JPH0589A - Method for inducing gene manifestation - Google Patents

Abstract

PURPOSE:To effect the manifestation of introduced gene with light irradiation by using a promoter of light-harvesting chlorophyll a/b bond. CONSTITUTION:A promoter of light-harvesting chlorophyll a/b bond is introduced into a gene and the gene is irradiated with light to induce the gene manifestation. The light-harvesting chlorophyll a/b bond protein gene can be separated by screening a genomic library of Japanese lawn grass using a CDNA of a light-harvesting chlorophyll a/b bond protein gene of rice as a probe.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene for a light-harvesting chlorophyll a / b binding protein relating to photosynthesis of plants and its promoter, and particularly to the development of new plant varieties utilizing gene recombination and its metabolites. It is used for modifying cultured cells to be produced.

[0002]

2. Description of the Related Art In recent years, it has become possible to use gene recombination technology in plants, and studies on dicotyledonous plants are being actively conducted. In particular, a vector system utilizing the Ti plasmid of the soil bacterium Agrobacterium tumefaciens has been prepared and used for plant transformation experiments. (H
oekema et al. (1985) Plant Molcu
lar Biology 5: 85-89, Velten
Et al. (1984) The EMBO Journal3:
2723-2730)

However, since the genus Agrobacterium only infects dicotyledonous plants and limited monocotyledonous plants, it was difficult to carry out transformation experiments on monocotyledonous plants. The electroporation method is a technique in which an electric stimulus is applied to protoplasts to make a tiny hole in the cell surface, a gene is introduced from the hole, and transformation is performed. This electroporation method has enabled transformation experiments in rice and the like. The promoter in the conventional vector system is a promoter derived from the gene of Ti plasmid of Agrobacterium tumefaciens,
Or most of them are promoters derived from the cauliflower mosaic virus (CaMV) gene.

[0004]

[Problems to be Solved by the Invention] These promoters have been used as promoters for efficiently expressing a gene in plant cells. It was a promoter that could always regulate the expression of genes regardless of the control. Currently, gene transfer technology into plant cells is established, and strong promoters are used for gene expression. However, when a plant is bred, a promoter that can be specifically expressed in the period of growth or an organ or a promoter that can be artificially controlled is desired.

[0005]

[Means for Solving Problems] The light-harvesting chlorophyll a / b binding protein (hereinafter abbreviated as LHCPII) present in the thicolide membrane of plant chloroplast binds to the light-harvesting chlorophyll of photosynthetic system II. Is a complex protein
It plays an important role in photosynthesis. The gene is encoded by nuclear DNA and is known to be induced by light and expressed in chloroplasts.

The inventors of the present invention thought that it would be possible to induce gene expression by light by utilizing the promoter of the LHCPII gene, and cloned the LHCPII gene and analyzed the promoter portion. 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 plant cells. It was confirmed that the β-glucuronidase gene was expressed by irradiating light in a callus (cell mass) obtained by culturing cells into which the gene was introduced, or a plant body redifferentiated therefrom. Therefore LHCPII
It was confirmed that the promoter of the gene was induced by light, and the present invention was completed.

Specific LHCPII gene isolation and plant transformation using a vector utilizing a promoter are as follows. The LHCPII gene is isolated as follows. Nuclear DNA is extracted from the above-ground parts of Noshiba by the CTAB method and ligated to a vector for cloning Escherichia coli to prepare a Noshiba genomic library. LHCP of rice
The genomic library is screened by using the cDNA of the II gene as a probe to isolate the LHCPII gene of Noshiba.

FIG. 1 shows the entire structure of the isolated LHCPII gene and the DNA base sequence of the promoter region. The portion surrounded by a frame shows the structural portion of the LHCPII gene. +1
Is the transcription start point of the LHCPII gene, and the A of +91
TG represents the nucleotide sequence encoding the first amino acid of the mature LHCPII protein. The TATA box required for gene transcription is found at -46.

