of sucrose, 2 mg / l of 2,4-PA as a plant hormone, and 8 g / l of agar to the inorganic component composition of the MS medium. Incubation was performed at 28 ° C. for 21 days, illuminated with 2000 lux of light for 16 hours per day. Callus was formed. These calli were cut out from the endosperm portion, and the calli were transferred to a medium having the same composition as described above and cultured for 3 days.

(6) Transgenic Agrobacterium (pUb-OSASA1D / LBA4404 or pUb-OSASAW1 / LBA4404) for gene transfer obtained as described above, A mushroom fungus solution was obtained in 30 ml of a liquid medium containing 20 g / l of sucrose, 2 mg / l of 2,4-PA, 0.2 mg / l of kinetin and 10 mg / l of acetosyringone added to the salt composition. This suspension was placed in a 9 cm petri dish, and 100 rice calli obtained in the above (5) were placed therein, and then immersed for 5 minutes. After soaking, the excess bacterial solution was removed with a paper towel, and then the sucrose 30 g / l, glucose 10 g / l, 2,4-PA 2 mg / l, gellite 2 g / l and acetosyringone 10 mg were added to the inorganic salt composition of the N6 medium. On the solid medium to which / l was added, 20 pieces of the above-mentioned calli were placed on each petri dish. The cells were cultured at 28 ° C. for 3 days in the dark to infect rice calli with Agrobacterium.

(7) Selection of callus transformed with the introduced vector The transformed callus into which the recombinant vector was introduced in the above (6) was washed with sterilized water to which 500 mg / l carbenicillin had been added to remove Agrobacterium bacteria. Removed. After removing excess water from the callus, the callus was added to the inorganic salt composition of the N6 medium with sucrose 30 g / l, 2,4-PA 2 mg / l, carbenicillin 500 mg / l, hygromycin 50 mg / l, and gellite 2 mg / l. 20 were transplanted per petri dish onto the added solid medium. Transformation callus resistant to hygromycin was obtained by culturing at 25 ° C. for 21 days while illuminating with 2000 lux of light for 16 hours per day.

(8) Reselection of transformed callus with vector pUb-OSASA1D Among the transformed callus that is resistant to hygromycin obtained above, a modified D sequence having a function contained in vector pUb-OSASA1D and expressed in plants is a modified D sequence. Only calli that have been sufficiently introduced and transformed as foreign genes are reselected. To do this, 15 transformed calli (5 mm in diameter) were added to the inorganic salt composition of the N6 medium with 30 g / l sucrose, 2 mg / l 2,4-PA, 250 mg / l carbenicillin, and 2 g / l gellite to inhibit cell growth. The cells were transplanted onto a solid medium to which 3 × 10 −4 M of 5-methyltryptophan (hereinafter referred to as 5MT) (an analog of tryptophan) acting as an agent was added. The cells were cultured at 25 ° C. for 21 days while illuminating with 2000 lux of light for 16 hours per day. Transformed calli containing the modified D sequence having the function of being expressed in the plant that was able to grow on the 5MT-supplemented medium were re-selected in this way.

(9) Regeneration of Plants from Transformed Callus Cells 14 to 15 transformed callus cultures having hygromycin resistance and 5MT resistance obtained as described above, 30 g / l of sucrose in the inorganic salt composition of the MS medium, The cells were transplanted to a solid medium containing 30 g / l of sorbitol, 2 g / l of casamino acid, 1 mg / l of NAA, 2 mg / l of BA, 50 mg / l of hygromycin, and 4 g / l of gellite. When cultured at 25 ° C for 30 days while irradiating with 2000 lux light for 16 hours per day, shoots and roots could be regenerated from the transformed calli. The regenerated germs (buds grown to a length of 10 to 30 mm) were transplanted into test tubes (diameter 45 mm, length 25 cm) containing an MS medium containing 30 g / l of sucrose and 2 g / l of gelrite. The transplanted germ was cultured for 20 days to obtain a transformed rice plant.

  By the above method, 10 rice plants could be regenerated from 15 transformed calli containing the OSASAW1 sequence of SEQ ID NO: 1 in the sequence listing according to the first invention as a foreign gene.

