JP4742258B2 - Firefly luciferase gene functioning in chloroplast and real-time monitoring of chloroplast gene expression using it - Google Patents

Description

  The present invention relates to a novel nucleic acid that can be used as a real-time reporter of chloroplast gene expression and a transformant incorporating the nucleic acid.

  The product of the firefly luciferase gene emits bioluminescence according to its expression level when a substrate is added from the outside. Moreover, since it is possible to emit light in living cells, changes in gene expression over time can be known in real time. In addition, since the measurement preparation work is very simple (only substrate is given), large-scale measurement is possible. Therefore, the firefly luciferase gene has been applied to a wide range of species as a reporter gene for examining the gene expression level. (Sherf, B. A., and Wood, K. V. 1994, Promega Notes 49: 14-21)

The circadian clock is a molecular device that almost all cells have, and regulates various intracellular life phenomena in order to adapt to changes in the environment of day and night in the outside world. The gene encoded in the chloroplast genome is also controlled by the circadian clock. As a result, photosynthesis, which is an important function of chloroplasts, shows a circadian rhythm that flourishes in the daytime and rests at night.
Sherf, BA, and Wood, KV 1994, Promega Notes 49: 14-21

  However, it is not known at all how the circadian clock regulates the chloroplast genome. The biggest factor is the absence of an effective real-time reporter gene like firefly luciferase. The chloroplast genome is often an AT-rich sequence, including the model plant Chlamydomonas. Therefore, the existing luciferase gene is difficult to function.

  Accordingly, an object of the present invention is to provide a firefly luciferase gene that functions in chloroplasts and to provide various measurements using the gene.

  In order to achieve the above object, the inventors focused on the frequency of codon usage in chloroplasts and, as a result of intensive research, have found the nucleic acid of the present invention and a transformant incorporating the nucleic acid.

The nucleic acid of the present invention comprises the following (a) or (b). That is,
(a) A nucleic acid having the base sequence represented by base sequence number 1-1653 shown in SEQ ID NO: 1 in the sequence listing.
(b) A nucleic acid in which a base sequence related to a codon that is used less frequently in the chloroplast genome than the existing luciferase gene in the base sequence of base sequence number 1-1653 is substituted.

  In a preferred embodiment of the nucleic acid of the present invention, the codon that is less frequently used in the chloroplast genome is 184 to 186, 342 to 344, 427 to 429, among the nucleotide sequences of SEQ ID NOs: 1 to 1653 in the Sequence Listing. 562-564, 757-759, 821-823, 955-957, 988-990, 1008-1010, 1024-1026, 1375-1377, 1588-1590, and 1615-1617 And

  The transformant of the present invention is characterized by incorporating the nucleic acid according to any one of claims 1 to 3.

  The method for measuring gene expression of the present invention comprises measuring the gene expression by incorporating the nucleic acid according to any one of claims 1 to 3 into a chloroplast genome as a reporter gene. .

  The circadian rhythm measurement method of the present invention comprises connecting the nucleic acid according to any one of claims 1 to 3 as a reporter gene downstream of a promoter of the chloroplast gene psbD and a 5 ′ untranslated region. The circadian rhythm is measured.

  The reporter vector of the present invention is characterized by incorporating the nucleic acid according to any one of claims 1 to 3 as a reporter gene.

  In the method for synthesizing a synthetic gene of the present invention, since the synthetic gene is expressed in a host or organelle, the frequency of use of the codon used in the host or organelle is higher than that of other codons. It is characterized by synthesizing genes using codons.

  In a preferred embodiment of the method for synthesizing a synthetic gene of the present invention, the gene is synthesized by substituting a codon whose appearance frequency is 1/1000 or less (in the host or organelle). Exceptions are those in which more frequently used codons encoding one amino acid are less than this number.

  According to the nucleic acid of the present invention, since a codon frequently used in the chloroplast genome is used, the luciferase gene is efficiently expressed, and the expression state of the chloroplast gene such as circadian rhythm is observed. It has the advantageous effect that it becomes possible.

  Further, according to the gene synthesis method of the present invention, it is possible to synthesize a desired synthetic gene that can be easily incorporated into a desired organ and expressed in an optimized form of codon usage. Has an effect.

