JPH0678777A - New vector derived from cyanobacteria - Google Patents

Abstract

PURPOSE:To provide a DNA sequence useful for surely and readily introducing another biological gene into a bacterium of the genus Cyanobacteria. CONSTITUTION:The objective DNA sequence of the formula or DNA sequence capable of manifesting the activity of a genetic region related to the replication substantial equal to that is provided. A DNA is recovered from a cultured microbial cell of Cyanobacteria PCC7002 to afford the whole plasmid by precipitating treatment with ethanol. The resultant whole plasmid is then separated into each plasmid fraction by the agarose gel electrophoresis and the objective plasmid pAQ1 is obtained from a DNA band corresponding to about 4 kilo base pairs. In order to determine the whole base sequence thereof, the plasmid pAQ1 cleaved in a restriction enzyme HindIII site is used to construct a shuttle vector and the DNA fragments derived from the plasmid pAQ1 are obtained in a large amount.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cyanobacterial PC.
C7002 (Cyanobacteria, Agmenellum
quadruplicatum PR-6) determining the entire nucleotide sequence of the endogenous plasmid pAQ1 and DNA derived therefrom, which has a gene region involved in replication substantially retaining its activity, an expression vector comprising the DNA, and The present invention relates to a transformant transformed with the expression vector. The present invention also relates to the use of a DNA encoding a gene region involved in replication derived from the endogenous plasmid pAQ1 of the above-mentioned cyanobacterial PCC7002, and a product obtained by using the DNA.

[0002]

2. Description of the Related Art It is known that proteins play an important role in sustaining life, and effective utilization of their functions is considered to have great significance for human beings. From this point of view, various technological developments have been made. Among these techniques, animal cells, plant cells, or microorganisms, particularly Escherichia coli, which utilize the progress of gene manipulation technology in recent years are used. Applied technologies including the synthesis of all proteins have been attracting attention. However,
At the same time, various problems associated with it, such as economic and efficiency problems, are occurring. Also, it has been pointed out that the increase of carbon dioxide concentration in the atmosphere is one of the causes of global warming in recent years. As one of the methods for recovering carbon dioxide, attention has been paid to a method utilizing photosynthesis of cyanobacteria, that is, a reaction of synthesizing oxygen and an organic substance such as sugar from carbon dioxide and water by solar energy. Cyanobacteria are prokaryotes that perform photosynthesis similar to higher plants. However, prokaryotes grow faster than higher plants and can be easily cultured in the presence of trace amounts of metal ions and nitrate ions, and thus are more economical and efficient in utilizing carbon dioxide in the atmosphere. Moreover, it is presumed that the gene translation mechanism of cyanobacteria is basically similar to that of Escherichia coli, and it is considered that the heterologous protein synthesis technology in Escherichia coli can be utilized. Despite the speculation of such advantages, the fact is that the host-vector system necessary for heterologous protein synthesis in cyanobacteria has hardly been constructed. A kind of cyanobacteria such as cyanobacteria P
It is known that CC7002 has at least a plurality of plasmids, among which the smallest plasmid is present (TM Robert, et a.
l. , Cell, 9, 551-557 (1976)).

[0003]

Cyanobacteria PCC7002 has at least 6 types of plasmids. Of these, the endogenous plasmid pAQ1 is the smallest plasmid, and is required for the heterologous protein synthesis in cyanobacteria based on this plasmid. The research was advanced to construct a new host-vector system. The present invention aims to develop means for more reliably and easily introducing a gene of another organism into cyanobacteria. In particular, for that purpose the cyanobacterial PCC700
The entire nucleotide sequence of the endogenous plasmid pAQ1 in 2 is analyzed, and the gene region involved in replication, which contributes to the development of a host-vector system necessary for heterologous protein synthesis using genetic engineering techniques, is It is to develop a DNA having an activity, an expression vector containing the DNA, and a transformant transformed with the expression vector.

[0004]

According to the present invention, the endogenous plasmid pAQ1 of the cyanobacterial PCC7002.
Of the whole nucleotide sequence of Escherichia coli was determined, and from its analysis, the DNA sequence having the gene region involved in replication substantially retained its activity was elucidated, and the gene region involved in replication was substantially activated. An expression vector is provided that has the DNA sequence retained intact. According to the present invention, there is particularly provided a DNA sequence represented by SEQ ID NO: 2 in the sequence listing or a DNA sequence exhibiting the activity of a gene region involved in replication substantially equivalent thereto, and the DNA sequence represented by SEQ ID NO: 2 An expression vector characterized by containing a sequence or a DNA sequence showing the activity of a gene region involved in replication substantially equivalent thereto has been developed and provided, and a transformant transformed with the expression vector is provided. Sent. In particular, the present invention provides improved techniques for performing genetic engineering techniques using E. coli or cyanobacteria as host cells. Further, according to the present invention, there is provided a means for controlling gene replication using a protein having the amino acid sequence represented by SEQ ID NO: 3 or a protein having an activity equivalent to replication substantially equivalent thereto.

