JP2003116553A - Method for expressing foreign gene and vector therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for expressing a foreign gene, capable of expressing the foreign gene stronger than conventional methods, and to provide a recombinant vector therefor. SOLUTION: This method for expressing the foreign gene comprises expressing the foreign gene in a cell by inserting the foreign gene into a downstream region of a promoter, wherein the foreign gene is expressed by inserting nucleic acid fragments having specified base sequences into an upstream region of the foreign gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for expressing a foreign gene and a recombinant vector therefor. More specifically, the present invention provides a method for expressing a foreign gene and a vector therefor, which can promote the expression of the foreign gene more than conventional methods when expressing the foreign gene in cells by a genetic engineering technique. Regarding

[0002]

2. Description of the Related Art Promoting the expression of foreign genes is one of the most needed techniques in genetic engineering techniques, especially when applying the genetic engineering techniques to plants.

As one of the methods, a method of inserting an intron-derived DNA fragment upstream of a foreign gene is known. For example, in Japanese Patent Laid-Open No. 3-103182,
It is described that the expression of the foreign gene is promoted by inserting an intron-derived DNA fragment of the himacatalase gene (CAT-1) upstream of the foreign gene to express the foreign gene. Similar phenomena have been reported for various intron-derived DNA fragments.

Although introns have been used for the purpose of promoting the expression of foreign genes, the use of multiple introns is not common and their effects have not been recognized. For example,
First of alcohol dehydrogenase-1 in maize
The intron and the 6th intron each independently have a gene expression promoting effect, but the effect of linking both is 6th intron.
Less than intron alone use (Mascarenhas et al.
Plant Mol. Biol., 15, 913-920 (1990)). In addition, when two third introns of maize actin are linked, the effect does not reach to single use (Luehrsen et al.
al., Mol. Gen. Genet., 225, 81-93 (1991)).

Although the conventional method of inserting a DNA fragment derived from an intron is effective, in many cases the effect of promoting expression is insufficient, and a method that enables stronger expression has been desired.

[0006]

Therefore, an object of the present invention is to provide a method for expressing a foreign gene and a recombinant vector therefor, which can express the foreign gene more strongly than conventional methods.

[0007]

Means for Solving the Problems As a result of earnest research, the inventors of the present invention have identified a plurality of intron-derived DNA fragments of the same or different which have the effect of promoting the expression of a foreign gene when inserted upstream of the foreign gene. The present invention has been completed by finding that the expression of a foreign gene is remarkably increased by inserting it upstream of the foreign gene, as compared with the conventional method of inserting one intron-derived DNA fragment.

That is, the present invention provides a method of inserting a foreign gene downstream of a promoter to express the foreign gene in a cell, and the effect of promoting the expression of the foreign gene upstream of the foreign gene. There is provided a method for expressing a foreign gene, which comprises inserting a plurality of identical or different intron-derived DNA fragments having the above-mentioned expression of the foreign gene.

The present invention also has the effect of promoting the expression of a foreign gene inserted upstream of the promoter, the foreign gene inserted downstream thereof, and the foreign gene.
Provided is a recombinant vector containing a plurality of DNA fragments derived from the same or different introns.

The present inventors have found that even when the intron sequence of the corn ubiquitin gene having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing is inserted alone upstream of the foreign gene. It was found that the present invention has an effect of promoting the expression of, and completed the second invention of the present application.

That is, the present invention also provides a method of expressing a foreign gene by inserting the foreign gene downstream of a promoter to express the foreign gene in a cell. Provided is a method for expressing a foreign gene, which comprises inserting the sequence shown or a functional variant thereof to express the foreign gene.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION According to the method of the present invention, two or more intron-derived DNA fragments having the effect of promoting the expression of a foreign gene are inserted upstream of the foreign gene to be expressed, thereby expressing the foreign gene. It is characterized by promoting.