The nucleic acid of the present invention has the following (a) or (b):
(a) a nucleic acid comprising the base sequence represented by base sequence number 1-1653 shown in SEQ ID NO: 1 in the sequence listing;
(b) consisting of a nucleic acid having a base sequence associated with a codon that is less frequently used in the chloroplast genome than the existing luciferase gene, among the base sequences of base sequence numbers 1 to 1653 Features. The nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing is a synthetic gene optimized so that it can be expressed in chloroplasts.

  Further, the nucleic acid of the present invention is a nucleic acid in which the base sequence related to a codon that is used less frequently in the chloroplast genome than the existing luciferase gene in the base sequence of base sequence number 1-1653 is substituted. It is characterized by comprising. The nucleic acid of the present invention is optimized so that luciferase protein can be expressed also in chloroplasts when the frequency of codon usage in the existing firefly luciferase gene and chloroplast genome is different. A foreign gene can be expressed by a nucleic acid in which a codon that is less frequently used in an extranuclear organ such as the chloroplast is appropriately substituted.

  In a preferred embodiment, codons that are not frequently used in the chloroplast are 184 to 186, 342 to 344, 427 to 429, 562 to 564, 757 to 759, 821 to 823, 955 to 957, 988 to 990, 1008 to 1010, 1024 to 1026, 1375 to 1377, 1588 to 1590, and 1615 to 1617 (shown with a red underline in FIG. 9) Part). These codons are less frequently used than existing luciferase genes, and the present inventors have speculated that the difference in these use frequencies may prevent smooth expression of foreign genes. This is an attempt to construct a synthetic gene encoding a target protein by replacing codons including codons that are not frequently used with codons that are frequently used in an organ in which a foreign gene is to be expressed.

  In chloroplasts, codon usage may be different from that of existing luciferase genes, making expression of foreign genes very difficult. According to the nucleic acid of the present invention, since the codon is optimized, it is possible to express a foreign gene, and thus, for example, a reporter gene for real-time measurement that is indispensable for large-scale and high-precision gene expression measurement. Can also be provided.

  Moreover, the transformant of the present invention is characterized by incorporating the nucleic acid according to any one of claims 1 to 3. The transformant of the present invention can be obtained by gene transfer by connecting the nucleic acid of the present invention to an appropriate promoter by a conventional method.

  Next, the method for using the nucleic acid of the present invention will be described as follows. First, the method for measuring the expression of the gene of the present invention is characterized by measuring the expression of the gene by incorporating the above-described nucleic acid of the present invention into a chloroplast genome as a reporter gene. The above description can be applied as it is to the “nucleic acid” of the present invention. Since the nucleic acid of the present invention is optimized so that it can be expressed in chloroplasts, it can be used as a reporter gene to observe the expression of an arbitrary gene. For example, preferably, the above-described nucleic acid of the present invention can be connected as a reporter gene downstream of the promoter of the chloroplast gene psbD and the 5 'untranslated region, and the circadian rhythm of gene expression can be measured. By using the nucleic acid of the present invention, it is possible to construct an experimental system that can easily and simultaneously test the rhythm of multiple samples. This can lead to dramatic acceleration of research speed.

  The reporter vector of the present invention is characterized by incorporating the above-described nucleic acid of the present invention as a reporter gene. The cassette can be transferred to a desired organelle targeting site by a conventional gene transfer method such as homologous recombination.

  Next, a method for synthesizing the synthetic gene of the present invention will be described. The synthesis method of the present invention is characterized in that in order to express a synthetic gene, the gene is synthesized by replacing it with a codon frequently used in a host or organelle. According to the synthesis method, it is possible to synthesize a gene that can accurately express a foreign gene that is difficult to express in the host or organelle. In a preferred embodiment, a gene is synthesized by substituting a codon having an appearance frequency of 25/1000 or less, more preferably 1/1000 or less in the host or organelle. Exceptions are those in which more frequently used codons encoding one amino acid are less than this number.

  EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not the intention limited to these Examples and interpreted.

  First, the present inventors paid attention to Chlamydomonas reinhardtii, which is one of eukaryotic algae. The Chlamydomonas is a unicellular organism with only one chloroplast in the cell. Because of this simplicity, it has been widely used as a model organism that leads chloroplast research. It also has a clear circadian rhythm and is suitable for circadian rhythm research.