Cyanobacteria PCC7 in the present invention
To determine the entire nucleotide sequence of the endogenous plasmid pAQ1 of 002, first the cyanobacterial PCC700
From the bacterial cells obtained after culturing and growing 2 in the usual manner,
The DNA is recovered and then subjected to ethanol precipitation to obtain all plasmids, which are then subjected to agarose gel electrophoresis to separate each plasmid fraction. From the DNA band corresponding to about 4 kilobase pairs, the desired plasmid p
Get AQ1. To determine the entire nucleotide sequence of plasmid PAQ1 thus obtained, to construct a shuttle vector using those cleaved with restriction enzyme Hin dIII site of plasmid PAQ1, to prepare a DNA fragment derived from plasmid PAQ1 large amount . Here, as a plasmid suitable for constructing this shuttle vector, a plasmid derived from Escherichia coli can be mentioned, for example, pUC
System, M13 system and the like plasmids. In order to determine the entire base sequence of the DNA fragment corresponding to the plasmid pAQ1, the shuttle vector prepared in large quantity was digested with an appropriate restriction enzyme, and then the base sequence was analyzed by the dilation mutant method using exonuclease. Regarding the region whose base sequence cannot be determined by the above operation, the + chain and − chain of the plasmid pAQ1 were directly synthesized using the primers and analyzed to determine the base sequence. The entire nucleotide sequence of the plasmid pAQ1 thus obtained is shown in SEQ ID NO: 1, and the plasmid pAQ1
Has a DNA sequence consisting of 4,809 base pairs. Since when utilizing a plasmid pAQ1, it is essential to allowed to cleave the plasmid pAQ1 with the restriction enzyme _Hin dIII, first restriction enzyme Hin
The plasmid pAQ1 was cleaved with dIII, and the fragment obtained from the plasmid was cloned.

The presence of at least three protein coding regions in the plasmid pAQ1 was predicted as a result of analysis,
A shuttle vector was constructed using each of the plasmids lacking the three types of protein coding regions, and each clone was prepared using Escherichia coli or the like. Then, the replication efficiency of each clone was used with the transformation efficiency in cyanobacteria PCC7002 as an index. Add consideration. Note that limiting the total DNA sequence information obtained for plasmid pAQ1 enzyme Hin dIII site, but it is estimated that by cutting the single protein coding region of which, with this restriction enzyme Hin dIII plasmid p
Cleavage of AQ1 is indispensable for using plasmid pAQ1. Therefore, whether or not it is involved in adversely affecting the gene region involved in replication is an important issue in the analysis. is there. The result of this analysis, the holding since the gene region with finding that the gene region consisting 2832 bases are involved in replication that is cleaved by the restriction enzyme _{Hin dIII,} a region of the gene involved in the replication substantially active remains We found that it was necessary to construct the DNA sequence described. Next, in order to develop an expression vector having a DNA sequence having a gene region involved in replication constructed in such a manner that it remains substantially active, the shuttle vector and the plasmid pAQ1 were cleaved with appropriate restriction enzymes and obtained. A new shuttle vector is constructed by using the DNA fragment obtained by further adding bases as necessary, and the gene region involved in replication is substantially examined while examining the replication efficiency with the transformation efficiency in the cyanobacteria PCC7002 as an index. A shuttle vector is constructed with the DNA sequence retained in the active state. Suitable plasmids for constructing this shuttle vector include Escherichia coli-derived plasmids, for example, pUC and M13 plasmids. Thus, an expression vector containing a DNA sequence derived from the endogenous plasmid pAQ1 of Cyanobacteria PCC7002 and having a gene region involved in replication substantially while retaining its activity is constructed. In addition, cyanobacteria PCC70
A usual method can be used to transform E. 02. S. Buzby, et al. ，
Science, 230, 805-807 (1985).
Can be performed according to the method described in.

According to the invention, in particular SEQ ID NO: 2 of the sequence listing
A DNA sequence showing the activity of the DNA sequence shown in or a gene region involved in replication substantially equivalent thereto has been constructed, and the DNA sequence shown in SEQ ID NO: 2 or a gene involved in replication substantially equivalent thereto An expression vector characterized by containing a DNA sequence showing the activity of the region is developed and provided, and a transformant transformed with the expression vector is provided. In particular, the present invention provides an improved technique for carrying out gene manipulation techniques using Escherichia coli or cyanobacteria as host cells. The DNA sequence shown in SEQ ID NO: 2 is a restriction enzyme.
Protein coding regions have been cleaved by _Hin dIII ORF1 (open reading frame 1)
It is constructed by cutting out the DNA sequence portion from the shuttle vector partially containing each of the above with an appropriate restriction enzyme and adding an appropriate modification. The DNA sequence thus obtained, which is represented by SEQ ID NO: 2, has a function of substituting or adding a base contained therein as long as it substantially has the function.
Alternatively, it can be deleted and altered to something other than the sequence. Examples of such a modification method include a method using an appropriate restriction enzyme and DNA synthase, and ligase, and an oligonucleotide-directed mutation method (oligonomic).
leotide directed mutagen
sis), for example M. Smith
And S. Gillam "Genetic Eng"
ineering (J. K. Setlow and
A. Hollaender eds. ), Vol,
3, P.P. 1 (1981), Methods in E
nzymology, Vol. 153-155 (19
1987), Academic Press, CA
Chemical modification methods such as those described in (1) or those described in the literature cited therein are cited. The DNA sequence thus obtained is the DNA shown in SEQ ID NO: 2.
As long as it shows the activity of the gene region involved in replication that is substantially equivalent to the sequence, it can be used in the same manner for the development of an expression vector containing it.

