摘要
为了确定少孢节丛孢菌内蒙古株丝氨酸蛋白酶Aoz1基因的潜在启动子区,试验采用透析膜培养的方法培养出少孢节丛孢菌内蒙古株并提取基因组DNA,与NCBI上Aoz1基因进行比对,选定Aoz1基因上游侧翼长度为2 000 bp的序列(命名为AP1)并据此设计引物,用PCR法扩增该序列并构建至平末端载体后测序,使用启动子在线预测软件Web Promoter Scan Service、NNPP以及CpG岛预测软件EMBOSS对AP1进行生物信息学分析。结果表明:所提取的基因组DNA完整性良好;成功构建出重组平末端质粒PEASY-AP1-Blunt,测序结果正确;通过相应软件预测出AP1含有的启动子基本元件,如TATA框、转录起始位点(TSS位点),以及参与Aoz1基因表达调控的转录因子结合位点Sp1、GHO、E2F、T-Ag和GCF等,同时确定了CpG岛的Obs/Exp值与GC含量的百分比分布。说明AP1序列具有启动子的基本结构和特点,初步确定了少孢节丛孢菌Aoz1基因的潜在启动子区。
The aim of the present study was to identify the potential promoter region of serine protease Aoz1 gene of Arthrobotrys oligospora in Inner Mongolia strains. The Inner Mongolia strain of Arthrospora oligosporium was cultured by dialysate membrane culture method and the genomic DNA was extracted. The sequence of 2 000 bp upstream flanking length of Aoz1 gene ( designated as AP1) was selected by comparison with Aoz1 gene on NCBI,and the primers were designed based on this sequence. The sequence was amplified by PCR and constructed into the flatterminal vector and sequenced. The promoter online prediction software Web Promoter Scan Service,NNPP and CpG island prediction software EMBOSS,were used for bioinformatics analysis of AP1. The results showed that the integrity of genomic DNA was good. The recombinant flatend plasmid PEASY-AP1-Blunt was successfully constructed and the sequencing results were correct. The basic promoter elements of AP1, such as TATA box,transcription initiation site ( TSS site) and transcription factor binding sites ( Sp1,GHO,E2F,T-Ag and GCF) involved in the regulation of Aoz1 gene expression were predicted by the corresponding software. At the same time,the percentage distribution of GC content and Obs /Exp value of CpG island was determined. The results indicated that the AP1 sequence had the basic structure and characteristics of the promoter,and the potential promoter region of the Aoz1 gene of Arthrospora oligosporium was preliminarily identified.
作者
王新芳
尹相吉
聂福贵
陈湘江
郭宏年
梁萌
罗小平
钱英红
郭少平
杨晓野
王瑞
WANG Xinfang;YIN Xiangji;NIE Fugui;CHEN Xiangjiang;GUO Hongnian;LIANG Meng;LUO Xiaoping;QIAN Yinghong;GUO Shaoping;YANG Xiaoye;WANG Rui(Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease,Ministry of Agriculture and Rural Affairs/ College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Zhucheng Animal Husbandry and Veterinary Administration Bureau of Shandong Province,Zhucheng 262200,China;Inner Mongolia of Agricuhural and Animal Husbandry Science,Hohhot 010031,China;Animal Husbandry arid Veterinary Working Station in Alashan League of Inner Mongolia,Alagxa 750306,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2018年第23期21-26,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家重点研发计划项目(2017YFD0501200)
国家自然科学基金项目(31460656)
内蒙古自然科学基金项目(2014MS0339
2015MS0308)
家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2017KFKT010
SKLVEB2015KFKT013)
