# Patent

## Title
Method of producing diacylglycerol and gene for inactivating function of gene which encodes diacylglycerol acyltransferase

## Abstract

The present invention provides a method of producing diacylglycerol characterized by inactivating the function of a gene which encodes diacylglycerol acyltransferase having the function of enzymatically reacting with diacylglycerol to create triacylglycerol in a seed, so as to allow diacylglycerol to be accumulated in the seed.

## Background
FIELD OF THE INVENTION
The present invention relates to a method of producing diacylglycerol according to gene recombination techniques, and a gene for inactivating the function of a gene which encodes diacylglycerol acyltransferase.
BACKGROUND OF THE INVENTION
Diacylglycerol has different nutritive values and physiochemical characteristics from triacylglycerol which is a major component of vegetable oil. Taking advantage of such characteristics, diacylglycerol has a wide range of prospective applications to edible oil, such as a salad oil, or chemical materials.
Heretofore, diacylglycerol has been produced from triacylglycerol obtained by compressing soybeans, rapeseeds or the like. These conventional methods include (1) a method based on an ester exchange reaction between oil and glycerol, and (2) a method based on an esterification reaction using fatty acid and glycerol. In these methods, either one of a chemical reaction using a catalyst of alkali (earth) metal hydroxide or an enzyme reaction has been used (see Japanese Patent Laid-Open Publication No. 2001-64671, WO 99/48378).
Such reactions have a disadvantage in terms of complicated production processes and/or relatively high cost because of (1) the need for a process of separating a desired product from by-products to purifying the desired product or (2) the needs for purified fatty acid as a material.
SUMMARY OF THE INVENTION
In order to improve the complicated production processes and/or relatively high cost in the conventional methods, it is an object of the present invention to provide a method of producing diacylglycerol, capable of directly producing diacylglycerol, which is hardly created from ordinary plants, by genetically modifying a lipid metabolic pathway of a plant, and to provide a gene sequence for inactivating the function of a gene which encodes diacylglycerol acyltransferase.
According to the present invention, there is provided a method of producing diacylglycerol characterized by inactivating the function of a gene which encodes diacylglycerol acyltransferase having the function of enzymatically reacting with diacylglycerol to create triacylglycerol in a seed, so as to allow diacylglycerol to be accumulated in the seed.
For example, in soybeans, all or a part of a diacylglycerol acyltransferase gene having a base sequence as described in claim 2 is used to suppressing the expression of the gene which encodes diacylglycerol acyltransferase having the function of creating triacylglycerol from diacylglycerol in the seed.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an explanatory diagram of a biosynthesis of triacylglycerol.



PREFERRED EMBODIMENT OF THE INVENTION
In an immature seed of a soybean, sesame or the like, triacylglycerol is synthesized from a glycerol-3-phosphate synthesized in cytoplasm during its growth process and a fatty acid synthesized in chloroplast and endoplasmic reticulum, through lysophosphatidic acid, phosphatidic acid, and diacylglycerol, by acylating and dephosphorylating reactions according to enzymes in the so-called Kennedy pathway residing in endoplasmic reticulum membrane, and the synthesized triacylglycerol is eventually accumulated as an oil body in the seed.
Diacylglycerol acyltransferase is an enzyme serving as a catalyst of the final step of the above series of enzymatic reactions or a reaction of transferring an acyl group to diacylglycerol, and it is considered that the catalysis of this enzyme acts as a rate-determining reaction in the biosynthetic pathway of triacylglycerol.
From this point of view, in the present invention, a diacylglycerol acyltransferase gene is recombined to inactivate its function or to suppress its catalytic action, so that diacylglycerol created during the course of synthesizing triacylglycerol is not synthesized as triacylglycerol, but sufficiently accumulated in a seed.
For example, in a soybean which is one of important oil crops, the gene sequence of diacylglycerol acyltransferase was obtained by screening a cDNA library derived from a soybean immature seed with the use of a probe designed based on a base sequence in a diacylglycerol acyltransferase cDNA of Arabidopsis thaliana. Further, the gene sequence of diacylglycerol acyltransferase clarified by determining a base sequence in the conventional manner was as described in claim 2. As a result, it was proved that the diacylglycerol acyltransferase gene of a soybean encodes a polypeptide composed of 500 amino acids, and exhibits 59.9% and 62.0% of homology in amino acids level, respectively, with the diacylglycerol acyltransferase of Arabidopsis thaliana and the diacylglycerol acyltransferase of Tobacco. Further, by analyzing the expression pattern of the gene encoding diacylglycerol acyltransferase based on the Northern blotting analysis, it was proved that the gene encoding diacylglycerol acyltransferase is highly expressed in an immature seed having a diameter of about 7 to 9 mm, and the expression is induced by an ethylene treatment.
According to the present invention, the original function of the gene encoding diacylglycerol acyltransferase can be inactivated by a gene recombination. The gene can be recombined by using conventional gene recombination techniques based on the development of vectors and the gene transfer.
A cDNA fragment to be transferred may have a full or a partial length. However, it is desirable to use a cDNA fragment derived from a plant seed into which the gene is transferred. The cDNA fragment is linked in the forward or backward direction to a promoter having a strong function in a plant, such as the cauliflower mosaic virus 35S promoter, and is then transferred into a plant by use of a conventional method, such as the Agrobacterium method or the particle gun method. During this process, the type of a tissue into which the gene is transferred is not limited to a specific cultured cell, lamina, growing point or the like, and any suitable tissue having means for obtaining a gene-recombined individual can be used. In addition, a marker for selecting the gene-transferred cell (individual) does not have any specific limitation as in various antibiotics or herbicides, phytohormone synthetic genes or the like.
A conventional technique can be used as the method for developing vectors. In pBI121 as a gene transfer vector for plants, this vector is used for the gene transfer using Agrobacterium, and a T-DNA region existing between an RB site and an LB site of pBI121 is transferred into a plant. A desired gene is generally transferred into a plant by using such a vector. When it is intended to X transfer a diacylglycerol acyltransferase gene, GUS gene may be cut out and the diacylglycerol acyltransferase gene may be integrated as a substitute for the cut-out GUS gene. For the cutting-out and integration, each recognition sequence of restriction enzyme existing at both sides of the GUS gene is used. Further, the transcription of the integrated gene is caused from one of the sides of the GUS gene to the other side, wherein this direction and the opposite direction are defined as forward and backward directions, respectively. The original functions of the plant gene in case of integrating the diacylglycerol acyltransferase gene in the forward direction and the backward direction can be suppressed by the cosuppression method and by the antisense method or RNA interference method, respectively.
The gene transfer is carried out by using a conventional method, such as the Agrobacterium method, particle gun method, or electroporation method. Instead of gene recombination, mutation may be used to obtain the gene for suppressing the original function of the gene which encodes diacylglycerol acyltransferase. A treatment for damaging DNA is performed to induce the mutation. For example, a treatment using nuclear-radiation or chemical mutagen (such as alkylating agent) or a treatment of incorporating nucleic acid analogues is performed to provide a mutant group, and a diacylglycerol accumulating type mutant is selected from the resulting mutant group.
These treatments can cause breakage, deletion or base substitution at an unspecified location of DNA to create a different gene from the original gene.
However, these mutations are caused definitely at random, and thereby the resulting mutations are almost independent of the desired gene. Thus, it is required to pick up a specific desired mutant gene from the mutant group. For example, in soybeans, the desired mutant can be only about one per tens of thousands to hundreds of thousands of individuals. Accordingly, it is necessary to provide a method for detecting a gene mutation efficiently and accurately.
The desired mutant gene can be detected by directly analyzing a base sequence of DNA because the mutations are caused on the DNA. The PCR method or Southern blotting method can be used for analyzing the DNA. Alternatively, the desired mutant gene can be determined by examining mRNA transcribed from the DNA or protein translated from the mRNA because diacylglycerol acyltransferase has the function of an enzyme. The Northern blotting method and the Western blotting method can be used for analyzing the RNA and the protein, respectively.
EXAMPLE
One example of gene recombination in soybeans will be described below.
1. Isolation (Cloning) of a Soybean Diacylglycerol Acyltransferase Gene