The expression vector of the present invention is not particularly limited as long as it contains a DNA sequence derived from the endogenous plasmid pAQ1 of the cyanobacteria PCC7002 and having a gene region involved in replication while substantially retaining its activity. There is no particular limitation as long as it is capable of autonomous replication in the host. In addition, the expression vector of the present invention may have bases substituted, added, or deleted in a region other than a DNA sequence in which a gene region involved in its replication is substantially retained while retaining its activity. Thus creating restriction enzyme sites for inserting appropriate foreign genes. Such restriction enzyme sites should be designed so that the expression of the foreign gene can be favorably performed. In particular, E as the restriction enzyme site in the expression vector of the present invention
co RI, Cla I, Kpn I, Sma I, Bam H
I, Pst I, and Xba I are included, and the second and third DNA sequences shown in SEQ ID NO: 1 in the sequence listing are also included.
Pst I, Sma I, and
Those with Eco RI are preferred. The expression vector may have various commonly used control sequences or control sequences in addition to the above DNA sequences. Examples of such a control or sequence for control include a promoter, a ribosome binding site (for example, SD sequence), a translation initiation site and a codon, a termination site and a codon, a terminator and the like. The expression vector may have an origin of replication, a selection marker and the like. Examples of selectable markers for this expression vector include various antibiotic resistance genes such as ampicillin resistance gene, tetracycline resistance gene and neomycin resistance gene. In addition, these are derived from various bacteria, derived from bacteriophage,
It may have various commonly used control sequences or control sequences derived from animal viruses including insect and mammalian cells. Examples of the control or sequences for control include those used in various viral vectors, various plasmid vectors, cosmid vectors, shuttle vectors and the like. Examples of these vectors include Escherichia coli, particularly EK type plasmid vector, λgt type phage vector, and cyanobacteria-derived vector. The promoter that can be used in the above vector can be used without particular limitation as long as it can be expressed in a host, but tryptophan (tr
p) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, bacteriophage-derived lambda (λ) PL promoter, N-gene ribosome binding site promoter,
SP01 promoter, SP02 promoter, GAP
A variety of promoters, PGK promoters, SV40 promoters, HSV1 TK promoters and the like, which are well known to those skilled in the art, can be used as long as they are not against the object of the present invention. These gene control sequences can be used in the construction of a vector for expressing a cDNA containing a nucleotide sequence encoding a foreign gene by appropriately combining them or chemically modifying them and incorporating them into a vector.
For example, the translation initiation codon ATG and the termination codon TAA,
TGA or TAG may be contained as a gene control sequence of the cDNA of the present invention, and they may be arranged in combination of one or more or in combination with other codons.

The expression vector of the present invention is introduced into a suitable host, particularly a host cell, according to a commonly known method to transform the host cell, and then the host cell thus transformed. By proliferating by using a method such as culture, cells having a large amount of the foreign gene called a transformant and having the ability to produce the protein encoded by it can be obtained. Gram-negative bacteria such as Escherichia coli or cyanobacteria can be preferably used as the host, particularly the host cell used here. As a method for introducing the gene expression vector of the present invention into the above-mentioned host, a method usually used in the field of gene recombination technology can be used, for example, mixing competent cells with the above vector, or Can be carried out by binding the vector to a carrier after incorporation into a protoplast, or by using calcium phosphate coprecipitation method, electric pulse method, virus vector method, microinjection method or the like. The transformant thus obtained is
After proliferating with the expression of the foreign gene suppressed,
It is also possible to induce expression of the gene. Proliferation or culture of this transformant can be carried out using various ordinary cell culture media, such as, for example, carbon sources, nitrogen sources, vitamins, amino acids, nucleobases,
Including inorganic salts and the like, as appropriate meat juice, peptone, casamino acid, yeast extract, fish meat extract, potato, wort, those added with antibiotics, suitable medium is generally widely commercially available. It can be used as it is or after being modified appropriately. In growing or culturing the above transformant, conditions suitable for growth of the transformant such as pH, temperature, aeration, stirring, light (illuminance), carbon dioxide concentration, and frequency of medium exchange are appropriately determined by experiments or the like. You can decide.

