家蚕滞育生物钟蛋白质EA4基因的cDNA克隆和序列分析

Cloning and Sequence Analysis of cDNA of Diapause Biological Clock Protein Esterase A4 of Bombyx mori

家蚕滞育生物钟是依赖卵内酯酶A4(EA4)的倒计时型生物钟。EA4是具时间间隔测定酶(time interval measuring enzyme,TIME)功能的蛋白质。根据家蚕EA4测定的氨基酸序列,以第3天龄雌蛹脂肪体和卵巢总RNA为模板,利用简并引物RT-PCR方法,得到308 bp的部分ea4序列,其1-144 nt和145-308 nt分别与家蚕全基因组鸟枪序列(whole genome shotgun sequence,WGS)AADK01019126.1的5595-5738 nt及WGS AADK01014467.1的8636-8473 nt有95%和98%的一致性。根据获得的ea4基因片段序列,利用生物信息学方法和家蚕基因组数据预测了ea4基因的完整cDNA序列,设计特异引物,以雌蛹脂肪体和卵巢RNA为模板,进行RT-PCR得到完整的cDNA。ea4基因的cDNA全长519 bp,其中1-48 bp为信号肽序列,49-519 bp编码的氨基酸与蛋白质纯化后测序EA4部分的一致性为98%,C末端存在3个氨基酸的差异,其中第156位为新确定氨基酸。

The diapause biological clock protein of Bombyx silkworm is a count down clock which depends on esterase A4 （EA4） in their eggs. EA4 was the first confirmed time interval measuring enzyme （TIME）. A 308 bp of ea4 cDNA sequence was generated by RT-PCR with degenerate primers designed according to the determined amino acids and using the total RNA of fat body and ovary from 3-day old female pupa as the template. The consistence of 1 -144 nt and 145 -308 nt of the sequence with 5 595 -5 738 nt of whole genome shotgun sequence （WGS） （AADK01019126. 1 ） and 8 636 -8 473 nt of WGS （AADK01014467. 1 ） were 95% and 98%, respectively. According to the sequence of the ea4 gene fragment obtained, ea4 gene integrity cDNA sequence was forecasted by method of bioinformatics and the genome data of Bombyx mori. Then, a pair of special primers was designed according to the forecasted cDNA sequence of ea4, and the complete cDNA sequence was obtained by RT-PCR using the total RNA of fat body and ovary of female pupa as the template. The cDNA of ea4 gene was 519 bp in length, in which 1 -48 bp was the signal peptide sequence. And the amino acid sequence encoded by 49 -519 bp showed 98% similarity with that of EA4 protein, with 3 amino acids difference at the C-terminal, in which the 156th was the first defined one.