ESTABLISHMENT AND APPLICATION OF REAL-TIME QUANTITATIVE PCR REACTION SYSTEM FOR STUDY IN GENE EXPRESSION OF 5 FATTY ACYL DESATURASE IN ABALONE (HALIOTIS DISCUS HANNAI INO)

LI Ming-Zhu; MAI Kang-Sen; HE Gen; AI Qing-Hui; XU Wei; ZHANG Wen-Bing

doi:10.7541/2014.47

Volume 38 Issue 2

Mar. 2014

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ACTA HYDROBIOLOGICA SINICA > 2014 > 38(2): 328-334. > DOI: 10.7541/2014.47

LI Ming-Zhu, MAI Kang-Sen, HE Gen, AI Qing-Hui, XU Wei, ZHANG Wen-Bing. ESTABLISHMENT AND APPLICATION OF REAL-TIME QUANTITATIVE PCR REACTION SYSTEM FOR STUDY IN GENE EXPRESSION OF 5 FATTY ACYL DESATURASE IN ABALONE (HALIOTIS DISCUS HANNAI INO)[J]. ACTA HYDROBIOLOGICA SINICA, 2014, 38(2): 328-334. DOI: 10.7541/2014.47

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ESTABLISHMENT AND APPLICATION OF REAL-TIME QUANTITATIVE PCR REACTION SYSTEM FOR STUDY IN GENE EXPRESSION OF 5 FATTY ACYL DESATURASE IN ABALONE (HALIOTIS DISCUS HANNAI INO)

Key Laboratory of Aquaculture Nutrition and Feeds (Ministry of Agriculture), Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, China

Received Date: January 28, 2013
Rev Recd Date: October 16, 2013
Published Date: March 24, 2014

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Abstract

Abstract

5 fatty acyl desaturase (Fad) is the key enzyme for the biosynthesis of long chain polyunsaturated fatty acids. Abalone, Haliotis discus hannai Ino, has two genes of 5 Fad (Hdhfad1 and Hdhfad2), of which the cDNA sequences are highly similar (96.82 %). In this study, an efficient Real-Time quantitative RCR (RT-qPCR) assay based on the 2-Ct method was established for measuring the expression levels of Hdhfad1 and Hdhfad2. Data were normalized to the gene expression levels of -actin and ribosomal protein S9 (RPS9). The effects of different dietary lipids on the expressions of Hdhfad1 and Hdhfad2 were studied in muscle tissue of abalone using this assay. We prepared three types of diets that contain different dietary lipids－tripalmitin (TP diet), ARA oil (AO diet) and EPA oil (EO diet). The results showed that primers designed for Hdhfad1, Hdhfad2, -actin and RPS9 were specific, with the amplifying efficiency of 1.05, 0.99, 0.97 and 0.98 respectively. The efficiency of these primers was suitable for the 2-Ct method. The optimal annealing temperature and reaction volume for the RT-qPCR amplification were 52℃ and 25 L respectively. We concluded that this assay was rapid and sensitive in analyzing the expressions of 5 Fad in abalone. This assay demonstrated that the expression levels of Hdhfad1 and Hdhfad2 were significantly decreased in abalone fed with EO or AO diet compared to those of TP group.
- Fatty acyl desaturase,
- Abalone,
- Real-Time quantitative PCR,
- Gene expression

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ESTABLISHMENT AND APPLICATION OF REAL-TIME QUANTITATIVE PCR REACTION SYSTEM FOR STUDY IN GENE EXPRESSION OF 5 FATTY ACYL DESATURASE IN ABALONE (HALIOTIS DISCUS HANNAI INO)

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ESTABLISHMENT AND APPLICATION OF REAL-TIME QUANTITATIVE PCR REACTION SYSTEM FOR STUDY IN GENE EXPRESSION OF 5 FATTY ACYL DESATURASE IN ABALONE (HALIOTIS DISCUS HANNAI INO)

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