  In addition, 10 rice plants could be regenerated from 14 5MT-resistant transformed calli containing the modified D sequence of SEQ ID NO: 12 according to the third invention as a foreign gene.

(10) Gene Analysis of Regenerated Plant of Transformed Rice Plant The DNA encoding ASA present in the rice plant regenerated as described above was analyzed by the PCR according to the following procedure.

  (i) Leaves were collected from the regenerated rice plant obtained in the above (9). 50 mg of the leaf was placed in a 1.5 ml microtube, and 300 μl of 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA was added thereto, and the mixture was ground. 20 ml of 20% SDS was added to the ground material, and the mixture was heated at 65 ° C. for 10 minutes. 100 μl of 5 M potassium acetate was added to the obtained mixture, and the mixture was placed on ice for 20 minutes, and then centrifuged at a centrifugal acceleration of 1,7000 × g for 20 minutes. 200 μl of isopropanol was added to the obtained supernatant, the mixture was inverted and stirred, and the mixture was centrifuged again at a centrifugal acceleration of 1,7000 × g for 20 minutes. The obtained DNA precipitate was dried under reduced pressure. The DNA was dissolved in 100 μl of TE buffer.

  (ii) As the primer for PCR, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 14 in the sequence listing and the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 15 were used.

  Next, using 1 μM of each of these two oligonucleotides as primers and using 5 μl of the DNA derived from the regenerated rice plant as a template, these were used as amplification reaction solutions (10 mM Tris-HCl (pH 8.3), 1.0 mM MgCl2, 50 mM KCl, 0.01% gelatin, pH 8.3, containing 0.2 mM each of dNTPs and 2.5 units of Taq DNA polymetalase) (100 μl). Then, an amplification reaction was performed. The amplification reaction solution used was prepared with a PCR kit (PCR Amplification Kit (manufactured by Takara Shuzo Co., Ltd.)).

  The amplification reaction was performed in a PCR reaction apparatus (Program Temp Control System PC-700 (manufactured by Aztec)) using denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 30 seconds, extension at 72 ° C and 1 minute for three times. The reaction operation was repeated 30 times.

  (iii) When the obtained PCR reaction solution was subjected to agarose electrophoresis according to a conventional method and analyzed, various DNA bands amplified from DNA extracted from the regenerated rice plant were confirmed.

  Furthermore, when those various DNA sequences were analyzed by a base sequence determination kit, the DNA sequence OSASA-W1 sequence according to the first invention (SEQ ID NO: 1) or the modified D sequence according to the third invention (SEQ ID NO: 12) It was confirmed that DNA fragments corresponding to the above existed in various DNA fragments extracted as described above from regenerated rice plants.

  The regenerated rice plants in which the introduced foreign genes could be confirmed by performing the gene analysis by the PCR method as described above were transplanted and cultivated in pots each containing culture soil. Since these regenerated rice plants grew normally, self-fertilized seeds could be obtained.

(11) Measurement of tryptophan content in regenerated transformant rice plant The recombinant vector pUb-OSASAW1 containing the OSASA-W1 sequence of SEQ ID NO: 1 according to the first invention obtained in the above section (10) was introduced. Transformed rice plant having a transformed plant cell, and a transformed rice plant having a plant cell into which a recombinant vector pUb-OSASA1D containing the modified D sequence of SEQ ID NO: 12 according to the third invention has been introduced. Green leaves were collected from the plants.

  1 g of each leaf thus collected was taken, and 100 mg of the leaf was placed in a 1.5-ml first tube, and 1 ml of 50% acetonitrile was further added thereto and ground. The trituration mixture was transferred to a new 1.5 ml second tube and centrifuged at a centrifugal acceleration of 17 000 Xg for 20 minutes. The resulting supernatant was again transferred to a new third tube, 1 ml of 50% acetonitrile was added to the third tube, the mixture was thoroughly inverted and stirred, and centrifuged again. The obtained supernatant was added to the first tube, and this operation was repeated three times. Thus, an acetonitrile extract containing tryptophan extracted from leaves was obtained.