The protein based on the expression of the foreign gene in the gene expression vector of the present invention produced by growing or culturing the above transformant or by inducing the expression of the gene is separated and collected by a usual operation. can do. Examples of this separation and collection method include ultrasonic crushing of cells, mechanical crushing, freezing and thawing,
In addition to a method using osmotic shock, a method of separating from the culture supernatant by, for example, precipitating using a protein precipitating agent and the like can be mentioned. In addition to the above separation method,
Further, the target protein can be purified by utilizing various separation and purification methods that are generally widely used by utilizing its physical properties and chemical properties. Such separation and purification methods include solubilization treatment by homogenizer, ultrasonic cell disruption, extraction treatment with buffer containing various salts, solubilization or precipitation treatment with acid or alkali, and extraction with organic solvent. Alternatively, precipitation treatment, salting out using a protein precipitating agent such as ammonium sulfate, dialysis, ultrafiltration using a membrane filter, gel filtration chromatography, adsorption chromatography, ion exchange chromatography, reverse phase chromatography, countercurrent distribution chromatography. Chromatography, affinity chromatography,
Examples include high performance liquid chromatography, isoelectric point and gel electrophoresis, which may be used alone or in appropriate combination. Furthermore, the protein collected from the transformant as described above may be appropriately denatured, if necessary.
It can be made active by a method known in the art, such as a regeneration treatment, for example, a treatment with a guanidine solution and then dialysis. As the modification method, a reduction method using dithiothreitol or the like can be used and dialysis can be performed. The protein obtained by purification and separation as described above is hydrolyzed with an acid such as hydrochloric acid, pepsin, chymotrypsin, a protease such as carboxypeptidase, etc., and the obtained peptide fragment is subjected to ion exchange chromatography or the like. It can be subjected to chromatography to analyze its amino acid composition and to determine its amino acid sequence. The method for analyzing the amino acid composition of such a protein will be described in more detail. First, the purified foreign protein is hydrolyzed with hydrochloric acid, and then phenylisothiocyanate (PITC) is reacted to convert the amino acid to the corresponding phenylthiocarbamyl. A method (PITC method) of converting to a derivative and quantifying it by reverse phase high performance liquid chromatography can be mentioned. Also,
According to the present invention, there is provided a means for controlling gene replication using a protein having the amino acid sequence represented by SEQ ID NO: 3 or a protein exhibiting replication-related activity that is substantially equivalent thereto. This protein can be treated in the same manner as the protein based on the expression of a foreign gene in the gene expression vector. The method used for the above gene recombination technology is described in T.W. Maniatis, et a
l. , Molecular Cloning. Cold
Spring Harbor Laborator
y. New York. 1982) or the methods described in the documents cited therein, and the methods obtained by appropriately modifying them, but they may be appropriately modified within the scope of achieving the intended effects of the present invention. It is also acceptable to apply.

EXAMPLES The present invention will be described in more detail with reference to the following examples.

Embodiment 1. Preparation of endogenous plasmid pAQ1 0.1 g of wet bacterial cells of cyanobacteria PCC7002 was freeze-thawed twice (-85 ° C., 15 minutes left → 37 ° C., 15 minutes left), and then 25 mM containing lysozyme (5 mg / ml)
Tris (pH 8.0), 50 mM glucose, 1
It was suspended in 1 ml of 0 mM EDTA solution. After standing at room temperature for 30 minutes, 0.1% sodium dodecyl sulfate, 0.2
2 ml of N NaOH solution was added and left in ice for 5 minutes. Next, 1.5 ml of 3M potassium acetate solution was added, and the mixture was allowed to stand in ice for 5 minutes, and then the supernatant was collected by centrifugation. 4.5 ml of isopropanol was added to the collected supernatant and the mixture was centrifuged to collect the precipitate (crude purified DNA). After suspending the precipitate in a TE solution (50 μl), an equal amount of 5M ammonium acetate was added, the mixture was allowed to stand for 30 minutes on ice, and the supernatant was recovered by centrifugation. 250 μl of cold ethanol to the collected supernatant
Was added and centrifuged, and the precipitate was washed with 500 μl of 70% cold ethanol. The precipitate was RNase (20 μg / m
After suspending in 50 μl of TE solution containing 1), the mixture was left at 37 ° C. for 30 minutes. Further 1/10 amount of Protenase K
(1 mg / ml) was added, and the mixture was reacted at 50 ° C for 2 hours.
After the reaction, about 4 μg of the entire plasmid was purified by ethanol precipitation, and each plasmid was separated by 1% low melting point agarose gel (Bezester Research Laboratory, Inc .: BRL) electrophoresis, and a DNA band at about 4 kilobase pairs was obtained. Was purified by known methods. About 0.7 µg of the plasmid was purified and confirmed to be a single band by 1% agarose gel (Sigma) electrophoresis. Restriction enzyme Hin dI was added to 0.1 μg of the purified plasmid.
II (Takara Shuzo Co., Ltd. (Ltd.)), Hin cII (Takara Shuzo Co., Ltd.)
After adding 2.5 units of each, the reaction was carried out at 37 ° C. for 1 hour. As a result of 1% agarose gel electrophoresis, the restriction enzyme Hin dIII site was 1 in the purified plasmid.
Locations, it was confirmed that the restriction enzyme Hin cII site exists three places. This was consistent with the number of restriction enzyme sites described in the literature for the endogenous plasmid pAQ1 (JS Buzby, et al., J. Bacte).
riol. , 154, 1446-1450 (198)
3)).

