JPH07108227B2 - Photosynthesis related gene and its promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a light-harvesting chlorophyll a / for plant photosynthesis.
The present invention relates to a gene for a b-binding protein and its promoter, and is used for the development of new plant varieties using genetic recombination and the modification of cultured cells for producing metabolites.

[Prior Art] In recent years, it has become possible to use gene recombination technology even in plants, and researches on dicotyledons are being actively conducted.
In particular, several vector systems using the T1 plasmid of the soil bacterium Agrobacterium tumefaciens have been constructed and used for plant transformation experiments (Hoekema
(1985) Plant Molecular Biology 5: 85-89. Velten
(1984) The EMBO Journal 3: 2723-2730 et al.). However, since the genus Agrobacterium infects only a very limited part of dicotyledonous plants and monocotyledonous plants, it was almost impossible to carry out transformation experiments on major crops such as rice. The technique that solved this problem is the electroporation method (Fromm et al. (1986) Nature 319: 791-793.
other). This method creates an extremely small hole in the cell surface by applying an electrical stimulus to the isolated protoplasts,
This is a technique for transforming by incorporating a gene from there. With the development of this method, it has become possible to carry out transformation experiments on major grains such as rice, and it is now being actively conducted.

The promoter in the vector system that has been conventionally used is mostly the promoter derived from the gene of Ti plasmid of Agrobacterium tumefaciens, or the promoter derived from the gene of cauliflower mosaic virus (CaMV). These promoters have been used as promoters for efficiently expressing genes in plants. However, these promoters always express the gene in the plant after gene transfer regardless of the growing season and plant parts, and were promoters that cannot be controlled.

[Problems to be Solved by the Invention] At present, a technique for efficiently inserting a gene into a plant cell has been completed, and a strong promoter for expressing the gene is used. However, in actual plant breeding, there is an inconvenience in a promoter that constantly expresses a gene. Therefore, a promoter that can be expressed in a growth stage-specific manner or an organ-specific manner or a promoter that can be artificially controlled is desired.

[Means for Solving the Problems] The light-harvesting chlorofail a / b binding protein (hereinafter sometimes abbreviated as LHCP II) present in the thylakoid membrane of plant chloroplasts is light-harvesting in photosynthetic system II. It is a complex protein that binds sex chlorophyll and plays an important role in photosynthesis. The gene is encoded in nuclear DNA,
It is known that it is induced by light and is expressed in chloroplasts.

The inventors of the present invention believe that by utilizing this LHCP II gene promoter, it may be possible to induce gene expression by light, that is, to artificially induce gene expression. The gene was cloned and the promoter portion was analyzed. Then, this promoter portion was cut out and connected to the β-glucuronidase gene derived from Escherichia coli, and this chimeric gene was introduced into the plant face. It was confirmed that the β-glucuronidase gene was expressed by irradiating with light in callus (cell mass) obtained by culturing cells into which the gene had been introduced, or a plant body redifferentiated therefrom. Therefore, we confirmed that the LHCP II gene promoter can be induced by light,
The present invention has been completed.

That is, the present invention provides a DNA fragment having the following nucleotide sequence or a DNA fragment comprising a part thereof having promoter activity.

Furthermore, the present invention provides a plant cell vector containing the DNA fragment of the present invention.

[Effect of the Invention] The present invention provides a novel DNA fragment containing a promoter whose expression can be controlled by light irradiation, and a novel plant cell vector containing the same. By using the vector developed by the present invention, it became possible to express the transgene by irradiating it with light. In addition, since the highest expression level was observed in leaves, it was suggested that the gene expression site may have specificity. Therefore, the present invention is considered to greatly contribute to the genetic engineering of plants.

[Detailed Description of the Invention] As described above, the DNA fragment of the present invention contains the promoter of the LHCP II gene. Although the origin of the promoter is not particularly limited, the rice LHCP II gene containing the promoter portion has been isolated in the following examples. The entire structure of the LHCP II gene isolated in the Examples below and the DNA base sequence of the promoter region are shown in FIG. The framed portion shows the structural portion of the LHCP II gene. +1
Is the transcription start point of the LHCP II gene, and ATG at +60 is mature
The nucleotide sequence encoding the first amino acid of the LHCP II protein is shown. It is a necessary element for gene transcription
CAT and TATA boxes are found at -91 and -29, respectively. There is also a light-sensitive box recently reported by Grob et al. At −64 (Grob et al. (1988) Nuclei
c Acids Resaerch 23: 9953-9973). In the following examples, the nucleotide sequence shown in FIG.
Since a DNA fragment having the th base sequence has been incorporated into a vector and expression of a foreign gene existing downstream of the DNA fragment has been successful, the promoter sequence is at least this DNA fragment.
It is clear that it is completely contained in the fragment.