The activity as a promoter is examined as follows. The portion that is considered to have promoter activity is 5 of the β-glucuronidase (GUS) gene.
Connect to the upstream side. 4-methylumbelliferone (4
-MU) is reacted with 4-MUG having glucuronic acid bound thereto as a substrate, and the reaction product 4-MU emits strong fluorescence for quantification. (Richard et al. (198
9) The EMBO. Journal 6: 3901-
3907). LHCPII gene promoter and GUS
The gene-connected plasmid pLH / GUS is introduced into plant protoplasts by the electroporation method. The introduced plant cells are selected with kanamycin. pL
The leaf NPTII gene is also contained in H / GUS, and when this enzyme is synthesized, it becomes resistant to kanamycin. Therefore,
Cells that survive in the presence of kanamycin are more likely to have a chimeric inheritance of the LHCPII promoter and GUS gene.

As described above, the plant is redifferentiated from the callus which is considered to have the foreign gene. Gene is expressed in the regenerated plant under light conditions, and the plant is stalked,
By measuring the GUS activity for each of the leaf and root organs, the highest expression level in the leaves of any plant is confirmed. Therefore, according to the method of the present invention, gene expression by light can be induced by utilizing the promoter of LHCPII gene. In addition, since it is easily expressed in a site having a photosynthetic organ such as a leaf, it has opened up the possibility of organ-specific gene expression.

[0011]

[Example] A noshiba genomic library was prepared as follows. CTAB method (Pla
nt Molecular B: Alogy (198)
DNA was extracted by 5) and 69). DNA10
μg was completely digested with a restriction enzyme EcoR | (for example, manufactured by Toyobo). A DNA fragment of about 6 kb was cloned into the phage containing phage to make a genomic library. The genomic library was screened using the rice LHCPII gene CDNA as a probe to obtain one positive clone, and the nucleotide sequence was determined by the dideoxy method. FIG. 1 shows the nucleotide sequences of Noshiba LHCPII gene and its promoter region.

Next, in order to examine the activity of the LHCPII gene promoter, the LHCPII gene promoter site was excised and connected to the GUS gene. Ie −
The portion from 265 to +78 was cut out, and the restriction enzyme xbaI linker was cut on the -265 side, and the restriction enzyme BamH | site was cut on the +78 side with a restriction enzyme, and the promoter region of the LHCPII gene (LHCP-P
ro) was ligated (plasmid pLH / GUS). A transformation experiment was performed using this plasmid pLH / GUS to assay the GUS activity of the transformed plant.

Prepare protoplasts from tobacco leaves,
The cells were suspended in a buffer solution at a concentration of 2 × 10 5 cells / ml. To this solution was added pLH / GUS plasmid DNA having the structure shown in FIG. 2 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, kanamylen 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, it was transplanted to a plant hormone-free medium and redifferentiated. In the leaves, stems, petals, and roots of the plant redifferentiated in this way, the enzyme that is the product of the GUS gene is 4MUG according to the method of Richard et al.
Was measured by 4 MU fluorescence of the decomposed product. The results are shown in FIG. 3. From this figure, it can be seen that the expression level of the GUS gene is the highest in the leaves in all of the cases, although there are differences among the individuals.

[0014]

EFFECTS OF THE INVENTION By using the promoter developed by the present invention, it becomes possible to express a transgene by irradiating it with light. Moreover, since the highest expression level was observed in the leaves, it became possible to give specificity to the site where the gene was expressed.

[Brief description of drawings]

FIG. 1 is a nucleotide sequence of Noshiba LHCPII gene and its promoter region.

FIG. 2 is a diagram showing a plant transformation vector.

FIG. 3 is a diagram showing the measurement results of GUS activity of regenerated plants.

Claims (3)

[Claims] 1. A method for inducing gene expression, which comprises performing light exposure on a promoter for light-harvesting chlorophyll a / b binding. 2. The method of inducing gene expression according to claim 1, which is carried out on Noshiba. 3. The method for inducing gene expression according to claim 1, wherein the light-harvesting chlorophyll a / b binding promoter is a DNA sequence.