【摘要】 F18大肠杆菌(Escherichia coli F18,E.coli F18)是养猪(Sus scrofa)业中发生最普遍、危害最大的病原菌之一,球系列鞘糖脂生物合成通路及通路中α-(1,2)岩藻糖转移酶1基因(alpha(1,2)fucose transferase 1,FUT1)对断奶仔猪F18抗性具有重要调控作用。本研究运用生物信息学技术挖掘课题组前期获得的断奶仔猪转录组测序结果,确定FUT1基因的转录起始位点和启动子区。同时对启动子区序列进行Cp G岛分析;采用双荧光素酶报告基因以及Ali Baba 2.0软件,分别分析启动子区活性和Cp G岛序列潜在的转录结合位点。通过比对人类(Homo sapiens)和猪的基因序列信息数据库,结果表明,FUT1基因转录起始区域具有5种可变剪接(AS-1,AS-2,AS-3,AS-4和AS-5)和2个启动子区域(启动子1和启动子2);双荧光素酶报告基因检测结果进一步显示,FUT1基因启动子2的转录活性极显著高于启动子1的转录活性(P<0.01),启动子2的活性是启动子1的2.75倍,根据结果可以推测启动子2在转录过程中起主导作用;Cp G岛分析显示,猪FUT1基因启动子1和启动子2分别存在一个Cp G岛。FUT1启动子1扩增片转录因子预测分析表明,FUT1基因启动子1存在20个潜在的转录因子结合位点,并且Sp1出现在多个转录结合位点处。本研究结果为猪FUT1基因的甲基化检测和调控机制分析提供一定的基础和依据。更多还原

【Abstract】 Escherichia coli F18(E. coli F18) is the major pathogen causing porcine edema disease(ED) and porcine post- weaning diarrhea(PWD),which leads to tremendous damage and loss to the pig(Sus scrofa)industry all around the world. Previous study pointed that glycosphingolipid biosynthesis- globo series pathway and alpha(1,2) fucose transferase 1(FUT1) as one of 7 key glycosyl transferase genes of pathway might play an important regulatory role to anti E. coli F18 in piglets. Therefore,given the importance of gene promoter in the regulation of gene transcription,the aim of this study was to identify the transcription initiation site and promoter of FUT1 gene by using bioinformatics to mine the RNA- seq results which obtained early. Besides,we analyzed the promoter Cp G islands of FUT1 through online software. Then,dual luciferase reporter gene technology and Ali Baba software were used for the analysis of promoter Cp G islands and prediction of putative transcription factor binding sites(TFBS),respectively. By comparing the gene sequences of human(Homo sapiens) and porcine information from Gen Bank database,we found that FUT1 transcription initiation region had 5 kinds of alternative splicing(AS- 1,AS- 2,AS- 3,AS- 4 and AS- 5) and 2promoter regions named as promoter 1 and promoter 2. Further research by dual luciferase reporter gene technology detected the transcriptional activity of the 2 promoters. The result showed that transcriptional activity of promoter 2 was extremely significantly higher than that of the promoter 1(P<0.01) and the activity of the promoter 2 was 2.75 times of the promoter 1,which illustrated that promoter 2 played leading role in the transcription process. Then,analyzing the Cp G island showed that FUT1 promoter 1 contained a Cp G island within 500 bp length and promoter 2 with 2 Cp G island. Using Alibaba version 2,we analyzed the identified Cp G island,and found 20 TFBSs in the FUT1 gene,and specificity protein 1(Sp1) located in many TFBS region,which might play important roles in the regulation of FUT1. All the results provide a deeper insight into the methylation detection and regulatory mechanism of FUT1 gene in piglets.更多还原