Therefore, the present inventors produced an artificial synthetic gene LUC ⁺ _cp (LUC ⁺ gene for Chlamydomonas chloroplast) in which the codon usage frequency of the modified firefly luciferase gene (LUC ⁺ ) is optimized for Chlamydomonas chloroplast genome. The reporter vector using was transferred into the chloroplast genome. Using the obtained transformant, we succeeded in real-time monitoring of the gene expression rhythm of the chloroplast genome as bioluminescence.

Materials and methods
Strains and cultures of Chlamydomonas strains and Escherichia coli strains and cultured Chlamydomonas reinhardtii wild strain 2137mt ⁺ (CC-1021) were purchased from Dr. Jun Minagawa (Hokkaido University) and used. Chlamydomonas culture is usually performed continuously at 24 ° C using Tris-acetate phosphate (TAP) liquid medium or TAP solid medium (Harris, 1989) containing 1.5% agar (INA AGAR BA70; INA Foods, Nagano, Japan). Bright, 30 μE / m ² sec, performed under white fluorescent lamp. In the case of liquid culture, swirl culture was performed at 180 rpm.

  As strains of Escherichia coli, DH5α (Takara, Kusatsu, Japan) and XL10 Gold (Stratagene Japan, Tokyo, Japan) were used. E. coli was cultured using LB medium (Sambrook et al., 1989), and final concentrations of 50 μg / ml ampicillin, 25 μg / ml chloramphenicol, and 100 μg / ml spectinomycin were added to the medium as needed.

DNA manipulation, base sequence determination and analysis Plasmid DNA extraction, DNA restriction enzyme digestion, blunting, ligation, polymerase chain reaction (PCR), etc. are performed according to the description of Sambrook et al. (Sambrook et al., 1989). It was. The DNA base sequence was determined using an ABI373S automatic sequencer system (Applied Biosystems Japan, Tokyo, Japan) and PRISM Dye-Terminator Cycle Sequencing Kit (Applied Biosystems Japan) according to the manufacturer's manual. The determined base sequence is analyzed using base sequence analysis software Analysis (Applied Biosystems Japan), AutoAssembler (Applied Biosystems Japan), GENETYX-MAC (GENETYX Corp., Tokyo, Japan) and GENETYX-WIN (GENETYX Corp.). Analyzed. In each process of DNA fragment and vector construction obtained by PCR cloning, the base sequence was confirmed.

Plasmid DNA
<Artificial synthesis of LUC ⁺ cp gene>
The artificially synthesized firefly luciferase gene LUC ⁺ _cp optimized for the codon usage of Chlamydomonas chloroplast genome was synthesized by the method of Stemmer et al. The codon usage frequency of Chlamydomonas was investigated using the database Codon Usage Database (http://www.kazusa.or.jp/codon/) of Kazusa DNA Research Institute.

<Preparation of chloroplast targeting vector (pCTS2XX)>
The stuI fragment of the chloroplast genome (6.3 kb; nucleotides 75838 to 82122 of GenBank / DDBJ / EMBL accession no. BK000554) was cloned into the plasmid vector pUC118 (TAKARA) and the polylinker (5'-cccgggatccactagtcgacgcatgcagatctaggcctgca-3 ') was I inserted it at site (79351 of BK000554). A selection marker aadA expression cassette was inserted into the polylinker site. When this marker is inserted into the genome, it is resistant to the antibiotics spectinomycin and streptomycin.

<Production of bioluminescent rhythm oscillation cassette>
The translation region of the artificially synthesized LUC ⁺ cp gene was cloned into pCR2.1-TOPO (Invitrogen) to prepare pCR2.1-TOPO / LUC ⁺ cp. During the artificial synthesis, an NdeI site was added to the 5 ′ end and an XbaI site was added to the 3 ′ end. The promoter of the chloroplast gene psbD and the 5 ′ untranslated region (175630-176055 of BK000554) were amplified by PCR using primers psbD-F1 and psbD-R1. It was cloned into pT7Blue-T (Novagen) to prepare pT7BT / P _psbD . The 3 ′ terminator region (1590005-160185 of BK000554) of the chloroplast gene atpB was amplified by PCR using primers atpB-F1M and atpB-R1M, and cloned into pT7Blue-T to prepare pT7BT / _TatpB .

Insert the 0.43kb SmaI-XbaI fragment of pT7BT / P _psbD to HincII / XbaI digested pT7BT / T _atpB to prepare a pT7BT / P _psbD :: T _atpB. pCR2.1-TOPO / LUC ⁺ cp 1.66 kb NdeI-XbaI fragment inserted into NdeI / XbaI digested pT7BT / P _psbD :: T _atpB , vector pT7BT / P _psbD :: LUC ⁺ cp :: T _atpB was prepared.