    (1) An mRNA is extracted from soybean immature seeds and purified according to a conventional manner.
    (2) Double-stranded cDNAs are synthesized by using the mRNA as a template, and are then integrated into a vector to prepare a cDNA library.
    (3) Using a primer designed based on the base sequence of a known diacylglycerol acyltransferase gene, a PCR is caused in the prepared library to amplify a part of the diacylglycerol acyltransferase gene.
    (4) After determining the base sequence of the gene fragment amplified by the PCR, the cDNA library is screened by using the fragment as a probe to obtain a full-length soybean diacylglycerol acyltransferase gene.

2. Development of an Expression Vector for an Antisense Diacylglycerol Acyltransferase Gene



After cutting out a cloned soybean diacylglycerol acyltransferase gene by using a restriction gene, the cloned soybean diacylglycerol acyltransferase gene is linked to an adequate promoter in the backward direction according to a conventional manner to create a plasmid having an arbitrary selection marker gene (preferably, this promoter provides a seed-specific gene expression. However, any other suitable promoter providing a steady gene expression, such as CaMV 35S promoter or actin promoter, may be used).
3. Transfer of an Antisense Gene Into a Soybean Cell.


    (1) The developed vector is coated over golden particles or the like, and the golden particles coated with the vector are transferred into cultured cells of a plant by using a particle gun.
    (2) The gene transfer cells are cultured by using a suitable agent while selecting the cultured cells to obtain a plant body through an adventitious embryo.


According to the present invention, a plant having an inactivated function of a gene which decodes diacylglycerol acyltransferase is used to allow desired diacylglycerol to be obtained immediately after an oil press process. This eliminates any need for the complicated synthesis process and the by-product removal process which have been inevitable in a conventional industrial production method, and thereby provides desirable supply of diacylglycerol with reduced cost and enhanced stability.
Further, the present invention allows lipid metabolic pathway in a plant body to be genetically modified, so as to accumulate a lager amount of diacylglycerol, which is hardly created by ordinary plants, in a seed. Thus, a desired diacylglycerol can be obtained directly from a plant without using any complicated processes as in the conventional method.
In addition, whereas the conventional method has produced diacylglycerol by processing oil/fats obtained through an oil press process and has not thereby been able to modify any oil/fats contained in grains, the present invention can freely change the ratio of triacylglycerol/diacylglycerol and can thereby provide enhanced nutritive value even in crops used directly for food.

## Claims

1. A method of accumulating diacylglycerol in a soybean seed comprising:
transforming a soybean plant with an RNA interference vector comprising a polynucleotide of SEQ ID NO: 1 in forward and backward orientation operably linked to a soybean seed specific promoter,
wherein expression of said vector suppresses expression of diacylglycerol acyltransferase in said soybean seed and diacylglycerol accumulates in the seed to an amount larger than in untransformed soybean seed.

2. An isolated soybean diacylglycerol acyltransferase polynucleotide comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 encodes SEQ ID NO: 2.