Cloning, expression and functional analysis of a delta 6-desaturase gene from the silkworm, Bombyx mori L.

Abstract

Delta 6-fatty acid desaturase is a membrane-bound enzyme, which is the rate-limiting factor in the biosynthesis of polyunsaturated fatty acids. In this study, a novel delta 6-desaturase gene was cloned from Bombyx mori (BmD6DES). Sequencing analysis revealed that BmD6DES has an open reading frame of 1357bp that encodes 448 amino acids. Heterologous expression in yeast demonstrated that BmD6DES could synthesize γ-linolenic acid (GLA, 18:3, Δ6,9,12) by utilizing the endogenous substrate linoleic acid (LA, 18:2, Δ9,12). We found that BmD6DES transcripts were distributed in almost all B. mori tissues, with high expression levels observed at the 5th instar larval, pupal, and adult moth stages. A functional analysis of BmD6DES was performed by measuring mRNA levels after temperature stress, fungal infection, and RNA interference (RNAi). The results indicated that the highest expression of BmD6DES was observed at low temperatures (0°C) and 6h to 36h after fungal infection. qPCR analysis demonstrated that BmD6DES mRNA levels in pupa after BmD6DES RNAi treatment were significantly reduced from 12h to 72h compared to those in the control group. Our findings suggest that BmD6DES not only induces the formation of the third carbon–carbon double bond in the LA carbon chain, but also leads to sensitivity to low-temperature stress and fungal infection. These results imply that BmD6DES is a key gene in the γ-linolenic acid pathway during B. mori development.