Cloning and Functional Characterization of a Fatty Acyl-Acyl Carrier Protein Thioesterase Gene (BnFatB) in Brassica napus L.

Plant fatty acyl-acyl carrier protein thioesterase (FAT) is a major enzyme regulating the amount and composition of fatty acids in lipids. In this study, one type of cDNA, corresponding to the fatty acyl-acyl carrier protein (acyl-ACP) thioesterase (Fat) enzyme, was isolated from the seed of Brassica napus cv. Ningyou۱۲. BLAST results revealed that the cDNA identified highly with the FatB class of plant thioesterases. The cDNA contained a ۱,۲۴۵ bp open reading frame (ORF), encoding a protein that contained ۴۱۴ amino acid residues. Subcellular localization results showed that the BnFatB protein was located in the chloroplast. The BnFatB (KC۴۴۵۲۴۳) gene was expressed in many tissues and was strongly expressed in seeds. Heterologous expression of the BnFatB gene in yeast cells was performed in order to ascertain the function of the BnFatB gene. Semi-quantitative RT-PCR results indicated that the expression level of the BnFatB gene in transformed yeast had significantly increased compared to the control. GC analysis of the fatty acid revealed that, when compared with the control, the content of C۱۶:۰ and C۱۸:۰ in yeast cells expressing BnFatB increased by ۴۵.۷ and ۲۱.۷%, respectively; while C۱۶:۱ and C۱۸:۱ decreased by ۱۵.۳ and ۳۰.۶%, respectively. This study demonstrated that the BnFatB gene had similar function as the FatB enzyme, preferentially releasing saturated fatty acid from the acyl carrier protein. It can therefore be used as a candidate target for fatty acid improvement in oilseed rape.

Keywords:

Fatty acid , GC analysis , Heterologous expression , Lipid , Yeast