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Transgenic fish with increased unsaturated fatty acid content

A fatty acid and genetically modified technology, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of no observed vitamin C production, no direct evidence, etc., and achieve high vitality and high contribution Effect

Inactive Publication Date: 2007-06-27
JAPAN SCI & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This study was about the introduction of the L-gulonate-γ-lactone oxidase (rGLO) gene that acts as a catalyst in the terminal reaction of vitamin C, but the production of vitamin C was not observed
Furthermore, attempts were made to improve carbohydrate metabolism in rainbow trout by simultaneous injection of constructs containing cDNAs of human glucose transporter protein (hGluT) and rabbit hexokinase II (rHK II), but no functionally relevant effects of rHKII and hGluT1 were observed. Direct evidence (for example, see Non-Patent Document 32)

Method used

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  • Transgenic fish with increased unsaturated fatty acid content
  • Transgenic fish with increased unsaturated fatty acid content
  • Transgenic fish with increased unsaturated fatty acid content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Breeding and food for zebrafish

[0092] Using zebrafish of the AB strain (walker et al., 1999), with several amendments to the abstract of Westerfield (1995), the fish were spawned and reared. Parent fish (broodstock) were reared at 28±1° C. in a 40 liter tank under a photoperiod of 14 hours light and 10 hours dark. The fish were fed with otohime (trade name: manufactured by Nisshin Corporation) and Artemia (manufactured by Salt Creek Corporation) nauplii every other day, respectively. In order to perform microinjection twice a day, 12 females and 8 males were placed in 4 water tanks. Divide these tanks into two groups so that one group spawns at 10:30am and the other at 14:00pm.

Embodiment 2

[0093] Example 2: Cloning of Δ6 desaturase cDNA

[0094] [Delta]6 desaturase-like cDNA was isolated from the liver of Masu salmon (Oncorhynchus masou) by PCR using two denatured primers based on the GenBank database (AB070444). These two denatured primers, forward and reverse are (5'-TACTCCATGGTTCAGCAAATGAATTGAACA-3') and (5'-TCGTCCATGGCCATTCACTGCTGACAAGGA-3') respectively. In order to facilitate the cloning of expression vectors, the two-day primers each contain an NcoI site ( offline part). PCR amplification was performed under the following conditions. 3 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 62°C, 1.5 minutes at 72°C. Then, the PCR amplification product of 1680 bp was subcloned into pGEM-TEasy vector (manufactured by Promega).

Embodiment 3

[0095] Embodiment 3: Construction of introducing gene expression

[0096]The 8.5 kb plasmid pArD6 (Fig. 1) was used for gene expression. That is, pRc / RSV (manufactured by Invitrogen) was treated with XhoI, and the extracted bovine growth hormone (BGH poly(A)) sequence was ligated to pBluescriptSK(+ / -) (manufactured by Clontech). The 3.7 kb medaka β-actin promoter (mβ-actin) (Takagi et al., 1994) was modified by PCR with pOBA-109, provided for the expression of pBluescriptSK containing BGH poly(A) ( + / -) linked EcoRI sites. The Δ6 desaturase-like gene (D6D) of the 1477kb masu salmon (O.masou) obtained in embodiment 2, as two oligonucleotide primers based on the GenBank database (AB070444), use the SalI recognition site containing Forward primer SalI-desF3 (5'-TTGTCGACGGTCTGAGTGGAGCAGAGAGAA-3'; sequence number 5) and reverse primer-des-OmaR (5'-ATCCAGGAAATGTCCTCTCTGTTCGCA-3' (sequence number 6) of point (underline part), at 94 3 minutes at 94°C, 30 seconds at 94°C, 30 seconds...

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Abstract

It is intended to utilize a vegetable fat, which is less expensive and can be easily controlled in qualities, thereby largely contributing to the reduction of cost and labor in fish nursery sites and, moreover, enabling the production of fish fry having a high vitality and being tolerant to handling and low temperature without resort to fish oil containing EPA and DHA as a formula feed for fish cultivation. Namely, fish with an increased unsaturated fatty acid content in which a fatty acid desaturase gene is expressed due to the transfer of the fatty acid desaturase gene thereinto. The fatty acid desaturase gene is one or more members selected from among Delta4 fatty acid desaturase gene, Delta5 fatty acid desaturase gene and Delta6 fatty acid desaturase gene, and it is preferable that these genes originate in a freshwater fish. It is preferable that the expression of a fatty acid desaturase gene originating in a freshwater fish is promoted by a beta-actin promoter originating in killifish.

Description

technical field [0001] The present invention relates to a fish whose unsaturated fatty acid content is increased by introducing a fatty acid desaturase gene, and a production method of the fish whose unsaturated fatty acid content is increased. Background technique [0002] Now being extensively studied, n-3 highly unsaturated fatty acids (HUFA), especially eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n -3) Excellent effects on human health and development (for example, refer to Non-Patent Documents 1, 2, 3, 4, 5). In humans, these fatty acids are mainly inoculated from seawater fish and fish oils of their extracts, but in recent years, as the number of fish caught in the sea has decreased, the stable supply of raw materials necessary for the production of EPA and DHA is being threatened (for example, , refer to Non-Patent Document 6). Furthermore, it is estimated that between 2005 and 2010, the supply of these became critical (for example, refe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/53C12N9/02C12N15/09C12N15/85
CPCC12N15/8509A01K2217/05C12N9/0083A01K2267/02A01K2227/40A01K67/0275A01K67/027C12N15/09
Inventor 吉崎悟朗竹内俊郎佐藤秀一奇龙维斯旺纳特阿利穆丁
Owner JAPAN SCI & TECH CORP
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