  After the acetonitrile extract was dried under reduced pressure, 1 ml of distilled water was added to obtain an aqueous solution. The aqueous solution was centrifuged at a centrifugal acceleration of 17 000 Xg for 20 minutes to obtain 0.5 ml of the supernatant. 100 μl of the supernatant was mixed with 100 μl of 5 mM DNFB (2,4-dinitro-1-fluorobenzene). The resulting mixture was reacted overnight. 200 μl of acetonitrile was added to the reaction solution, stirred, and then centrifuged at a centrifugal acceleration of 17,000 × g for 20 minutes. An extract of tryptophan was obtained as a supernatant. This was subjected to a high performance liquid chromatography (HPLC) apparatus (manufactured by Tosoh Corporation, Model 8020) to measure the free tryptophan content. The column used for this HPLC was CAPCELL PAK-C18 (manufactured by Shiseido Co., Ltd.), and the developing solvent was acetonitrile-water with a concentration gradient of 60% to 72% of acetonitrile, and the flow rate was 0.8 ml / min, 350 nm. Was measured to determine the tryptophan content.

A normal rice plant (variety: Nipponbare) which had not been transformed was used as a control rice plant. The obtained measurement results are shown in Table 1 below.

  As is clear from the results shown in Table 1 above, when the novel modified DNA sequence according to the third invention is used as a foreign gene and a recombinant vector having a promoter that can be expressed in plant cells, It was confirmed that the introduction of the novel modified DNA sequence according to the present invention can increase the content of free tryptophan produced in rice plants.

Example 4
This example is directed to a method for selecting transformed plant cells according to the ninth invention (claim 8) and a tenth invention (claim), comprising introducing the modified DNA sequence according to the third invention into a rice plant as a foreign gene. The method for producing a transformed plant according to 9) will be exemplified.

(1) Preparation of Recombinant Vector for Introduction of Foreign Gene As a recombinant vector for introduction of a foreign gene, the recombinant vector pUb-OSASA1D described in Example 3, (3) (iii) (modification of SEQ ID NO: 12) Or the recombinant vector pUBdD described in Example 3, (1) (iii) (having the modified D sequence DNA of SEQ ID NO: 12).

(2) Introduction of foreign genes into rice callus cells and selection of transformed cells by Agrobacterium bacterial method or whisker method
(i) Agrobacterium bacteria method Using the recombinant vector pUb-OSASA1D shown in (1) above, Agrobacterium bacteria were prepared by the same method as in (4) of Example 3, and Example 3 A rice callus cell was prepared by the same method as in (5) of Example 3, and an operation of introducing it into rice callus cells was performed by the same method as in (6) of Example 3.

  The transformed callus into which the recombinant vector was introduced was washed with sterilized water to which 500 mg / l carbenicillin had been added to remove Agrobacterium bacteria. After removing excess water from the callus, the callus was sucrose 30 g / l, 2,4-PA 2 mg / l, carbenicillin 500 mg / l, gellite 2 g / l, and 5MT3 A total of 500 cells were transferred on a solid medium to which × 10−4 M was added, 20 cells per petri dish. The cells were cultured at 25 ° C. for 1 month while illuminating with 2000 lux light for 16 hours per day.

  After one month of culture, transformed calli were selected and the results of counting the number of calli obtained are shown in Table 2 below. In the control, a solid medium supplemented with 50 mg / l of hygromycin instead of 5MT was used.

  Transformed callus obtained by selection on the basis of 5MT resistance by the above culture, sucrose 30g / l, sorbitol 30g / l, casamino acid 2g / l, NAA 1mg / l, the inorganic salt composition of the MS medium The cells were transferred to a solid regeneration medium to which BA 2 mg / l, gellite 4 g / l and 5MT3 × 10 −4 M were added. The cells were cultured at 25 ° C. for 30 days while illuminating with 2000 lux of light for 16 hours per day. The results of counting the number of regenerated transformed plants regenerated from the transformed calli are shown in Table 2 below. In the control, a solid medium supplemented with 50 mg / l of hygromycin instead of 5MT was used.