Here, "an intron-derived DNA fragment having an effect of promoting the expression of a foreign gene" means that the expression of the foreign gene can be detected as compared with the case of expressing the foreign gene without inserting it. It means an intron-derived DNA fragment that has the effect of increasing to a certain degree. Various intron-derived DNA fragments themselves are known, and for example, the first intron of the castor catalase gene (CAT-1) of castor bean (JP-A-3-103182, Tanaka et al.
L. Nucleic Acids Res. 18, 6767-6770 (1990)), a maize UDP-glucose: flavonol glycosyltransferase intron (Callis et al., G.
enes & Develop. 1, 1183-1200 (1987)), corn alcohol dehydrogenase-1 first intron (Ca
llis et al., Genes & Develop. 1, 1183-1200 (198
7)), corn alcohol dehydrogenase-1
Second and sixth introns (Mascarenhas et al., Plant
Mol. Biol. 15, 913-920 (1990)), maize shrunken-1 first intron (Vasil et al., Plant Ph.
ysiol. 91, 1575-1579 (1989)), a translation elongation factor (transl) of Arabidopsis thaliana.
ation elongation factor) EF-1α 1st intron (Curi
e et al. Nucleic Acids Res. 19, 1305-1310 (1991))
And the first intron of rice actin (McElroy et al.
Plant Cell 2, 163-171 (1990)) and the like. However, the intron-derived DNA fragment that can be used in the present invention is not limited to these,
Any one may be used as long as it can promote the expression of a foreign gene located downstream thereof.

The inventors of the present invention have previously reported that the rice phospholipase D (PLD) gene cDNA and genomic DNA.
The intron of the rice PLD gene was found by comparing the nucleotide sequences of, and one of these introns was found to have the effect of significantly promoting the expression of the gene downstream thereof, and a patent application was filed (PCT / JP96 / 00812).
The base sequence of this intron is shown in SEQ ID NO: 1 in the sequence listing. In the present invention, the intron-derived DNA fragment shown in SEQ ID NO: 1 can also be used. Moreover, the intron sequence of the maize ubiquitin gene represented by SEQ ID NO: 3 and the himacatalase intron sequence represented by SEQ ID NO: 2 in the sequence listing can also be preferably used.

Generally, in a DNA sequence having a physiological action, one or more nucleotides are added, inserted,
It is widely accepted by those skilled in the art that the physiological activity may be maintained even if it is deleted or substituted. In the present invention, the above-mentioned known intron-derived DNA fragment and the intron-derived DNA fragment represented by SEQ ID NO: 1 are modified as described above and have a function of promoting expression of a downstream gene.
The NA fragment is also included in the "intron-derived DNA fragment" in the present invention. That is, in the known intron-derived DNA fragment and the intron-derived DNA fragment represented by SEQ ID NO: 1 in the Sequence Listing, one or more nucleotides have been added, deleted or substituted, and the expression of a gene existing downstream thereof can be expressed. A DNA fragment having a promoting action is also included in the "intron-derived DNA fragment" of the present invention. Here, such a modified intron-derived DNA fragment is the same as the original intron-derived DNA fragment.
It is preferable that the homology is 0% or more, more preferably 90% or more. Similarly, the "functional variant" described in the claims of the present application also promotes the expression of a gene having one or more nucleotides added, deleted or substituted in the original sequence and located downstream thereof. It has a homologous action, and preferably has 70% or more, more preferably 90% or more homology with the original sequence. For example, "a functional variant of the sequence shown in SEQ ID NO: 1" refers to a gene having one or more nucleotides of the sequence shown in SEQ ID NO: 1 added, deleted or substituted and located downstream thereof. It means a sequence having an action of promoting expression and having a homology of preferably 70% or more, more preferably 90% or more with the original sequence.

Each of the above intron-derived DNA fragments has a known base sequence and its origin, and thus can be easily prepared by a conventional PCR method. Further, if the length is about 200 bp or less, it is possible to perform chemical synthesis. In addition, the above modified intron-derived DNA
The fragment can also be easily prepared by a conventional site-directed mutagenesis method or chemical synthesis method.

In the method of the present invention, the intron-derived D is located upstream of the foreign gene to be expressed, that is, at the transcription region of the foreign gene, and more preferably at the 5'end side of the transcription region.
A plurality of NA fragments are inserted. The intron-derived DNA fragment is preferably inserted between the promoter and the foreign gene. The intron-derived DNA fragment may be inserted immediately upstream of the foreign gene to be expressed, but other sequences may be interposed between the intron-derived DNA fragment and the foreign gene. The length of the intervening sequence is not particularly limited, but is usually about 0 to 1000 bp. Further, the promoter and the intron-derived DNA fragment may be directly linked, or another sequence may be interposed therebetween. The length of the intervening sequence is not particularly limited, but is usually about 0 to 1000 bp.

