KR100747151B1 - -6 Delta-6 desaturase gene from Phytophthora citrophthora - Google Patents

Abstract

A delta-6 desaturase gene isolated from Phytophthora citrophthora is provided to produce gamma-linolenic acid which lowers blood cholesterol level and improves blood circulation from the plants, so that the added value of plant is increased. The delta-6 desaturase gene isolated from Phytophthora citrophthora has the nucleotide sequence of SEQ ID NO:1. A vector which produces gamma-linolenic acid contains the delta-6 desaturase gene has the nucleotide sequence of SEQ ID NO:2. A transformed Saccharomyces cerevisiae(KACC 93044P) capable of producing gamma-linolenic acid is produced by inserting the delta-6 desaturase gene into Saccharomyces cerevisiae.

Description

Delta-6 desaturase gene from Phytophthora citrophthora}

Figure 1 shows the cloning results of the delta 6 desaturase gene by RACE PCR.

Figure 2 shows the result of cloning delta 6 desaturase gene of full size.

Figure 3 shows the nucleotide sequence and the estimated amino acid sequence of the delta-6 desaturase gene isolated from the fungus.

Figure 4 shows the result of identifying the Delta 6 desaturase gene expression yeast carrier.

Figure 5 shows the fatty acid analysis of Delta-6 unsaturated enzyme gene transgenic yeast.

Figure 6 shows the fatty acid analysis chromatogram of pYES2 and pYES2-D6 transgenic yeast.

The present invention is a technique for separating a functional gene and confirming the function of the gene. Until now, delta-6 desaturase genes isolated from animals and microorganisms have many functions in phythium, Mucor, and Phyzopus, but most fungi have caused disease in animals, The potential risk of developing transgenic crops has not been reported in Phytophthora, which is lower than other fungi.

Since the present invention has only 64% identity at the amino acid level with genes isolated from the existing phythium genus and has less than 50% homology with genes isolated from other plants and fungi, it is different from those previously reported. Isolate Delta-6 desaturase gene to check its function, and use it to transform functional crops of perilla, soybean, and so on.

The gene of the present invention is the first isolated gene in Phytophthora, which has a homology of 60% or less with other previously reported genes, which are similar in function but different in structure.

The nucleotide sequence of the gene was determined by selecting a delta-6 desaturase gene clone from the fungus (Phytophthora) using the RACE PCR method. The isolated gene was inserted into pYES2 carrier, which is a yeast expression carrier, and transformed into yeast to assay the function of the gene expression product.

Hereinafter, the present invention will be described in detail with reference to examples, but the protection scope of the present invention is not limited to the following examples.

Example  1: mold Phytophthora Origin delta -6 Desaturase Gene Isolation and Sequencing

     Using a delta 6 desaturase gene involved in the production of gamma-rollenoic acid in a phytophthora, 5'-RACE, 3-RACE method (Figure 1) using the RACE cDNA Amplification kit (Figure 1) to secure full length clones (Figure 2) ) And the nucleotide sequence shown in SEQ ID NO: 1 (Fig. 3).

Example  2: Delta Production and transformation of yeast-expressing transgenic carriers of --6 unsaturated enzyme gene

To confirm the function of the Delta-6 desaturase gene, the isolated gene was inserted into the yeast transformation carrier pYES2 / CT. pYES2 / CT is a selection marker in Escherichia coli and yeast and has ampicillin resistance gene and uracil biosynthesis gene, respectively.

Delta-6 desaturase gene was inserted into HindIII and BamHI site of multicloning site between GAL1 promoter and CYC1 terminator. Carrier creation was confirmed after insertion of the delta 6 desaturase gene in Escherichia coli and cleaved with HindIII / BamHI, and the linkage of the nucleotide sequence was confirmed by sequencing (FIG. 4). Yeast Expression Carrier Saccharomyces by Lithium Acetate Cerevisiae was transformed and selected using a minimal medium lacking uracil and deposited as a new strain to the Agricultural Biotechnology Research Institute (KACC 93044P).

Example  3: Delta Gamma-linolenic Acid Biosynthesis Assay in a 6-6 Unsaturated Enzyme Gene Transformed Yeast

In order to confirm gene function using yeast inserted with Delta-6 desaturase gene, transgenic yeast was seed cultured at 30 ° C. in medium containing raffinose and tergitol NP-40 added to uracil deficient medium. Seed cultured yeast was inoculated in fresh medium, and then cultured to OD ₆₀₀ = 0.5-0.6 and linolenic acid (18: 2, Δ ^9,12 ) and alpha-linolenic acid (18: 3, Δ ^9,12,15 ) were used as substrates. In addition, galactose was added to the inducer, and the expression of the genes was induced while incubating at 25 ° C. for 2 days. The control group used yeast transformed with only the pYES2 carrier. The activity of the delta-6 desaturase gene product induced by galactose was found in the yeast uptake substrates of linolenic acid (18: 2, ^Δ9,12 ) and alpha-linolenic acid (18: 3, ^Δ9,12,15 ). Whether or not a double bond can be formed on the first carbon was confirmed using gas chromatography.

The added linolenic acid (18: 2, Δ ^9,12 ) was converted to gamma-linolenic acid (18: 3, Δ ^{, 6,9,12} ) and alpha-linolenic acid (18: 3, Δ ^9,12,15 ). Was confirmed to be converted to stearonic acid (stearidonic acid) (18: 4, Δ ^{6,9,, 12,15} ). In addition, it was confirmed that the 16: 1 (Δ ⁹ ) fatty acid inherent in the yeast was converted to a 16: 2 (Δ ^6,9 ) fatty acid (FIGS. 5 and 6). As a result, the delta-6 desaturase gene product isolated from the fungus was confirmed to be involved in the biosynthesis of gamma-linolenic acid (18: 3, Δ ^6,9,12 ).

The delta-6 desaturase gene was constructed to be controlled by the yeast expression carrier, pYES2 galactose-induced promoter, and transformed into yeast. It was confirmed that this was synthesized. Gamma-linolenic acid is well known for its bioactive functions and is currently marketed as a high value added health food. Therefore, if the function of the delta-6 desaturase gene isolated by the present invention is confirmed and used for oily crops such as perilla, oily crops that produce high-value functional gamma-linolenic acid will be developed to contribute to raising farm income.

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Claims (3)

The delta-6 desaturase gene of SEQ ID NO: 1 derived from a fungus (Phytophthora). A gamma-linolenic acid production vehicle set forth in SEQ ID NO: 2 comprising the gene of claim 1. Saccharomyces for production of gamma-linolenic acid prepared by inserting the gene of claim 1 cerevisiae ) transformants (KACC 93044P).

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