(ii) Direct introduction method using whisker Rice hulls were threshed from mature seeds of rice (variety: Nipponbare). The seeds with the outer skin obtained were immersed in a 70% ethanol solution for 60 seconds and then in a sodium hypochlorite solution containing about 1% of available chlorine for 6 minutes to sterilize the seeds. The seeds were further washed with sterile water.

  The rice seeds sterilized as described above were placed on a medium obtained by adding 30 g / l of sucrose, 2 mg / l of 2,4-PA as a plant hormone, and 8 g / l of agar to the inorganic component composition of the MS medium. Incubation was performed at 28 ° C. for 45 days, illuminated with 2000 lux of light for 16 hours per day. Callus was formed. These calli were cut out from the endosperm portion, and callus having a diameter of 1 mm or less was obtained as a PCV (Packed Cell Volume) in a volume of 3 ml using a stainless mesh sieve having a hole of 1 mm.

  5 g of potassium titanate whisker (LS20, manufactured by Titanium Industry Co., Ltd.) was placed in a 1.5 ml tube, 0.5 ml of ethanol was added, and the mixture was allowed to stand overnight. Ethanol was completely evaporated to obtain sterilized whiskers. 1 ml of sterile water was added to the tube containing the whiskers and stirred well. The whisker and the sterilized water were centrifuged, and the supernatant water was discarded. The whisker was thus washed. This whisker cleaning operation was performed three times. Thereafter, 0.5 ml of R2 liquid medium was added into the tube to obtain a whisker suspension.

  250 μl of callus of 1 mm or less was put into the tube containing the whisker suspension obtained above and stirred. The resulting mixture was centrifuged at 1000 rpm / min for 10 seconds to precipitate callus and whiskers. The supernatant was discarded. A mixture of callus and whisker was obtained.

  To the tube containing the mixture of callus and whisker, 10 μl of the recombinant vector (ie, the recombinant vector pUBdD described above) (containing 10 μg of DNA) was added. A well-shaken homogeneous mixture was obtained.

  The tube containing the homogeneous mixture was then centrifuged at 18000 xg for 5 minutes. The centrifuged mixture was shaken again. This operation of centrifugation and the operation of shaking again were repeated three times.

  The tube containing the callus cells, whiskers, and the recombinant vector containing the modified DNA sequence of SEQ ID NO: 12 according to the third invention obtained as described above was placed in a bathtub of an ultrasonic generator. Was installed so that it could be fully immersed. Ultrasonic waves with a frequency of 40 kHz were irradiated at an intensity of 0.25 W / cm2 for 1 minute. After irradiation, the sonicated mixture was incubated at 4 ° C. for 30 minutes.

  The mixture thus sonicated was washed with an R2 liquid medium to obtain a desired transformed callus cell into which the recombinant vector pUBdD was introduced.

  The callus having the transformed cells obtained by introducing the recombinant vector above was placed in a 3.5 cm petri dish. Further, 3 ml of a liquid medium obtained by adding 30 g / l of sucrose and 2 mg / l of 2,4-PA to the inorganic component composition of the R2 medium was added. Thereafter, callus cells were cultured on a rotary shaker (50 rpm) while irradiating at 2,000 lux of light at 28 ° C. for 16 hours per day to obtain dividing cells.

  On the third day of the culture, 3 ml of the obtained dividing cell suspension (number of dividing cells: 2000) was added to the inorganic component composition of the N6 medium with sucrose 30 g / l, 2,4-PA 2 mg / l, and gellite 3 g. / l and 5MT 10-4M as a selection agent were uniformly spread on a medium. The cells on the medium were cultured for 1 month at 28 ° C. with 2,000 lux of light for 16 hours per day.

  After one month of culture, calli composed of transformed cells could be selected. Table 2 shows the results of calculating the number of callus obtained in this manner. In the control, a solid medium supplemented with 50 mg / l of hygromycin instead of 5MT was used.