In the method of the present invention, the intron-derived D
A plurality of NA fragments, preferably 2 to 5, and more preferably 2 or 3 NA fragments are inserted. Intron-derived DN to insert
The A fragments may have the same sequence or different sequences. The plurality of intron-derived DNA fragments to be inserted may be directly linked to each other, or other sequences may be interposed between them. The length of the intervening sequence is not particularly limited, but is usually about 0 to 1000 bp.

As the promoter, any promoter capable of expressing a downstream foreign gene can be used, and a preferred example is the 35S promoter, but it is not limited thereto.

The present invention also provides a recombinant vector to which the above-mentioned method of the present invention is applied. That is, the present invention provides a promoter, a foreign gene to be expressed linked to the downstream thereof, and a plurality of introns derived from the same or different, which are inserted upstream of the foreign gene and have an effect of promoting the expression of the foreign gene. A recombinant vector containing a DNA fragment is provided. Such a recombinant vector can be obtained by inserting the plurality of intron-derived DNA fragments and a foreign gene into an appropriate expression vector.
Since the nucleotide sequence of the cloning site of the expression vector is known, this insertion can be easily performed by using an appropriate restriction enzyme and a linker if necessary.

Various types of such expression vectors are well known in the art and are commercially available. These expression vectors include at least a replication origin for replication in a host cell, a promoter, a cloning site that provides a restriction enzyme site for inserting a foreign gene, and a selectable marker such as a drug resistance gene.
It contains a terminator for stable termination of transcription and an SD sequence when the host is a bacterium. In the method of the present invention,
Any of these known expression vectors can be adopted.

The present inventors have found that even when the intron sequence of the maize ubiquitin gene having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing is inserted alone upstream of the foreign gene. It was found that it has an effect of promoting the expression of Therefore, it is within the scope of the present invention to insert only one sequence shown in SEQ ID NO: 3 or a functional derivative thereof in place of the above plural intron-derived sequences. However, even in the case of the sequence shown in SEQ ID NO: 3, it is more effective to insert a plurality, especially
The effect is large when it is inserted together with the rice PLD intron sequence shown in SEQ ID NO: 1.

[0023]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 The rice PLD gene has a 173 bp first intron at the position corresponding to the 5'untranslated region of mRNA (SEQ ID NO: 1, WO 95/09234). The effect of the intron on gene expression in plant cells was investigated. 20mer primer containing 10 bases each of exon (5'-TCACCACCCGGTAAGCCCAG-3 ', 3'-CCCCCGCGTC
CATCCCGCTC-5 ′) was synthesized, and PCR was performed using the rice genomic clone as a template. The PCR reaction consisted of 50 pmol of each primer mixture, 200 μM dATP, dCTP, dGTP.
And dTTP, 1 x PCR buffer (Takara Shuzo) and 2.5
U AmpliTaq DNA polymerase (Takara Shuzo) was used in a total volume of 50 μl. The reaction was repeated 30 cycles according to the following temperature conditions; in a DNA thermocycler (Perkin Elmer Cetus): 94 ° C for 1 minute, 40 ° C for 1 minute, and 72 ° C for 2 minutes 30 seconds.

The PCR product was subcloned into a PCRII vector (Invitrogen), and Eco
The fragment cut out with RI was treated with blunt ends, and then the plasmid pBI221 (35S promoter (hereinafter
The vector pBI221P (35S pr) in which the β-glucuronidase gene (hereinafter referred to as “GUS”) is inserted into the SmaI site of the downstream of (35S pro).
o, PLD intron, GUS) was prepared. Further, the plasmid was digested with BamHI and treated with blunt ends, and then the above fragment was incorporated into the vector pBI221PP (35S pr
o, PLD intron x 2, GUS) was prepared.

Further, pIG221 described in Japanese Patent Laid-Open No. 03-103182 (Himacatalase intron (SEQ ID NO: 2 in the sequence listing) and GUS in this order in the downstream of 35S pro)
Was digested with XbaI and treated with blunt ends, and then the DNA fragment having the sequence of the PLD intron was inserted into the vector pIG221P (35S pro, PLD intron, Catalase intron,
GUS) was prepared.