Example 2. Construction of shuttle vector Restriction enzyme for purified plasmid pAQ1 (1 μg)
The Hin dIII was added 10 units, it was carried out for 2 hours at 37 ° C.. Similarly, 1 μg of Escherichia coli-derived plasmid pUC19 (Takara Shuzo Co., Ltd.) was supplemented with 1 restriction enzyme Hin dIII.
0 unit was added and the reaction was carried out at 37 ° C. for 2 hours. After heat treatment (70 ° C, 15 minutes), ethanol precipitation was performed.
Each was suspended in 10 μl of TE solution (0.1 μg / μ
l). pAQ1 / Hin dIII 0.3μg to the p
UC19 / Hin dIII0.01μg added, 16 at 16 ° C. using a ligation kit (Takara Shuzo)
The reaction was carried out over time. After the reaction, ethanol precipitation was performed and TE
Suspended in 10 μl of solution. J for 10 μl of DNA solution
M109 competent cell (Takara Shuzo Co., Ltd.) 100 μl
Was added and transformation was performed according to the method described in the instruction manual of the ligation kit. For transformants, the boiling method (J. Sambrook, et al.
l. , Molecular Cloning 2nd
screening), the shuttle vectors pAQJ-1 and pAQJ-2
(The difference from pAQJ-1 is that pAQ1 is inserted in the opposite direction).

Embodiment 3. Shuttle vector pAQJ-
1. Large-scale preparation of pAQJ-2 Inoculated with JM109 colonies into which shuttle vectors pAQJ-1 and pAQJ-2 were introduced, respectively, and LB
100 ml of medium (containing 50 μg / ml ampicillin)
The cells were cultured overnight at 37 ° C. After harvesting the cells, the alkali-SDS method (J. Sambrook, et al., Molcu.
lar Cloning 2nd edition
n), and density gradient centrifugation with cesium chloride and ethidium bromide (J. Sambrook, et a.
l. , Molecular Cloning 2nd
about 100 μg of plasmid DNA was prepared by the following.

Example 4. Determination of complete nucleotide sequence of endogenous plasmid pAQ1 To 20 μg of each of pAQJ-1 and pAQJ-2, 200 units of a restriction enzyme Kpn I (Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. for 3 hours. After confirming complete digestion by agarose gel electrophoresis, heat treatment (60 ° C,
15 minutes), 200 units of restriction enzyme Xba I (Takara Shuzo Co., Ltd.) was further added, and the reaction was carried out at 37 ° C. for 3 hours. After the reaction, phenol treatment was performed twice, and the DNA was concentrated and purified by ethanol precipitation. Purified DNA (2
0 μg) was suspended in 20 μl of sterilized water (1 μg)
/ Μl). For the determination of the nucleotide sequence of pAQ1, the dilation mutant method (Attachment-1 of the kit (BRL)) by exonuclease III (Bezesta Research Laboratory: BRL) was used. In addition, for screening of mutation mutants,
Restriction enzymes Pvu II (Takara Shuzo Co., Ltd.) and Sac I
(Takara Shuzo Co., Ltd.) was used. As a result, 25 mutants of pAQJ-1 and 18 clones of PAQJ-2 were selected, and plasmid DNA was again prepared from 3 ml of the culture solution by the alkali-SDS method. For the determination of the base sequence, a fluorescent sequencer (GENESIS2 from DuPont) was used for pAQJ-1.
000), a fluorescent sequencer (373A) manufactured by Applied Biosystems was used for pAQJ-2.
However, since there were regions where the base sequence could not be determined at 4 positions for PAQJ-1 and at 3 positions for PAQJ-2, a total of 9 primers were synthesized and the total base sequence of pAQ1 was determined independently for the + strand and the − strand.

Example 5. Analysis of gene region involved in replication (1) As a result of analysis of the nucleotide sequence of pAQ1, a gene region encoding at least three kinds of proteins, that is, 2
ORF consisting of 832 bases OR consisting of 279 bases
It was inferred to have ORF3 consisting of F2 and 156 bases. But in shuttle vector pAQJ-1 and pAQJ-2 was constructed ORF1 the restriction enzyme Hin d
The region consisting of 2466 bases, which is divided at the III site, was designated as ORF1A, and the region consisting of 366 bases was designated as ORF1B. In order to determine the gene region involved in replication, exonuclease III was isolated from pAQJ-2.
(Bezesta Research Laboratory Inc .: BRL Inc.), ORF1A-only clone, OR
A clone lacking F1A and ORF2, and a clone lacking ORF1A, ORF2, and ORF3 were prepared, respectively. Transformation of cyanobacteria PCC7002 with each clone resulted in ORF
The deletion of 1A reduced the transformation efficiency by about 30 times, and other regions did not affect the transformation efficiency.
The method for transforming cyanobacteria PCC7002 was according to the method described in the literature (J.S.
Buzby, et al. , Science, 230,
805-807 (1985)).