Callus consisting of transformed cells obtained by selection based on 5MT resistance by the above culture, sucrose 30g / l, sorbitol 30g / l, casamino acid 2g / l, NAA in the inorganic salt composition of MS medium The cells were transferred to a solid regeneration medium to which 1 mg / l, BA 2 mg / l, gellite 4 g / l and 5MT3 × 10 −4 M were added. The cells were cultured at 25 ° C. for 30 days while illuminating with 2000 lux light for 16 hours per day. A transformed plant regenerated from the transformed callus was obtained. Table 2 shows the results of counting the number of regenerated plants. In the control, a solid medium supplemented with 50 mg / l of hygromycin instead of 5MT was used.

Example 5
This example illustrates a method for obtaining DNA corresponding to the promoter gene of the rice ASA gene.

(1) Preparation of genomic DNA from rice 2 mg of genomic DNA was extracted from 2 g of calli of rice (variety: Nipponbare) according to the known CTAB method (cloning and sequence: 1989, pp. 262 to 264, Rural Bunkasha). did.

(2) Construction of rice genomic DNA library
After 10 micrograms of the genomic DNA was partially digested with a restriction enzyme EcoRI, the resulting DNA fragment was electrophoresed on a 0.8% agarose gel. The DNA fragments fractionated by electrophoresis were transferred to a nylon membrane (Amersham) Hibond N. The DNA thus transferred to the nylon membrane was treated with an alkali denaturing solution (1.5 M NaCl, 2.0 M NaOH) and a neutralizing solution (1.0 M Tris-HCl pH5, 2.0 M NaCl) for 10 minutes, and then at 80 ° C. For 2 hours to fix the DNA to a nylon membrane.

  Next, the labeled probe DNA obtained by labeling with DIG obtained in (5) of Example 1 was used. This labeled probe DNA was hybridized to the above nylon membrane. This operation was performed in the same manner as in Example 1, (5).

  As a result, a DNA which strongly emits a signal on the X-ray film was detected near a DNA size of 6 kb on the nylon membrane. Therefore, these DNAs were separated, and the DNAs were partially digested with the restriction enzyme EcoRI in the same manner as in Example 1, (5). Purification was performed using II Kit (manufactured by Bio 101). The obtained DNA fragment (purified product) was dissolved in 20 μl of TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). Thus, fractionated rice genomic DNA was obtained. This was ligated to a vector using a cloning kit (Lambda ZAPII / EcoRI / CIP Cloning Kit (manufactured by STATGENE)). The obtained recombinant vector was packaged on lambda phage using GigapackII Gold Packaging Extract. Escherichia coli XL1-Blue MRF ′ was infected with the obtained recombinant λ phage to obtain a large number of the above-mentioned recombinant phages. These obtained recombinant phages were used as a fractionated genomic DNA library of rice.

(3) Selection of promoter gene from rice genomic DNA library From the recombinant phage which is the rice genomic DNA library prepared in (2) above, use of the labeled probe DNA prepared in (5) of Example 1 Thus, a recombinant phage incorporating a DNA sequence corresponding to the promoter gene for expression of the rice ASA gene is screened.

  For the purpose of this screening, the recombinant phage was immobilized on a nylon membrane in the same manner as in Example 1 (5), and the labeled probe DNA was hybridized. Thus, a phage into which the desired DNA sequence was integrated was selected. As a result, three phage plaques which seem to have the ASA gene promoter gene incorporated therein were isolated and selected from 100,000 phage plaques. From these three recombinant phages, three types of λ DNA were separately isolated in the same manner as in Example 1 (6).

  Using each of the three types of phage DNA isolated as described above, 5 μl thereof was digested with 10 units of the restriction enzyme EcoRI, and the digested solution was analyzed. The RcoRI-DNA fragments of the three isolated phage DNAs were found to have the same base sequence.

(4) Cloning of genomic DNA containing a DNA sequence corresponding to the rice ASA gene promoter gene As a DNA fragment determined to contain a DNA sequence corresponding to the rice ASA gene as described above, three types of DNA A 6.2 kb-sized DNA fragment cut out from each of the phages with the restriction enzyme EcoRI was inserted into the EcoRI cleavage site of the plasmid vector pBluewscriptIISK (+) using a DNA ligation kit, and ligated. A recombinant plasmid vector was constructed. Escherichia coli XL1-BlueMRF ′ was transformed by introducing the recombinant plasmid vector thus constructed. The obtained transformant was named Escherichia coli XL1-Blue MRF ′ (Os-asa # 7), which was deposited with the Institute of Biotechnology and Industrial Technology on August 18, 1997 as FERM P-16387, Since August 7, 1998, it has been deposited under the terms of the Budapest Treaty under the accession number FERM BP-6452.