[0027] Cultured rice cells were prepared from immature embryos of Japanese rice varieties (Hiei et al. The Plant Journal,
6,271-282 (1994)), previous report (Shimamoto et al. Natur
e, 338, 274-276 (1989)), and the β-glucuronidase (GUS) activity was measured after introducing the above gene. The results are shown in Table 1.

As shown in Table 1, by introducing two homologous or heterologous introns, a remarkable increase in GUS activity was recognized as compared with the case of using them alone. At the same time, PLD
It has been clarified that the DNA fragment having the intron base sequence is a DNA fragment that can obtain a gene expression promoting effect when a plurality of DNA fragments having the intron base sequence are used.

[0029]

[Table 1] Table 1

Example 2 Maize Ubiquitin Gene (Ubi-1: Christensen
AH et al. Plant Mol. Biol., 18, 675-689 (1982))
The intron was also examined for its promoting effect on the expression of foreign genes. There are two types of vectors used.

First, a vector for investigating the effect of single use was prepared by the following method. The promoter and intron portion of the ubiquitin gene was cut out with PstI (SEQ ID NO: 3) and inserted into the PstI site of pUC18 vector. The intron portion was cut out with BglII and BamHI and inserted into the BamHI site of the pBI221 vector to obtain the vector pBI221U (35S pro, Ubiquitin intron, GUS).

Next, a vector for investigating the effect of multiple use was prepared by the following method. Bg intron part
lBI and BamHI, and after blunt-end treatment, pBI2
It was inserted into the SmaI site of 21 vector. Furthermore, the BamHI site of the vector is treated with blunt ends,
By inserting the described PLD intron, the vector pBI221PU (35S pro, PLD intron, Ubiquitin intron,
GUS). These vectors were introduced into protoplasts by the method described above, and GUS activity was measured.

As shown in Table 2, the effect of promoting the expression of GUS was recognized by using the ubiquitin intron alone. Further, the combined use of the PLD intron and the ubiquitin intron showed a stronger promoting effect as compared with the case where each intron was used alone.

[0034]

[Table 2] Table 2

[0035]

INDUSTRIAL APPLICABILITY The present invention provides a method for expressing a foreign gene in which the expression of the foreign gene is significantly promoted as compared with conventional methods, and a recombinant vector therefor. According to the present invention, the expression of a foreign gene introduced by a genetic engineering method is promoted, and thus the present invention is expected to greatly contribute to the field of genetic engineering.

[0036]

[Sequence list] SEQUENCE LISTING <110> JAPAN TOBACCO INC. <120> Method for Expressing Foreign Genes and Vectors Therefor <130> PF152-7-DA <160> 3

[0037] <210> 1 <211> 173 <212> DNA <213> Oryza sativa <400> 1 gtaagcccag tgtgcttagg ctaagcgcac tagagcttct tgctcgcttg cttcttctcc 60 gctcagatct gcttgcttgc ttgcttcgct agaaccctac tctgtgctgc gagtgtcgct 120 gcttcgtctt ccttcctcaa gttcgatctg attgtgtgtg tgggggggcg cag 173

[0038] <210> 2 <211> 217 <212> DNA <213> castor-oil plant <400> 2 acatggatcc ctacagggta aatttctagt ttttctcctt cattttcttg gttaggaccc 60 ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat tttttaattg 120 attggttatg gtgtaaatat tacatagctt taactgataa tctgattact ttatttcgtg 180 tgtctatgat gatgatgata gttacagaac cgtcgac 217

[0039] <210> 3 <211> 1010 <212> DNA <213> maize <400> 3 gtacgccgct cgtcctcccc ccccccccct ctctaccttc tctagatcgg cgttccggtc 60 catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt 120 tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat 180 tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga 240 cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct 300 ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc 360 ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt 420 caaactacct ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata 480 gttacgaatt gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc 540 gggttttact gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg 600 tggttgggcg gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg 660 tgtatttatt aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta 720 agatggatgg aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc 780 atatacatga tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat 840 tataataaac aagtatgttt tataattatt ttgatcttga tatacttgga tgatggcata 900 tgcagcagct atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt 960 actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag 1010