Example 6. pAQJ-3 (ORF1A / p
Construction of UC19) (1) Restriction enzyme Stu for pAQJ-2 (10 μg)
100 units of I (Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. for 2 hours. After confirming complete digestion by agarose gel electrophoresis, heat treatment (60 ° C, 15 minutes) is performed,
Further, 100 units of restriction enzyme Eco RI (Takara Shuzo Co., Ltd.) was added, and the reaction was carried out at 37 ° C. for 2 hours. After the reaction, phenol treatment was performed twice, and the DNA was concentrated and purified by ethanol precipitation. Next, OR generated by restriction enzyme cleavage
A DNA fragment of about 2.9 kilobase pairs containing F1A was recovered using 1% low melting point agarose gel (Bezester Research Laboratory, Inc .: BRL) electrophoresis according to a known method, and suspended in 10 μl of TE solution. Done (0.5 μg
/ Μl). (2) Also, the plasmid pUC19 (1 μm) derived from Escherichia coli
Add 10 units of restriction enzyme Hin dIII to g), and add 3
The reaction was carried out at 7 ° C for 2 hours. After confirming complete digestion by agarose gel electrophoresis, ethanol precipitation was performed and the cells were suspended in 8 μl of sterilized water. 10 times more concentrated T4 DN
The A polymerase reaction solution (1 μl) was added and the mixture was kept at 70 ° C. for 5 minutes and then left at 37 ° C. for 10 minutes. Add 1 μl (5 units) of T4 DNA polymerase (Takara Shuzo) at 37 ° C
The restriction enzyme site was smoothed by performing the reaction for 5 minutes. After the reaction, the DNA was purified by phenol treatment and ethanol precipitation, and the restriction enzyme Eco RI was added.
10 units of Takara Shuzo Co., Ltd. was added, and the reaction was carried out at 37 ° C. for 2 hours. After the reaction, ethanol precipitation was performed and TE solution 10
The cells were suspended in μl (0.9 μg / μl). (3) pAQJ-2 / Stu I, Eco RI 0.5μ
g with respect to pUC19 / Hin dIII, Eco RI
0.01 μg was added, and the reaction was performed at 16 ° C. for 16 hours using a ligation kit (Takara Shuzo Co., Ltd.). After the reaction, 100 μl of HB101 competent cell (Takara Shuzo Co., Ltd.) was added to 12 μl of the DNA solution, and transformation was performed according to the method described in the instruction manual of the ligation kit. Boiling method (J. Sambrook
k, et al. , Molecular Clonin
plasmid DNA is prepared from the transformant by using g 2nd edition) and the restriction enzyme Eco RI,
Hin dIII, a result of screening with Pvu II, to confirm the shuttle vector pAQJ-3.

Embodiment 7. Shuttle vector pAQJ-3
Large-scale preparation of HB101 colonies introduced with shuttle vector pAQJ-3 were inoculated and cultured in 100 ml of LB medium (containing 50 μg / ml ampicillin) at 37 ° C. overnight. After collecting the bacteria, the alkali-SDS method (J. Sambrook, et.
al. , Molecular Cloning 2n
decimation) and density gradient centrifugation with cesium chloride and ethidium bromide (J. Sambr
ook, et al. , Molecular Clon
ing 2nd education) about 10
0 μg of plasmid DNA was prepared.

Example 8. pUC19-SH (ORF1B
/ PUC19) Construction 100 units of the restriction enzyme Sac I (Takara Shuzo Co., Ltd.) was added to pAQJ-2 (10 μg), and the reaction was carried out at 37 ° C. for 2 hours. Heat treatment after confirming complete digestion by agarose gel electrophoresis (60 ° C., 15 minutes) is performed, further restriction enzyme Hin dIII (Takara Shuzo) was added 100 units, 37 ° C., it was carried out for 2 hours. After the reaction, phenol treatment was performed twice, and the DNA was concentrated and purified by ethanol precipitation. Next, ORF generated by restriction enzyme cleavage
1% of about 0.4 kilobase pair DNA fragment containing 1B and about 2.9 kilobase pair DNA fragment derived from plasmid pUC19.
Each was recovered by low-melting point agarose gel (Bezesta Research Laboratory, Inc .: BRL) according to a known method and suspended in 10 μl of a TE solution (about 2.9 kilobase pair DNA fragment 0.5 μg / μl, about 0.4 kilobase pair DNA fragment 0.05 μg / μl).
About 0.4 kilobase pair DNA fragment 0.2μg pU
Approximately 2.9 kilobase pair DNA fragment derived from C19 0.02
μg was added, and a reaction was performed at 16 ° C. for 3 hours using a ligation kit (Takara Shuzo Co., Ltd.). After the reaction, 100 μl of JM109 competent cell (Takara Shuzo Co., Ltd.) was added to 20 μl of the DNA solution, and transformation was performed according to the method described in the instruction manual of the kit. Boiling method (J. Sambrook, et al., Mole
color Cloning 2nd edicati
Plasmid DNA was prepared from the transformant using on), restriction enzymes Sac I, results of screened using Hin dIII, a shuttle vector pUC19-
SH was confirmed.

