关键词: 家蚕, 3-磷酸甘油脱氢酶, 基因克隆, 结构特征, 表达谱

Abstract: Glycerol-3-phosphate dehydrogenase （GPDH） is a soluble cytosolic NAD-dependent enzyme present in eukaryotic organisms and has some important functions, such as energy transfer. Bombyx mori is a central model animal to research metamorphosis and energy metabolism. In order to determine the role of GPDH in B. mori, we cloned a complete gene of BmGpd based on protein （lethal （2） k05713） sequence of Drosophila melanogaster from NCBI （GenBank accession number: NP_725495.1）, which is 27 983 bp in full length and contains 16 exons and 15 introns（GenBank accession number is EF154335）. The two complete mRNA transcripts from B. mori strain C108 are 3 456 bp and 2 979 bp in full length （GenBank accession numbers are GQ865685 and GQ865686, respectively）, both including a 2 166 bp ORF that encodes 721 amino acids （Mw 82.1 kD, pI 6.43）. BmGPD protein includes several membrane-spanning regions and a signal peptide sequence with a length of 20 aa. The results of phylogenetic analysis and motif search revealed that BmGPD belonged to glycerol-3-phosphate dehydrogenase （GPDH） family, which was not reported in B. mori and could be abundantly inducible expression in Escherichia coli. Semi-quantitative RT-PCR analysis indicated that a high-level of BmGpd mRNA occurred in tissues （organs） of the midgut, silk gland, gonad and fat body, and it occurred at the middle stage of the 5th instar larva; however, a low level in the blood, as well as on day 3 of the pupal stage, and a lower level occurred during the diapause. The result of microarray-based gene expression profile showed that the expression abundance of BmGpd was very high in head and integument, while low in fat body and Malpighian tubule on day 3 of the 5th instar larva, and there were no obvious differences between the male and female strains. Expression profile of ESTs indicated that the expression abundance was the highest in the midgut on day 2 of the 4th instar larva. RNAi result suggested that there are strong metabolic compensation pathways to make up for the silence of BmGpd gene. This study provides a basis for further investigating the expression regulation of GPDH and its relationship with metamorphosis and energy metabolism in B. mori.

Key words: Bombyx mori, glycerol-3-phosphate dehydrogenase, gene cloning, structure characteristics, expression profile