Primers used
psbD-F1: 5'-tatgaaattaaatggatatt-3 '
psbD-R1: 5'- catatg gtgtatctccaaaa-3 '
atpB-F1M: 5'-gct tctaga aaagctgcttcattaaaataa-3 '
atpB-R1M: 5'-t cccggg acgtttcctttagttttttgctg-3 '
* Underline indicates restriction enzyme sites added to facilitate subsequent cloning steps.

<Preparation of reporter vector pCTS208>
The bioluminescence rhythm oscillation cassette excised from pT7BT / P _psbD :: LUC ⁺ cp :: T _{atpB with} BamHI was inserted into the BglII site at the polylinker site of the chloroplast targeting vector. At this time, it was inserted in the opposite direction to the selection marker aadA expression cassette. When this vector is inserted into the genome, it has the ability to oscillate spectinomycin / streptomycin resistance and bioluminescence rhythm.

Introducing Chlamydomonas Chloroplast Genome Introducing Chlamydomonas chloroplast genome, biolistic transformation method using particle inflow gun (produced by Dr. Jun Minagawa) according to the description of Finer et al. (Finer et al., 1992). I went there. Tungsten particles (BIO-RAD M-17; BIO-RAD Japan, Tokyo, Japan) were used as carriers for introducing vector DNA into chloroplasts. Transformants were selected on a TAP solid medium supplemented with a final concentration of 50 μg / ml spectinomycin sulfate (BIOMOL, USA) using the aadA gene, which is a spectinomycin resistance gene, as a selection marker. The colonies of the transformants that appeared were used for experiments after three times of passage in a TAP solid medium containing a final concentration of 100 μg / ml spectinomycin to promote homoplasmic transformation of the transformants. Transformants were maintained in TAP solid medium containing 100 μg / ml spectinomycin.

Southern Blot Analysis Chlamydomonas genomic DNA was extracted according to the method of Rochaix (Rochaix, 1980). A DNA fragment (78658 to 79007, GenBank accession No. BK000554) obtained by PCR was used as a probe for Southern blot analysis.

Bioluminescence measurement of Chlamydomonas luminescence reporter strain Bioluminescence measurement of the transformant in which the luminescence reporter gene was transferred into the chloroplast genome was performed according to the following procedure. First, the Chlamydomonas transformant was inoculated on a TAP and HSM solid medium, and cultured at 24 ° C., continuous light, 30 μmol / m ² sec under a white fluorescent lamp for 5 days to form colonies. The formed colony was cut out together with the medium with a glass tube having an inner diameter of 6 mm, and this was left in a well of a 96-well microplate. Administer 25 μl of 1 mM D-Luciferin K-salt dissolved in TAP medium to each well (final concentration 150 mM; BIOSYNTH, Naperville, IL), then plate seal (Plate Seal T, Sanplatec Corp., Osaka, Japan) Sealed with. Then, after synchronizing the circadian rhythm by giving a light / dark cycle of 12 hours light period / 12 hours dark period (30 μmol / m ² sec in the light period, white fluorescent lamp turned on) once, continuous light (30 μmol / m ² sec) Bioluminescence was measured every 60 minutes in continuous darkness. In order to attenuate the delayed fluorescence of chlorophyll, a dark treatment for 3.5 minutes was performed immediately before the measurement. The amount of bioluminescence was measured using the bioluminescence measuring device (Ishiura, Okamoto, Kouchi, Furusawa, Japanese Patent Application 2003-384577) developed in our laboratory. Real-time monitoring of bioluminescence and analysis of measurement results were performed using the analysis program RAP (Japanese Patent Application 2003-061203).

result
Development of targeting sites in the chloroplast genome In Chlamydomonas chloroplasts , homologous genes transferred from outside undergo recombination with the homologous regions in the genome, so-called homologous recombination occurs (Rochaix et al., 1998). Therefore, by using homologous recombination, it is possible to incorporate a gene into a specific site of the chloroplast genome (gene targeting). When incorporating a luminescent reporter gene by gene transfer using this homologous recombination, a selection marker gene and a luminescent reporter gene are placed in a region (targeting site) that is unlikely to affect gene expression on the chloroplast genome. It is important to incorporate. Therefore, the region where the transcription termination region of the gene and the transcription termination region of another gene face each other was searched on the chloroplast gene map. From the results, it was determined that the region between the psbT gene and the psbN gene was appropriate, and the region was named targeting site 2 (cTS2) (FIG. 1).

