关键词: 家蚕, 淀粉酶基因, 组织表达, 实时定量PCR

Abstract: Amylase is one of the important digestive enzymes in silkworm and known for hydrolyzing starch and glycogen. The mRNA expression level of the Bmamy7 and Bmamy5 in different tissues of 5th3d larva was analyzed by real- time PCR. Absolute quantitative analysis results indicated, Bmamy7 expressed in midgut was up to 3×109 copies/μg total RNA, its expression level in the malpighian was low, only 1.9×106 copies/μg total RNA; however, Bmamy5 was only expressed in midgut and the mRNA expression level  was 1.5×109 copies/μg total RNA. The specific primers were designed according to the cDNA sequence predicted encoding amylase in silkworm transcript database, two amylase genes were cloned by RT-PCR. The complete sequence of Bmamy7 is 1 608 bp, ORF consists 748 bp, encodes 503aa. Amino acid sequence analysis showed that Bmamy7 had a typical structure domain of α-amylase. The complete sequence of Bmamy5 is 1 196 bp，ORF consists 738 bp and encodes 245aa. Bmamy5 and Bmamy7 gene was expressed in prokaryotic expression respectively. The activity of recombinant Bmamy7 protein was very weak. The expression product of Bmamy5 was not detected by SDS-PAGE. The results provided references for research and application of amylase gene in the silkworm.

Key words: Bombyx mori, amylase gene, tissue-specific expression, real-time quantitative PCR