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

摘要： 淀粉酶是家蚕体内比较重要的一种消化酶。利用real-time PCR技术对Bmamy5和Bmamy7基因在家蚕5龄3 d不同组织的mRNA转录 水平进行了分析，结果显示Bmamy7只在家蚕5龄3 d中肠和马氏管组织中有转录，在中肠组织的转录高达3×109个拷贝/μg总 RNA，在马氏管组织中的转录丰度较低，只有1.9×106拷贝/μg总RNA；Bmamy5只在中肠组织中有转录，转录水平为1.5×109个 拷贝/μg总RNA。根据在家蚕转录组数据库中预测的编码家蚕淀粉酶的cDNA序列设计引物，利用RT-PCR技术克隆到两个家蚕淀 粉酶基因Bmamy5和Bmamy7，Bmamy7基因长1 608 bp，ORF长1 512 bp，编码503个氨基酸；氨基酸序列分析表明Bmamy7具有典型 的α-淀粉酶结构域。Bmamy5全长1 196bp，ORF长738 bp，编码245个氨基酸。对Bmamy5和Bmamy7进行了原核表达，Bmamy7重 组蛋活性较弱，但未检测到Bmamy5重组蛋白的目的条带。研究结果为家蚕淀粉酶基因的研究与应用提供了参考。

关键词: 家蚕, 淀粉酶基因, 组织表达, 实时定量PCR