摘要
目的:从福寿螺胃组织细胞中克隆多功能纤维素酶基因(mfc)。方法:设计引物,采用RT-PCR方法从福寿螺胃组织细胞中扩增多功能纤维素酶基因,与pGEMT-easy载体连接,转化大肠杆菌DH5α。结果:该基因全长为1225bp,上游5′端非翻译区19bp,3′端非编码区21bp,开放阅读框(Open reading frame,ORF)1185bp,编码395个氨基酸,推测等电点pI为6.57,分子质量45.8kDa。推测的多功能纤维素酶具有糖苷水解酶保守的氨基酸序列,属于糖苷水解酶第10家族。同源性比对结果显示,该基因及推测的氨基酸序列与egx(Gen-Bank Accession No.AAP31839)的同源性分别为99.3%和99.8%,在8个位点出现核苷酸差异,在1个位点出现氨基酸差异。通过软件分析并预测,该基因编码的蛋白共存在14个丝氨酸、苏氨酸和酪氨酸磷酸化位点,没有信号肽序列。结论:成功克隆出福寿螺多功能纤维素酶基因。
Objective： Multi-functional eellulase gene （mfc）was cloned from stomach tissue of Ampullaria crossean. Methods： Designing primers, RT-PCR was employed to amplify a multi-functional cellulase gene （mfc） from the stomach tissue of Ampullaria crossean. Mfc was linked with pGEM T-easy, transferred to E.coli DH5 α. Results： DNA sequencing showed that the amplified mfc had 1 225 bp including 19 bp of 5′ end of upstream un-translated region, 21 bp of 3′ end of non-coding region, and 1 185 bp of an open reading frame. Sequence analysis indicated that the amplified rhfc coded 395 amino acid residues with predicted 45.8 kDa of molecular mass and 6.57 of pI （isoelectric point）. Blastn and Blastx alignment results demonstrated that the nucleotide sequence and its deduced amino acid sequence of the amplified mfc possessed 99.3% and 99.8% of similarities to those of egx （GenBank Accession number： AAP31839）. There were 8 sites of differences in nucleotide and 1 site of difference in amino acid sequence between the amplified mfc and egx. Bioinformatics analysis revealed that there were 14 phosphorylation sites of serine, threonine, tyrosine residues and no signal peptide sequence was found in the amplified mfc sequence. The deduced amino acid sequence （MFC） from the amplified mfc contained the conserved regions of glycoside hydrolase and should belong to number 10 family of glycoside hydrolase families. Conclusion： Multi-functional cellulase gene for A. crossean was cloned successfully.
出处
《中国食品学报》
EI
CAS
CSCD
2008年第4期21-27,共7页
Journal of Chinese Institute Of Food Science and Technology
基金
国家高技术研究发展计划(863计划)(2006AA10Z301)资助项目
关键词
福寿螺
多功能纤维素酶基因
克隆
序列分析
Ampullaria crossean Multi-functional eellulase gene（mfc） Cloning Sequence analysis