Example 9. Shuttle vector pUC19-
Large-scale preparation of SH JM10 with shuttle vector pUC19-SH introduced
9 colonies were inoculated and cultured in 100 ml of LB medium (containing 50 μg / ml ampicillin) at 37 ° C. overnight.
After harvesting, the alkali-SDS method (J. Sambrook,
et al. , Molecular Cloning
2nd education), and density gradient centrifugation with cesium chloride and ethidium bromide (J. Sam
Brook, et al. , Molecular Cl
about 100 μg of plasmid DNA was prepared by oning 2nd education.

Example 10. pAQJ-4 (ORF1 / p
Construction of UC19) (1) Restriction enzyme Hi for pUC19-SH (5 μg)
n dIII (Takara Shuzo) and 50 units added, and the mixture was reacted for 2 hours at 37 ° C.. After confirming complete digestion by agarose gel electrophoresis, heat treatment (70 ° C, 15 minutes) was performed, and further restriction enzyme Eco RI (Takara Shuzo Co., Ltd.) was added to 5
0 unit was added and the reaction was carried out at 37 ° C. for 2 hours. After the reaction, phenol treatment was performed twice and DNA was precipitated by ethanol precipitation.
Was concentrated and purified. Next, a DNA fragment of about 0.4 kilobase pairs generated by restriction enzyme digestion was converted into a 1% low melting point agarose gel (Bezesta Research Laboratory: BRL).
Company) using electrophoretic method according to a known method,
Suspended in 10 μl of TE solution (0.1 μg / μl). (2) Also, a restriction enzyme Hi for pAQJ-3 (5 μg)
n dIII (Takara Shuzo) and 50 units added, 37 ° C.,
The reaction was carried out for 2 hours. After confirming complete digestion by agarose gel electrophoresis, heat treatment (70 ° C, 15 minutes) was performed, and further restriction enzyme Eco RI (Takara Shuzo Co., Ltd.) was added to 5
0 unit was added and the reaction was carried out at 37 ° C. for 2 hours. After the reaction, phenol treatment was performed twice and DNA was precipitated by ethanol precipitation.
Was concentrated and purified. Next, a DNA fragment of about 5.8 kilobase pairs generated by restriction enzyme digestion was treated with a 1% low melting point agarose gel (Bezesta Research Laboratory: BRL).
Company) using electrophoretic method according to a known method,
Suspended in 10 μl of TE solution (0.3 μg / μl). (3) About 0.4 kilobase pair DNA obtained in (1) above
About 5.8 obtained in (2) above for 0.1 μg of fragment
0.05 μg of a kilobase pair DNA fragment was added, and a reaction was performed at 16 ° C. for 30 minutes using a ligation kit (Takara Shuzo Co., Ltd.). After reaction, JM for 9 μl of DNA solution
100 μl of 109 competent cells (Takara Shuzo Co., Ltd.) was added, and transformation was performed according to the method described in the instruction manual of the ligation kit. Boiling method (J. Sambrook, et al., Molcu
lar Cloning 2nd edition
Plasmid DNA was prepared from the transformant using n), restriction enzyme Pvu II, a result of screened using Hin dIII, was confirmed shuttle vector pAQJ-4 of ORF1A and ORF1B bound.

Embodiment 11. Shuttle vector pAQJ-
4. Large-scale preparation of 4 JM109 colonies introduced with shuttle vector pAQJ-4 were inoculated and cultured in 100 ml of LB medium (containing 50 μg / ml ampicillin) at 37 ° C. overnight. After collecting the bacteria, the alkali-SDS method (J. Sambrook, et.
al. , Molecular Cloning 2n
decimation) and density gradient centrifugation with cesium chloride and ethidium bromide (J. Sambr
ook, et al. , Molecular Clon
ing 2nd education) about 10
0 μg of plasmid DNA was prepared.

Embodiment 12. Analysis of gene regions involved in replication (2) Large amounts of pAQJ-1, pAQJ-2, pAQJ-
3, pAQJ-4 and pUC19-SH were used to construct cyanobacterial PC
Transformation of C7002 was performed. For each shuttle vector, 5 medium A plates (SE Ste
vens, et al. J. Phycol. , 9, 4
27-430 (1973)) with the transformation efficiency as an index, pAQJ-2 was 1.3, p
0.8 for AQJ-3, 1.5 for pAQJ-4, pUC1
9-SH became 0 (the efficiency of pAQJ-1 was set to 1). The transformation method of cyanobacteria PCC7002 was according to the method described in the literature (J.
S. Buzby, et al. , Science, 23
0,805-807 (1985)). Vector pAQJ
-4 was found to have very good transformation efficiency.