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shinji Morioka             2-2-1 Toranomon, Minato-ku, Tokyo, Japan             Bako Sangyo Co., Ltd. (72) Inventor Yoshiki Kuraya             Tobacco, Toyota-cho, Iwata-gun, Shizuoka 700 Tobacco, Japan             Sangyo Co., Ltd. Genetic Breeding Research Institute F term (reference) 4B024 AA08 AA20 CA01 DA01 EA04                       FA01 FA20 GA11

Claims (22)

[Claims] 1. A method for expressing a foreign gene, which comprises inserting the foreign gene downstream of a promoter and expressing the foreign gene in a cell, having the effect of promoting the expression of the foreign gene upstream of the foreign gene. Alternatively, a method for expressing a foreign gene is characterized by inserting a plurality of different intron-derived DNA fragments to express the foreign gene. 2. The method according to claim 1, wherein the plurality of intron-derived DNA fragments are inserted between the promoter and the foreign gene. 3. The method according to claim 1, wherein the cells are plant cells. 4. The method according to any one of claims 1 to 3, wherein at least one of the plurality of intron-derived DNA fragments contains a sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof. Method. 5. The method according to claim 4, wherein the plurality of intron-derived DNA fragments include two sequences represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof. 6. The method according to any one of claims 1 to 4, wherein at least one of the plurality of intron-derived DNA fragments contains the sequence represented by SEQ ID NO: 2 in the sequence listing or a functional variant thereof. . 7. The plurality of intron-derived DNA fragments respectively include the sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof and the sequence represented by SEQ ID NO: 2 in the sequence listing or a functional variant thereof. The method of claim 6 including. 8. The method according to any one of claims 1 to 3, wherein at least one of the plurality of intron-derived DNA fragments contains a sequence represented by SEQ ID NO: 3 in the sequence listing or a functional variant thereof. Method. 9. The plurality of intron-derived DNA fragments respectively comprises the sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof and the sequence represented by SEQ ID NO: 3 in the sequence listing or a functional variant thereof, respectively. 9. The method of claim 8 including. 10. A promoter, a foreign gene to be expressed which is linked to the downstream thereof, and a plurality of intron-derived DNAs, which are inserted upstream of the foreign gene and which have the effect of promoting the expression of the foreign gene, And a recombinant vector containing the fragment. 11. The recombinant vector according to claim 10, wherein the plurality of intron-derived DNA fragments are inserted between the promoter and the foreign gene. 12. The recombinant vector according to claim 10 or 11, which is replicable in plant cells. 13. At least one of the plurality of intron-derived DNA fragments contains a sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof.
The recombinant vector according to any one of 2 above. 14. The recombinant vector according to claim 13, comprising, as the plurality of intron-derived DNA fragments, two sequences represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof. 15. The method according to claim 10, wherein at least one of the plurality of intron-derived DNA fragments contains the sequence represented by SEQ ID NO: 2 in the sequence listing or a functional variant thereof.
The recombinant vector according to any one of 2 above. 16. The plurality of intron-derived DNA fragments respectively comprises a sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof and a sequence represented by SEQ ID NO: 2 in the sequence listing or a functional variant thereof. The recombinant vector according to claim 15, which comprises. 17. The method according to claim 10, wherein at least one of the plurality of intron-derived DNA fragments contains the sequence represented by SEQ ID NO: 3 in the sequence listing or a functional variant thereof.
The recombinant vector according to any one of 2 above. 18. The plurality of intron-derived DNA fragments respectively comprises the sequence represented by SEQ ID NO: 1 in the sequence listing or a functional variant thereof and the sequence represented by SEQ ID NO: 3 in the sequence listing or a functional variant thereof, respectively. The recombinant vector according to claim 17, which comprises. 19. In a method of expressing a foreign gene by inserting the foreign gene downstream of a promoter and expressing the foreign gene in a cell, the sequence represented by SEQ ID NO: 3 in the sequence listing or the sequence thereof is provided upstream of the foreign gene. A method for expressing a foreign gene, which comprises inserting a functional mutant to express the foreign gene. 20. The method according to claim 19, wherein the sequence represented by SEQ ID NO: 3 in the sequence listing is inserted. 21. The method according to claim 19 or 20, wherein the sequence represented by SEQ ID NO: 3 in the sequence listing or a functional variant thereof is inserted between the promoter and the foreign gene. 22. The cell is a plant cell.
22. The method according to any one of claims 21 to 21.