The frequency of codon usage of the artificially synthesized firefly luciferase gene LUC ⁺ _{cp in} the chloroplast genome of the artificially synthesized Chlamydomonas is extremely unique, which makes it difficult to express foreign genes. For example, the codon usage of the firefly luciferase gene, which is one of the commonly used luminescent reporter genes, is significantly different from the codon usage of the Chlamydomonas chloroplast genome. Therefore, an artificially synthesized firefly luciferase gene, LUC ⁺ _cp (LUC ⁺ gene for Chlamydomonas chloroplast), optimized for codon usage in the chloroplast genome was completely synthesized. The amino acid sequence of the protein translated from LUC ⁺ _cp was designed to be exactly the same as the protein translated from the LUC ⁺ gene. The method for preparing the artificially synthesized gene is shown in FIG. 2, the nucleotide sequence of the LUC ⁺ _cp gene is shown in FIG. 3, and the list of synthetic oligonucleotides used for the artificial synthesis is shown in Table 1. The name and sequence of the oligonucleotide used for the artificial synthesis of the LUC ⁺ _cp gene are shown.

Production of Chloroplast Luminescent Reporter Strains Using Artificial Synthetic Gene LUC ⁺ _cp psbD, a Chlamydomonas chloroplast gene, encodes photosystem I D2 protein and is known to have diurnal mRNA levels (Rochaix et al., 1998). Therefore, an artificially synthesized firefly luciferase gene LUC ⁺ _cp optimized for the Chlamydomonas chloroplast genome was connected to the promoter region (P _psbD ) of the psbD gene to produce a luminescent reporter gene cassette P _psbD :: LUC ⁺ _cp . The p _psbD :: LUC ⁺ _cp gene cassette and the selectable marker gene cassette P _psbA :: aadA were incorporated into a cTS2 targeting vector to prepare pCTS208. The structure of pCTS208 is shown in FIG. 4A.

pCTS208 was transferred to cTS2 on Chlamydomonas chloroplasts by biolistic method to prepare psbD :: LUC ⁺ cp strain. There were approximately 80 copies of Chlamydomonas chloroplast genome, and in order to confirm that all of them were transformed, Southern blot analysis of genomic DNA digested with restriction enzymes was performed. As a result, the chloroplast genomic DNA of the psbD :: LUC + cp strain was almost all copy-transformed. (Fig. 4B, C)

Measurement of Bioluminescence Rhythm of Chloroplast Luminescent Reporter Strain As a result of measuring the bioluminescence of the completed psbD :: LUC ⁺ cp strain under continuous bright conditions, significant luminescence was observed over 20 hours. On the other hand, no significant luminescence was detected in the wild strain.

Next, we examined whether the bioluminescence of psbD :: LUC ⁺ cp strain is circadian rhythm. The circadian rhythm is defined by the following three phenomena. 1: A fluctuation (rhythm) of a period of about 24 hours continues under a constant condition. 2: The rhythm is reset in the light / dark cycle. 3: The rhythm cycle has temperature compensation.

First, in order to verify one point, after giving a light-dark cycle, the bioluminescence of the psbD :: LUC ⁺ cp strain was measured under constant conditions. As a result, a bioluminescence rhythm was observed over 5 days under constant conditions (FIGS. 5 and 6). The rhythm cycle was 23.7 ± 0.6 h (n = 47, TAP medium) and 24.8 ± 0.6 h (n = 44, HSM medium) under continuous light conditions (FIG. 5). Under continuous dark conditions, they were 23.5 ± 0.3 h (n = 34, TAP medium) and 24.1 ± 0.2 h (n = 33, HSM medium) (FIG. 6).

  Next, in order to verify the point 2, bioluminescence was measured under continuous light conditions after giving a light / dark cycle shifted by half a day (12 hours) to cells cultured under the same conditions. As a result, the observed phase of bioluminescence synchronized with the light-dark cycle as expected, and showed a rhythm shifted by about half a day (FIG. 7).