Example 13. Analysis of gene regions involved in replication (3) To observe the retention of the shuttle vector in cyanobacteria PCC7002, each shuttle vector (p
For each of AQJ-1, 2, 3 and 4), three transformants were selected and subjected to Southern blotting.
The method for preparing plasmid DNA will be described below. 12 in total
Wet and thaw treatment of 0.05 g of wet transformants (ampicillin-resistant cyanobacteria) of two kinds (-85
After leaving it for 15 minutes at 37 ° C. → leaving it for 15 minutes at 37 ° C.), 25 mM Tris containing lysozyme (5 mg / ml) (pH 8.
0), 50 mM glucose, it was suspended in 1 ml of a 10 mM EDTA solution. 0.1% after standing for 30 minutes at room temperature
Sodium dodecyl sulfate, 0.2N NaOH solution 2m
1 was added and left for 5 minutes in ice. Next, 1.5 ml of 3M potassium acetate solution was added, and the mixture was allowed to stand in ice for 5 minutes, and then the supernatant was collected by centrifugation. 4.5 ml of isopropanol was added to the collected supernatant and the mixture was centrifuged to collect the precipitate (crude purified DNA). The precipitate was suspended in TE solution (50 μl), an equal volume of 5M ammonium acetate was added, and the mixture was mixed in ice to 3 times.
After leaving it for 0 minutes, the supernatant was collected by centrifugation. To the collected supernatant, 250 μl of cold ethanol was added and centrifuged, and the precipitate was washed with 500 μl of 70% cold ethanol. TE solution containing RNase (20 μg / ml) 50
After suspending in μl, the mixture was left at 37 ° C. for 30 minutes. Further 1 /
Ten amounts of Protenase K (1 mg / ml) were added and reacted at 50 ° C for 2 hours. After the reaction, about 2 μg of all plasmids were purified by ethanol precipitation.

0.4 μg of plasmid DNA was separated into each plasmid by 1% agarose gel (14 cm × 12.5 cm) electrophoresis, and the plasmid DNA was subjected to a nylon membrane (Hybond) according to a known method.
-N + (manufactured by Amersham) (J. Samb)
look, et al. , Molecular Clo
2nd education). After transcription, the plasmid DNA was immobilized on the membrane using UV Cross linker (Stratogene). The immobilized membrane was treated with 25 ml of a prehybridization solution (solution composition: 6 * SSPE, 5 * Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 50%).
% Formamide, 100 μg / ml salmon sperm DNA) and left at 42 ° C. for 9 hours. On the other hand, as a probe,
6 obtained by digesting pUC19 with the restriction enzyme Dra I
92 base pairs (part of ampicillin resistance gene) DNA
The fragment was used. α- ³² PdCTP, and random
The probe of ^9.4 * 10 7 cpm / 100ng using primers DNA labeling kit (Takara Shuzo Co., Ltd.) was prepared. After heat denaturation (95 ° C, 3 minutes), the whole amount was mixed with the prehybridization solution, and hybridization was carried out at 42 ° C for 16 hours. After hybridization, the membrane was washed 3 times with 2 * SSC, 0.1% SDS at room temperature for 10 minutes to remove the non-specifically bound probe, and then 0.1 * SSC, 0.1% SD.
S was washed twice at 65 ° C. for 10 minutes. After washing and drying, an autoradiogram (one night) was performed to analyze the shuttle vector. As a result of analysis using an autoradiogram, a shuttle vector p having an uncut ORF1 region
All of the AQJ-4 transformants were placed at the target position.
There was an NA band. However, all the transformants with other shuttle vectors were inserted into other plasmids by homologous recombination and the like, and no DNA band was present at the desired position.

INDUSTRIAL APPLICABILITY Expression of genes of other organisms in cyanobacteria can be carried out more reliably and more easily, and carbon dioxide excessively present in the atmosphere can be effectively used.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:01) (C12P 21/02 (C12R 1:19) (C12P 21/02 C12R 1:01) (72) Inventor Hideo Akiyama 1111 Tehiro, Kamakura City, Kanagawa Prefecture Toray Research Center, Inc., Department of Biological Sciences (72) Inventor Shozo Kanai Kamakura City, Kanagawa Prefecture 1111 Tehiro, Toray Research Center, Inc.

Claims (5)

[Claims] 1. A DNA sequence represented by SEQ ID NO: 2 or a DNA sequence showing an activity of a gene region involved in replication substantially equivalent thereto. 2. An expression vector containing the DNA sequence shown in SEQ ID NO: 2 or a DNA sequence showing activity of a gene region involved in replication substantially equivalent thereto. 3. A transformant transformed with an expression vector containing the DNA sequence represented by SEQ ID NO: 2 or a DNA sequence having an activity of a gene region involved in replication which is substantially equivalent thereto. 4. The transformant is obtained by transforming Escherichia coli or cyanobacteria.
The transformant described. 5. A protein having the amino acid sequence shown by SEQ ID NO: 3 or a protein showing a replication-related activity substantially equivalent thereto.