Generally, it is widely recognized by those skilled in the art that, in a DNA sequence having a specific function, there are cases in which even if a small number of nucleotides are substituted, deleted or added, the same function may be exerted. It is being done. Therefore,
Promoter DNA contained in the DNA fragment shown in FIG.
Of the sequence, a small number of nucleotides are substituted, even if it is a DNA fragment having a deleted or added sequence, if the sequence functions as a promoter, the sequence is referred to in the claims of the present application. A DNA fragment containing such a sequence shall be construed to be included in the scope of the present invention.

The present invention further provides a plant cell vector containing the above DNA fragment of the present invention. The vector of the present invention can be obtained by incorporating the above-mentioned DNA fragment of the present invention into a plasmid having a replication origin for autonomously replicating in a host cell and preferably an appropriate selection marker such as drug resistance. You can Such plasmids include:
Known vectors for plant cells, for example pBI101
(Manufactured by Clontech) can be used, and the vector of the present invention can be obtained by incorporating the DNA fragment of the present invention into the cloning site of such a known vector by a conventional method. In the following examples, the vector of the present invention was constructed by incorporating a fragment having the nucleotide sequence of −785th to + 59th of the sequence shown in FIG. 1 into the digested product of pBI101 with Xba I- Bam HI. In the vector of the present invention, a structural gene encoding a desired protein is incorporated downstream of the DNA fragment of the present invention. In the following examples, the vector pLH / GUS having the β-glucuronidase gene inserted as a structural gene was obtained.

Next, the method for preparing the above-mentioned DNA fragment and vector of the present invention will be described by taking rice as an example. The method for preparing the DNA fragment of the present invention is not limited to the following.

First, a rice cDNA library is prepared. In the case of rice, for example, it is preferable to extract total RNA from the seedlings that have been about 2 weeks after germination. Only mRNA is isolated from total RNA, and cDNA is synthesized by a conventional method using a commercially available kit or the like. The synthesized cDNA is connected to a cloning vector for E. coli, for example, to complete a cDNA library.

The LHCP II gene-specific sequence can be synthesized as a probe and screened by colony hybridization to obtain the LHCP II gene cDNA.

Next, from the rice germinated in the dark, the CTAB method (Plant Molecular
DNA can be extracted by Biology (1985) 5:69) and ligated to a vector for cloning Escherichia coli to prepare a rice genomic library.

LHCP II gene of rice can be isolated by screening genomic library using cDNA of LHCP II gene as a probe.

The examination of the activity as a promoter may be carried out as follows. For example, a part that is considered to have promoter activity is connected to the 5'-upstream side of the β-glucuronidase (GUS) gene. β-glucuronidase originally does not exist in plants, and 4-methylumbelliferone (4
-MU) with glucuronic acid bound 4-MUG as a substrate and the reaction product 4-MU emits strong fluorescence (Richard et al. (1986) The EMBO Journa
l 6: 3901-3907). LHCP II gene promoter and GUS
A recombinant plasmid in which a gene is incorporated into a plant cell vector is introduced into plant protoplasts by electroporation. After the introduced plant cells are cultured, they are selected according to the selection marker of the vector. For example, the promoter-containing DNA fragment of the present invention can be used as pBI101 having a GUS gene.
, It can be selected based on kanamycin resistance of pBI101. That is, pBI101 has NP
It also contains the T II gene, which makes it resistant to kanamycin when it is synthesized. Therefore, cells that survive in the presence of kanamycin are more likely to have a chimeric gene for the LHCP II promoter and GUS gene.

In this way, a plant is redifferentiated from a callus (cell mass) that is considered to have a foreign gene according to a conventional method. Gene expression of redifferentiated plants under light conditions and measurement of GUS activity in each plant of leaves, stems, petals and roots showed the greatest expression in leaves of any plant. The quantity is confirmed. Therefore, it is confirmed that the LHCP II promoter is capable of expressing a gene in the presence of light, and that its expression site is also likely to be expressed in a site where a photosynthetic open organ such as a leaf is present.

Therefore, by using the LHCP II gene promoter according to the present invention, gene expression can be induced by light. It was also suggested that the gene may be expressed in an organ-specific manner because it is easily expressed in a part having a photosynthetic organ such as a leaf.

The LHCP II promoter obtained in the following examples was a promoter that can be expressed not only in monocots such as rice, but also in dicots such as tobacco.