Finally, as a verification of point 3, the change in the cycle of the bioluminescence rhythm accompanying the change in temperature was examined. As a result of measurement under various temperature conditions, the period of this rhythm is 23.7 ± 0.6 h (n = 47,24 ° C), 23.1 ± 0.8 h (n = 34,19 ° C), 22.4 ± 0.4 h (n = 41, 14 ° C.) (FIG. 8). Temperature coefficient Q ₁₀ obtained by calculating the inverse of the period as the reaction rate was 0.95. For biological processes involving normal chemical reactions, Q ₁₀ is 2-3, and a value of 0.95 means that it is very insensitive to temperature changes.

From the above experimental results, the bioluminescence rhythm of the psbD :: LUC ⁺ cp strain was 1: continued under a constant condition, 2: reset with a light / dark cycle, and 3: the cycle was temperature compensated. Therefore, we concluded that this rhythm is a circadian rhythm.

Discussion In this example, an attempt was made to develop a luminescent reporter system in the chloroplast genome of Chlamydomonas. We succeeded in real-time measurement of bioluminescence rhythm of psbD :: LUC ⁺ cp strain by artificially synthesizing LUC ⁺ _cp gene optimized for codon usage of chloroplast genome and using it as luminescent reporter gene ( Figures 5-8). This bioluminescence rhythm met three conditions of circadian rhythm. That is, under constant conditions, it showed a clear rhythm with a period of about 24 hours (FIGS. 5 and 6), reset with a light-dark cycle (FIG. 7), and the rhythm cycle was temperature compensated (FIG. 8). Therefore, it was concluded that the bioluminescence rhythm of psbD :: LUC ⁺ cp strain was circadian rhythm. This is the world's first real-time monitoring of circadian rhythm in the chloroplast genome. This result is expected to elucidate the mechanism by which the circadian clock controls the chloroplast genome.

Cited references
Bruce, VG (1972), Genetics, 70: 537-548.
Finer, JJ, Vain, P, Jones, MW, and McMullen, MD (1992), Plant Cell Rep., 11: 323-328.
Harris, EH (1989), The Chlamydomonas Soucebook., Academic Press, San Diego.
Harris, EH (1999), Annu. Rev. Plant Mol. Biol., 52: 363-406.
Lumsden, PJ and Millar, AJ (1998), Biological Rhythms and Photoperiodism in Plants., BIOS Scientific Publishers, Oxford, UK.
Nelson, W, Tong, YL, Lee, JK, and Halberg, F (1979), Chronobiologia, 6: 305-323.
Rochaix, JD, Goldschmidt-Clermont, M, and Merchant, S (1998), The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas., Kluwer Academic Publisher, Netherlands.
Rochaix, JD (1980), Methods Enzymol., 65: 785-795.
Sambrook, J., Fritsch, EF, Maniatis, T (1989), Molecular cloning., Cold Spring Harbor Laboratory, Cold Sprong Horbor, New York.
Stemmer, WP, Crameri, A., Ha, KD, Brennan, TM & Heyneker, HL (1995), Gene 164, 49-53.
Shimogawara, K, Fujiwara, S, Grossman, A, and Usuda, H (1998), Genetics, 148: 1821-1828.

  The bioluminescence real-time measurement method (Fig. 1A), which continuously measures gene expression fluctuations in living cells after incorporating a luminescent gene into an organism, comprehensively covers mutants involved in controlling the expression of any key gene. It is an extremely effective experimental method for separation, and is a trump card for comprehensive genome function analysis in the post-genome era. According to the present invention, such analysis is possible in the chloroplast genome.

