家蚕淀粉酶基因的克隆、表达与分析  

Cloning,Expression and Sequence Analysis of Amylase Gene in the Silkworm,Bombyx mori

作　　者：吕言娜 陈晓妍[1,2] 邵丽萍[1,2] 李轶女[2] 胡小元[2] 张志芳[2] 易咏竹[1] 

机构地区：[1]江苏科技大学,江苏镇江212018 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《生物技术进展》2016年第3期193-199,共7页Current Biotechnology

基　　金：国家863计划项目(2011AA100603);国家973计划项目(2012CB114600)资助

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3×10^9个拷贝/μg总RNA,在马氏管组织中的转录丰度较低,只有1.9×10^6拷贝/μg总RNA;Bmamy5只在中肠组织中有转录,转录水平为1.5×10^9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c DNA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。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 5th3 d larva was analyzed by real-time PCR. Absolute quantitative analysis results indicated,Bmamy7 expressed in midgut was up to 3×10^9copies / μg total RNA,its expression level in the malpighian was low,only 1.9×10^6copies / μg total RNA; however,Bmamy5 was only expressed in midgut and the mRNA expression level was 1.5×10^9copies / μg total RNA. The specific primers were designed according to the c DNA 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 503 aa. 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 245 aa.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.

关 键 词：家蚕 淀粉酶基因 组织表达 实时定量PCR 

分 类 号：Q78[生物学—分子生物学]