JPH01235527A - Dwarfing of normal plant using 5-azacitidine - Google Patents

Abstract

PURPOSE:To accomplish dwarfing a plant over the next generation by immersing the germination-hastened seeds thereof in normal growth in a 5-azacitidine solution. CONSTITUTION:The germination-hastened seeds of a plant in normal growth are immersed in a 5-azacitidine solution for 12-36hr and washed with water to remove the 5-azacitidine attached thereto, thus dwarfing the plant in normal growth along with inheriting the dwarf nature to the next generation.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to genetic engineering of cultivated plants, and is particularly used for making cultivated plants such as rice and corn intelligent.

(Prior Art) Intelligence in cultivated plants is a very useful trait for agriculture. The mechanisms by which intelligence is expressed are diverse, and even though they are the same variety, there are quite a few intelligent plants that appear to be morphologically completely different species. The state of individual gene expression is also very different from that of the normal strain.

When attempting to elucidate such intelligence phenomena using molecular biological methods, methods to identify the primary cause of intelligence (
The former method and the latter method are considered to be methods of searching for the gene expression pattern of intelligent plants as a whole.Research on the known phenomena of intelligence has been carried out mainly by the former method, and a considerable amount of research has been done so far. Results have been obtained.In other words, through physiological analysis using short-lived mutant strains, it has been determined that abnormalities in endogenous hormones are one of the main causes.Looking at it schematically, it is the expression of intelligence. It is thought that this is caused by hormonal abnormalities, and that it is linked to all aspects of the plant's life cycle, including cell division, chest wall synthesis, metabolism, and water absorption.

Among them, defects in gibberellin biosynthetic threads have been found in some dwarf plants, rice, and maize.

Abnormalities in auxin biosynthetic threads were discovered in barley. Other growth hormone receptors that are thought to have abnormalities include corn D8 and wheat Rht. Since these mutant strains exhibit simple Mendelian inheritance, they are thought to be monogenic mutations, and research has been carried out on plants with abnormalities in the gibberellin biosynthesis system in order to search for the genetic locus.

Almost no results have been found based on the latter method, that is, research on the gene expression pattern of the whole rectangular plant. Recently, a method for searching for plant genes (hereinafter referred to as DNA) (hereinafter referred to as RIFLP) has been developed.

Attempts are being made to find genes and use them as genetic markers for various breeds. Attempts have been made to apply this method to the DNA of ppLpf'n plants to discover intelligence-specific mutations. Studies in animal cells show that actively mRNA
It has been found that e-transcribed DNA has a lower amount of methylated synthon (hereinafter referred to as mO) than inactive 7jl (7) DNA. DNA methylation and gene expression are inversely correlated. Although there is little research on this point in plant cells, it is suggested that no may act as a switch for gene expression.

(Problem to be solved by the invention) In view of the above-mentioned conventional research progress, we investigated the difference in gene expression between normal and orthogonal cases as a genetic marker,
If there is a difference in mC distribution, an attempt is made to forcibly lower endogenous m1Ufi using a methylation inhibitor called 5-azacytidine, thereby making the plant more orthogonal.

(Means for solving the problem) Germinated seeds of fines and maize, that is, seeds in the early stage of cell division, were added to 5-azacytidine solutions of various concentrations.
By soaking for about 2 to 66 hours, then washing with water to remove the chemicals, and cultivating normally, the plants are silicified and passed on to the next generation plants.

(Effect) In the chain consisting of adenine, guanine, cytosine, and thymine in the DNA of normal strains of rice and maize,
5- methylated by intracellular DNA methylase
Methylation of methylcytosine (hereinafter also referred to as mC) is inhibited by 5-azacytidine, participates in the DNA expression thread, and forms a form called orthogonalization, which is inherited by the next generation.

(Example λ The research results of the present invention will be described in detail below in place of Examples.

