Amplification of the active site of BnLIP3 gene of Brassica napus L. during germination

Abstract

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method to amplify a partial sequence of the lipase class 3 family designated BnLIP3 gene of Brassica napus L. in order to follow its expression and analyze its role during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the BnLIP3 family genes. Subsequently, to sequence determination of the 582 bp fragment, we deduced BnLIP3 specific primers for a nested RTPCR application. The deduced 194 amino acid sequence (Genbank 1160264) was found to share 85% of identity with lipase from Arabidopsis thaliana class 3 family. The GxSxG consensus motif near the catalytic triad at the active serine site was also identified. The peptidic sequence showed little homology with mammalian and microbial lipases. RT-PCR analysis indicated that BnLIP3 gene was expressed during B. napus seed germination.