FIG. 1 shows the gene structure of Chlamydomonas chloroplast targeting site cTS2 and its surrounding region. The transcription directions of the psbT and psbN genes on Chlamydomonas chloroplasts are reversed. In order to use the region sandwiched between these two genes as a targeting site cTS2 for gene transfer, a targeting vector system pCTS2XX was prepared in which the left region (cTS2L) and the right region (cTS2R) of cTS2 were incorporated. Arrows indicate the transcription direction of each gene. P, promoter region; Ter, transcription termination region. FIG. 2-1 shows a method for synthesizing the artificially synthesized firefly luciferase gene LUC ⁺ _cp . The synthesis method of firefly luciferase gene LUC ⁺ _cp optimized for codon usage of Chlamydomonas chloroplast genome was shown. The total length of the approximately 1.6 kb gene was divided into 4 parts and synthesized, and then the 4 regions were combined and amplified by PCR. Each region was synthesized by polymerization of a long oligonucleotide and amplification by PCR. FIG. 2-2 shows a method for synthesizing the artificially synthesized firefly luciferase gene LUC ⁺ _cp . The synthesis method of firefly luciferase gene LUC ⁺ _cp optimized for codon usage of Chlamydomonas chloroplast genome was shown. The total length of the approximately 1.6 kb gene was divided into 4 parts and synthesized, and then the 4 regions were combined and amplified by PCR. Each region was synthesized by polymerization of a long oligonucleotide and amplification by PCR. FIG. 3 shows the base sequence of the artificially synthesized firefly luciferase gene LUC ⁺ _cp . The base sequence of the firefly luciferase gene LUC ⁺ _cp optimized for the codon usage of Chlamydomonas chloroplast genome is shown. The base sequence is shown separated for each codon. The amino acid corresponding to each codon is written in one-letter code below the base sequence. The number above the base sequence represents the number of bases from A of the start codon ATG. FIG. 4 shows the structure of the luminescent reporter vector pCTS208 and Southern blot analysis of the psbD :: LUC + cp strain. A: The structure of the luminescent reporter vector pCTS208 in Chlamydomonas chloroplasts using LUC ⁺ _cp was shown. The origin of plasmid replication and antibiotic resistance genes in E. coli cells are omitted. cTS2L, Chlamydomonas chloroplast targeting site cTS2 left region; cTS2R, Chlamydomonas chloroplast targeting site cTS2 right region; P _psbA , promoter region of Chlamydomonas chloroplast gene psbA; aadA, spectinomycin streptomycin resistance gene; T _atpB , Transcription termination region of Chlamydomonas chloroplast gene atpB; P _psbD , promoter region of Chlamydomonas chloroplast gene psbD; LUC ⁺ _cp , artificially synthesized firefly luciferase gene optimized for Chlamydomonas chloroplast genome. B: The restriction enzyme site in the vicinity of cTS2 in the wild-type (2137mt +) and transformed chloroplast genomes and the length of the excised fragment are shown. The blue bar indicates the position of the probe used for Southern hybridization. C: Southern hybridization results of wild strain (WT) and psbD :: LUC + cp strain (208) are shown. The restriction enzyme used and the sample name are shown above. FIG. 5 shows the luminescence rhythm of the psbD :: LUC + cp strain under continuous bright conditions. The result of measuring the light emission rhythm over 5 days under continuous light conditions after one light-dark cycle was shown. The bar at the top of the graph shows the light and dark conditions. White part, light period. Black part, dark period. FIG. 6 shows the luminescence rhythm of the psbD :: LUC + cp strain under continuous dark conditions. The results of measuring the luminescence rhythm over 5 days under continuous dark conditions after two light-dark cycles were shown. FIG. 7 shows the tuning of the light emission rhythm to the light / dark cycle. The result of measuring the light emission rhythm under continuous light conditions after giving a light / dark cycle with the phase shifted by 12 hours is shown. HSM solid medium was used for the measurement. The blue graph corresponds to the upper and lower light bars, and the red graph corresponds to the lower light and dark bars. The graph plots the mean and standard deviation of 12-20 samples of data. FIG. 8 shows the temperature compensation of the light emission rhythm period. The bioluminescence rhythm was measured under continuous light conditions at 24 ° C, 19 ° C, and 14 ° C, and the period was examined. TAP solid medium was used for the measurement. In the graph, the mean value of the cycle ± standard deviation is plotted. FIG. 9 is a diagram showing the base changed from the existing luciferase gene LUC in one embodiment of the present invention in black. LUC has codons that are very infrequently used in Chlamydomonas chloroplasts, which are indicated by a red underline.

Claims (5)

The following (a) or Ranaru nucleic acid.
(a) A nucleic acid having the base sequence represented by base sequence number 1-1653 shown in SEQ ID NO: 1 in the sequence listing . A transformant incorporating the nucleic acid according to claim 1 . A measurement method for measuring gene expression by incorporating the nucleic acid according to claim 1 into a chloroplast genome as a reporter gene. A method for measuring circadian rhythm, wherein the nucleic acid according to claim 1 is connected as a reporter gene downstream of the promoter of the chloroplast gene psbD and the 5 ′ untranslated region, and circadian rhythm is measured. A reporter vector incorporating the nucleic acid according to claim 1 as a reporter gene.