›› 2006, Vol. 49 ›› Issue (5): 726-732.

• 研究论文 • 上一篇    下一篇

一个新的家蚕转录相关锌带蛋白基因的克隆及序列分析

冯姗,张耀洲*   

  1. (浙江大学生物化学研究所,杭州310029)
  • 出版日期:2006-11-07 发布日期:2006-10-20
  • 通讯作者: 张耀洲

Cloning and sequence analysis of a novel transcription-associated zinc ribbon protein gene from silkworm, Bombyx mori

FENG Shan, ZHANG Yao-Zhou   

  1. Institute of Biochemistry, Zhejiang SciTec University, Hangzhou 310018, China
  • Online:2006-11-07 Published:2006-10-20
  • Contact: ZHANG Yao-Zhou

摘要:

锌带蛋白(zinc ribbon protein )是锌指类蛋白的一种,它的Cys4 Zn(2+)结合位点由3个β2片层折叠而成,而不是α螺旋结构。锌带结构与锌指结构同为转录因子结合核酸的结构域,锌带蛋白作为转录相关因子在调节基因表达活性等方面具有重要作用。在对家蚕 Bombyx mori蛹cDNA文库测序中,发现一个新的编码家蚕锌带蛋白基因的EST序列(GenBank 登录号DY230964),以此序列为信息探针检索家蚕EST数据库,通过同源筛选,获得一个新的家蚕锌带蛋白基因cDNA全序列并经RT-PCR检测和克隆、测序验证,结果表明与电子克隆序列完全一致。我们将其命名为 BmZNRD1 (Zinc Ribbon Domain Containing 1)(GenBank登录号DQ432055)。该基因全长为675 bp,由363 bp的开放阅读框序列(ORF)、10 bp的5′端非翻译区序列(5′UTR)和302 bp 的3′端非编码区序列(3′ UTR)组成,其编码的120个氨基酸序列与其他真核生物间具有较高的同源性(达60%左右),预测分子量为13.54 kD, 等电点为6.8。BmZNRD1编码的氨基酸序列是一种锌带蛋白,推测有2个功能结构域,分别是位于N-端的Cx2Cx14Cx2C和C-端的Cx2Cx24Cx2C,其中C-端保守氨基酸序列Cx2Cx6Yx3QxRSADEx2TxFx2Cx2C在生物进化中保守性很高,从酵母、果蝇、线虫到两栖类、哺乳类都有发现该结构域的存在,与酵母RNA聚合酶A亚单位9和转录相关蛋白有很高的相似性,推测其具有相同的功能。将BmZNRD1基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有3个外显子,2个内含子,外显子/内含子边界符合经典的GT-AG规则。
关键词:  家蚕; 锌带蛋白基因; 电子克隆; 基因克隆; 序列分析

关键词: 家蚕, 锌带蛋白基因, 电子克隆, 基因克隆, 序列分析

Abstract:

The zinc ribbons are one group of diverse zinc finger proteins. Most zinc-ribbon domain is actually folded as three-stranded antiparallel β-sheets in their structure instead of finger-like helices. The zinc ribbon is a domain that functions as interaction modules binding to nucleic acids. The zinc ribbon proteins perform a broad range of functions such as replication and repair, transcription and translation as a transcriptional repressor.Through searching cDNA bank of silkworm pupae, a unique EST (GenBank accession no. DY230964) encoding the zinc ribbon protein was identified. In order to obtain full-length cDNA in Bombyx mori, we used this sequence as a probe and performed in silico cloning based on the B. mori EST database. A contig was thus assembled on the basis of several ESTs. It was also verified by RT-PCR. PCR product was purified and ligated intopGEM-T-easy vector. The recombinant clones were sequenced, which had the same sequence as the contig. We nominated this novel gene encoding zinc ribbon domain containing protein as BmZNRD1 (GenBank accession no. DQ432055). The full-length of the gene is 675 bp. It contains an ORF of 363 bp, 10 bp nucleotide sequence in 5′ UTR (untranslated region) and 302 bp nucleotide sequence in 3′ UTR (untranslated region). It encodes 120 amino acids and is highly homologous to those of eukaryotic organisms previously reported (about 60%), with their predicted mass 13.54 kD and isoelectric point 6.8. The deduced amino acid sequence is one of zinc ribbon proteins with two putative functional domains, the N-terminal domain Cx2Cx14Cx2C and the C-terminal domain Cx2Cx24Cx2C. The highly conserved amino acid sequence within the C-terminal domain is Cx2Cx6Yx3QxRSADEx2TxFx2Cx2C, which is well conserved through evolution,in organisms such as yeasts, Drosophila, nematodes, amphibians and mammals, and highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins. An alignment of the cDNA sequence with the silkworm genome sequences revealed that there were 3 exons and 2 introns in the gene of  BmZNRD1, and all of them conformed the canonical GT-AG rules.

Key words: Bombyx mori, zinc ribbon protein gene, in silico cloning, gene cloning, sequence analysis