[Examples] The present invention will be specifically described by way of examples, but the examples of the present invention are not limited thereto. In addition, unless otherwise specified in the following examples, each operation is performed by Maniatis et al.
It was performed based on the method described in Molecular Cloning, Cold Spring Harbor Lab.

First, a cDNA library was prepared from seedlings by the following method. That is, seedlings 2 weeks after seedlings 30
Total RNA was extracted from g by the guanidine thiocyanate method. MRNA was isolated from the extracted total RNA by an oligo dT cellulose column. The yield of mRNA was almost 1.5% of total RNA. For the synthesis of cDNA from mRNA, a commercially available synthesis kit (manufactured by Amersham) was used. The synthesized cDNA was ligated to the Eco RI site of pUC8, a commercially available cloning vector for E. coli, and E. coli was transformed to obtain a cDNA library.

Next, a synthetic probe of 17 bases (base sequence: GCCCATCTGCAGTGGAT) was prepared from the structural part of the known LHCP II gene,
Positive clones were obtained by the colony hybridization method. When this is analyzed, the known LHCP II
It was similar to the gene structure and was determined to be the rice LHCP II gene.

Next, a genomic library was prepared. That is,
One week after germination in the dark, rice seedlings 30g C
DNA was extracted by the TAB method (Plant Molecular Biology (1985) 5:69). 10 μg of DNA was partially digested with the restriction enzyme Sau 3 AI (Takara Shuzo). This is the vector EMBL3
Connect to Bam HI site of (Stratagene, Inc.), were allowed to infect E. coli to prepare a genomic library.

Part of cDNA (from 5'untranslated region to mature protein N
The genomic library was screened using about 260 bases up to the end) as a probe to obtain four polyclonal clones. When these nucleotide sequences were determined by the dideoxy method, it was confirmed that they were all LHCP II genes having the same sequence. What is shown in FIG. 1 is the nucleotide sequence of the LHCP II gene and its promoter region.

Next, in order to examine the activity of the LHCP II gene promoter, the LHCP II gene promoter site was excised and G
Connected to US gene. That is, -78 of the sequence shown in FIG.
The PCR method (Saiki et al., (198
8), Science 239 ; 482-491), a linker of restriction enzyme Xba I on the −785 side and a restriction enzyme B on the +59 side.
Am HI linker was connected. Next, the Xba I site and the Bam HI site of pBI101 were cleaved with restriction enzymes, and L
The above promoter-containing fragment of the HCP II gene (LHCP-pr
o) was ligated (plasmid pLH / GUS). The partial gene map of the obtained plasmid pLH / GUS is shown in FIG. Using this plasmid pLH / GUS, the following transformation experiments were carried out to test the GUS activity of the transformed plants.

Protoplasts were prepared from tobacco leaves by a conventional method. The protoplasts were purified and then suspended in a buffer solution at a concentration of 2 × 10 ⁵ cells / ml. This solution has the structure shown in Figure 2.
pLH / GUS plasmid DNA was added at a concentration of 20 μg / ml. After this suspension was transferred to a container for electroporation and subjected to electrical stimulation, protoplasts were collected and cultured in MS medium. One week after the start of the culture, kanamycin was added to the medium at a concentration of 50 μg / ml to further grow the cells. When the callus had a diameter of 5 to 10 mm, the callus was transplanted to a plant hormone-free medium for regeneration. The leaves, stems, petals, and roots of the plant thus redifferentiated according to the method of Richard et al.
The expression level of the US gene was measured. Specifically, the enzyme that is the product of the GUS gene measures the fluorescence of 4MU of the product obtained by degrading 4MUG.

The results are shown in Table 1. As is clear from Table 1, although there was a difference depending on each individual, GUS was found in the leaves in each case.
Gene expression was highest.

[Brief description of drawings]

FIG. 1 shows the entire structure of the rice LHCP II gene and the DNA sequence on the 5'side (promoter part). FIG. 2 shows β contained in pBI101, which is a plant vector, which is obtained by cutting out the promoter portion of the LHCP II gene shown in FIG.
-Shows a genetic map of the plasmid pLH / GUS constructed by connecting upstream of the glucuronidase (GUS) gene.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Muneaki Samejima 5-242-1, 545-2, Matsushiro Tsukuba, Ibaraki Prefecture (56) References Annual Meeting of the American Social of Plant Physiologistics, 1987 (1987) , P .; 19-23

Claims (3)

[Claims] 1. A DNA fragment having the following nucleotide sequence or a DNA fragment comprising a part thereof having promoter activity. 2. The DNA according to claim 1, which is derived from rice.
fragment. 3. The DN according to claim 1 or 2.
A plant cell vector containing an A fragment.