  The operations for constructing the recombinant plasmid vector and introducing it into Escherichia coli were performed in the same manner as in Example 1 (6).

  Plasmids were separated and purified from Escherichia coli into which the recombinant plasmid vector had been introduced, using a plasmid purification kit (QIA filter Plasmid Midi Kit, manufactured by QIAGEN). By this purification, 50 μg (50 μl) of three types of plasmid DNAs were obtained from the transformed Escherichia coli obtained above.

  About 1.5 kb of DNA contained in the above-mentioned three types of plasmids obtained by cloning from each of the isolated recombinant phages and flanked by the restriction enzymes EcoRI and BamHI is its base. The following operations were performed to analyze the sequence.

(5) Sequence analysis of cloned DNA When the above-mentioned three types of cloned recombinant plasmids are treated with a commercially available nucleotide sequencing kit, the entire nucleotide sequence of the DNA fragment can be determined. Then, a DNA sequence corresponding to the promoter gene of the rice ASA gene contained in the DNA fragment can be determined. The above base sequence was determined in the same manner as in Example 1 (7).

  As a result of determining the nucleotide sequence of the above-mentioned DNA, it was found that the three types of cloned recombinant plasmids were all identical.

  The DNA of the promoter of the ASA gene thus obtained was confirmed to have the nucleotide sequence described in SEQ ID NO: 7 in the sequence listing described later. The nucleotide sequence of SEQ ID NO: 7 obtained here contained an intron, and the portion encoding the protein was completely identical to the cDNA clone. Among the nucleotide sequences determined for the DNA of the obtained recombinant plasmid, SEQ ID NO: 3 shows the nucleotide sequence of the DNA sequence including the promoter portion of the ASA gene and the exon and intron portions of the DNA.

  In the sequence listing, SEQ ID NO: 3 also shows the partial amino acid sequence of a protein that is the α subunit of the first isozyme of rice ASA. SEQ ID NO: 4 in the sequence listing shows the amino acid sequence of the amino acid coding portion of the nucleotide sequence shown in SEQ ID NO: 3. Further, SEQ ID NO: 5 shows the nucleotide sequence of exon-1 in the DNA of SEQ ID NO: 3, and SEQ ID NO: 6 shows the partial sequence of exon-2 in the DNA of SEQ ID NO: 3.

(6) Promoter activity assay
(i) Preparation of Recombinant Vector for Assay In order to examine the promoter activity of the promoter DNA (SEQ ID NO: 7) obtained above, this DNA (EcoRI-BamHI fragment obtained in (4) above) was The transformation vector pGUS # 3 was obtained by integrating the transformation vector pBI101 (manufactured by Clontech) into the upstream portion of the β-glucuronidase (GUS) gene.

  In the preparation of the transformation vector, the DNA (10 μg) of a plasmid vector pBI101 (manufactured by Clontech) having a size of 12.2 kb containing the NOS terminator of the GUS gene and the kanamycin resistance gene was treated with the restriction enzyme HindIII in M buffer. Digested in 10 units. DNA was precipitated from the digestion reaction, centrifuged and dried. After dissolving the obtained dried product in 10 μl of sterilized water, subsequently, using a DNA planting kit (manufactured by Takara Shuzo Co., Ltd.) to smooth the cleavage site, a DNA purification kit ( Purification was performed using Gene Clean II Kit (manufactured by Bio 101). The purified DNA was dissolved in 10 μl of sterile water. This lysate was digested with 10 units of the restriction enzyme BamHI in K buffer. DNA was precipitated from the digestion reaction, centrifuged and dried. In this way, a vector fragment having a size of 12.2 kb and having blunt ends and BamHI cleavage sites at both ends was obtained (see the left side of FIG. 3 in the attached drawings).