For rice, dxlay and other normal types were used. Total DNA was extracted from the plant body, and its base composition and frequency of adjacent bases were investigated. mo appeared most frequently in OpG, a proximal base frequency analysis. In maize, +780% of the total OpG was mopG, and in rice it was about 40%. OpG in intelligence corn
methylation decreased by about 4-5%, and no difference was observed in rice. These results indicate that there is not much difference in mC content as a whole between normal and normal DNA. However, if the decrease in moi observed in corn is true, then per 600 bp of DNA - some no.
This can be said to be a fairly large difference at the genetic level.

mo in specific sequences mC in individual genes
The Southern hybridization method was used to measure the . Total DNA was digested with restriction enzymes that can distinguish between CpG and m0pG, and the molecular shields of the excised DNA fragments were compared. For detection, cDNA of alcohol dehydratase obtained from corn and 960 bp DNA obtained from rice were used. When alcohol dehydratase was used, there was a clear difference in OpG between normal and orthogonal in both rice and corn. Similar results were obtained when 960 bp DNA was used for detection.

This suggests that there is a difference in n0 distribution between Masamachi and sowing plants, and that each is involved in unique gene expression.

If normal plants become intelligent with 5-azacytidine I! IC
is really involved in gene expression in plants, and if there is a difference in its content among intelligent strains, then artificially the normal strain's mo
e? By e-cultivation, a plant exhibiting a morphology close to an intelligent type can be obtained. For this reason, I constructed the following experimental thread.

5-azacytidine is the most specific DNA methylation inhibitor currently known. To this, normal rice and corn seeds were soaked immediately after germination and late at 3ffi L. After completely removing the chemical with water, can you sow the seeds normally and let them grow?
It was measured. As a result, the total height of the 5-azacytidine-treated strain was reduced by about 15%. When we established the tT length of this plant, it showed a direction of about 1, similar to that of the intelligent type.

The same results were obtained with corn. These data are I
) This shows that NA methylation is involved in gene expression in some way in plants, and is ultimately involved in morphogenesis.

Hypomethylation of gθnomic DNA by 5-azacytidine Pre-objects morphologically intellectualized by 5-azacytidine in DNA! ! 1c (1) was decreased using the Southern blot method.

The method is as described above, using a restriction enzyme that can distinguish between no.
After digesting A, the presence or absence of no in the detection sequence was examined by appropriate detection. In the case of maize, it contains DNA with the same sequence as rice 960bP satellite DNA, and this I
) A part of geanine-cytosine-guanine-cytosine in the NA fragment was demethylated by treatment with 5-azacytidine. In the case of rice, it was also significantly demethylated. These results indicate that the treatment-induced intelligent morphological changes are parallel to DNA demethylation.

F1 gills were obtained by self-pollination from cultivated corn. These seeds were sown normally and growth was determined. The total height of plants obtained from treated parents is clearly lower than that obtained from untreated parents. In other words, it was shown that the morphological changes induced by the treatment were inherited by the offspring.

The above is a summary of the experimental results, but discussion of the experiment and future issues are omitted.

Although rice and corn B are mentioned in the examples, it is assumed that similar results can be obtained with edible plants of the Chigiaceae family, such as wheat, millet, and barnyard grass. Furthermore, the concentrations of the 5-azacytidine aqueous solution were 0%, 1 mM, Q, and 3mM in the experiment, but are not limited thereto. Furthermore, the immersion time of the germinated seeds in the same aqueous solution was not limited to overnight (12 hours), but could be as long as 66 hours if the concentration was low.

(Effects of the invention) According to the present invention, intelligent plants can be obtained very easily, and the intellectualization is inherited and reproducible to the next generation of plants. It is expected that stocks can be created.

Claims (1)

[Claims] By immersing germinated seeds of normally cultivated plants in a 5-azacytidine solution, then removing the drug and performing normal cultivation, the plants become dwarfed and the dwarfism is inherited by the next generation plants. Method for dwarfing normal plants using 5-azacytidine.