  On the other hand, the plasmid vector DNA (10 μg) containing the above-mentioned rice ASA gene promoter DNA fragment was digested with 10 units of the restriction enzyme EcoRI in H buffer. DNA was precipitated from the digestion reaction, centrifuged and dried. After dissolving the obtained dried product in 10 μl of sterilized water, subsequently, using a DNA planting kit (manufactured by Takara Shuzo Co., Ltd.) to smooth the cleavage site, a DNA purification kit ( Purification was performed using Gene Clean II Kit (manufactured by Bio 101). The purified DNA was dissolved in 10 μl of sterile water. This lysate was digested with 10 units of the restriction enzyme BamHI in K buffer. About 1.5 kb of the digested solution was cut out from the gel by agarose electrophoresis, purified using a DNA purification kit, and dissolved in 10 μl of sterilized water. Thus, a promoter DNA fragment having a size of 1.5 kb and having a blunt end and a BamHI cleavage site at both ends was obtained (see the right side of FIG. 3).

  After mixing 5 μl of the above plasmid vector fragment solution and 5 μl of the above promoter DNA fragment solution, the mixture was treated with a DNA ligation kit to ligate DNA.

  Thus, a circular recombinant vector was prepared in which the blunt-end-BamHI-fragment of the rice ASA gene promoter DNA was ligated to the blunt-end-BamHI-cut vector fragment of the plasmid vector pBI101. This recombinant vector is called pGUS # 3 (see lower part of FIG. 3).

  The vector pGUS # 3 has a GUS gene downstream of the rice ASA gene promoter DNA, has a downstream NOS terminator linked thereto, and has a structure containing a kanamycin resistance gene, and has a size of 13.7 kb. I had.

(ii) Introduction of Recombinant Vector for Assay into Rice Callus Cells The recombinant vector pGUS # 3 obtained above was introduced into rice callus cells in the same manner as in Example 3 (6). Thus, rice callus cells transformed with the recombinant vector pGUS # 3 were obtained.

(iii) Measurement of GUS activity The rice callus transformed with the recombinant vector pGUS # 3 obtained above was cultured at 28 ° C for 3 days, and then about 0.5 g of the callus was placed in a mortar and extracted with 500 μl. Buffer (50 mM NaPO4 (pH 7), 10 mM EDTA, 0.1% Triton X100, 0.1% Sarkosyl, 10 mM β-mercaptoethanol) and triturated. The triturated solution was transferred to a 1.5 ml tube and centrifuged at 17,000 × g for 20 minutes. Of the obtained supernatant, 10 µl was transferred to a new tube, and 100 µl of a 1 mM 4-methylamperiferyl glucuronide (4-MUG) solution as a substrate was added and mixed. The obtained mixture was reacted at 37 ° C. for 3 hours. 1.8 ml of a 3% sodium hydrogen carbonate solution was added to the reaction solution and mixed. It was applied to a fluorescence spectrophotometer (F2000, manufactured by Hitachi, Ltd.) to perform fluorescence absorption measurement. The GUS activity was evaluated under the conditions of 365 nm excitation light and 455 nm emission at that time.

The results of measuring the GUS activity are shown in Table 3 below. The numerical values indicate the amount of 4-methylambelliferone (4-MU) produced by the action of the GUS enzyme per hour per g of protein extracted from callus. As a control, callus into which a pBI101 vector without a promoter was introduced was used.

  The expression of GUS activity as described above proves that the promoter region confirmed from the results of the nucleotide sequence analysis functioned as a promoter.

  The modified rice DNA encoding the α subunit of the first ASA of rice provided by the third invention can be used alone or in combination with other genes to introduce tryptophan, an essential amino acid in the plant, into the plant. And is useful for breeding plants with high nutritional value. Furthermore, the above-mentioned DNA variants are useful for selecting transformed cells and transformed plants by introducing them into plant cells.

FIG. 1 shows a recombinant vector for introducing a foreign gene used for transforming a rice callus cell by a direct introduction method of a foreign gene by a whisker method, and has a base sequence of SEQ ID NO: 12 in the sequence listing. The procedure used in (1) of Example 3 to construct the recombinant vector pUBdD containing the modified D sequence of the third invention therein from the vector pUBA is shown schematically. FIG. 2 is a recombinant vector for introducing a foreign gene used for transforming rice callus cells by a method for introducing a foreign gene by the Agrobacterium method, which contains the above-mentioned modified D sequence therein. FIG. 7 schematically shows the procedure used in (3) of Example 3 to create the vector pUb-OSASA1D from the vector plG121-Hm. FIG. 3 shows a recombinant vector containing the promoter sequence as a foreign gene to be used to transform rice callus cells in a test for assaying the activity of the promoter sequence for rice ASA, comprising the sequence listing A recombinant vector pGUS # 3 ligated with a DNA fragment # 3 containing a promoter sequence having the base sequence shown in SEQ ID NO: 3 was prepared from the vector pBI101 in Example 5, (6). The procedure used is shown schematically.

Claims (9)

  Encodes a protein having the amino acid sequence of SEQ ID NO: 13 in the sequence listing, and having the activity of the α-subunit of the first isozyme of anthranilate synthase, but being insensitive to feedback suppression by tryptophan DNA.   The DNA according to claim 1, which is a DNA having the nucleotide sequence of SEQ ID NO: 12 in the sequence listing and designated as a modified D sequence, and which encodes a protein that is insensitive to feedback suppression by tryptophan. DNA as described.   A DNA encoding a protein having the activity of the α subunit of the first isozyme of rice anthranilate synthase, but which is insensitive to feedback suppression by tryptophan, having the nucleotide sequence of SEQ ID NO: 12 in the sequence listing. A plant cell transformed with the recombinant vector carrying the DNA of claim 2 designated as the modified D sequence, and wherein the introduced DNA can be expressed, Transformed plant.   A seed collected from the transformed plant according to claim 3.   A recombinant vector having a DNA fragment carrying the DNA sequence according to claim 2 having the nucleotide sequence of SEQ ID NO: 12 in the sequence listing and designated as a modified D sequence, wherein the recombinant vector is contained in a host cell. A recombinant vector capable of expressing the DNA sequence.   A recombinant vector having the nucleotide sequence of SEQ ID NO: 12 in the sequence listing and having the DNA fragment carrying the DNA sequence according to claim 2 designated as a modified D sequence, wherein the recombinant vector is contained in a host cell. E. coli transformed with a recombinant vector capable of expressing a DNA sequence.   DNA encoding a protein that is insensitive to feedback suppression by tryptophan and has the activity of the α-subunit of the first isozyme of anthranilate synthase, which is modified to have the nucleotide sequence of SEQ ID NO: 12 in the sequence listing A recombinant vector carrying the DNA of claim 2, designated as the D sequence, which is capable of expressing said DNA in a plant, is introduced into a plant cell callus, whereby A method for increasing the tryptophan content of a plant, comprising obtaining a plant cell callus transformed with DNA, and regenerating the callus into a plant.   DNA encoding a protein that is insensitive to feedback suppression by tryptophan and has the activity of the α-subunit of the first isozyme of anthranilate synthase, which is modified to have the nucleotide sequence of SEQ ID NO: 12 in the sequence listing A recombinant vector carrying the DNA of claim 2 designated as D sequence and an antibiotic resistance gene, wherein said DNA is capable of being expressed in a plant, and introduced into a plant cell; This provides the plant cells with resistance to a tryptophan analog that inhibits the growth of the plant cells, and further comprises selecting cells expressing the resistance to the tryptophan analog, to obtain a transformed plant cell. Selection method.   DNA encoding a protein that is insensitive to feedback suppression by tryptophan and has the activity of the α-subunit of the first isozyme of anthranilate synthase, which is modified to have the nucleotide sequence of SEQ ID NO: 12 in the sequence listing A recombinant vector carrying the DNA of claim 2 designated as D sequence and an antibiotic resistance gene, wherein said DNA is capable of being expressed in a plant, and introduced into a plant cell; This imparts resistance to tryptophan analogs that inhibit the growth of plant cells to plant cells, selects cells expressing resistance to the tryptophan analogs, and further transforms plant cells from the thus selected transformed cells. And producing a transformed plant having an increased tryptophan content.

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