大黄鱼slitrk3基因启动子克隆及转录调控分析

Cloning and transcriptional regulation of slitrk3 gene promoter in large yellow croaker(Larimichthys crocea)

神经突触相关蛋白Slitrk3具有调节抑制性突触发育的作用。研究大黄鱼(Larimichthys crocea)神经突触黏附分子slitrk3基因的转录调控机制,可为解决大黄鱼养殖面临的生长、应激及抗逆等问题提供新思路。通过生物信息学方法对大黄鱼及其他物种的Slitrk3氨基酸进行多重序列比对和系统进化树分析,并预测大黄鱼slitrk3基因启动子区域相关的调控元件,采用双荧光素酶报告基因系统检测大黄鱼slitrk3基因启动子区域的转录活性。生物信息学分析结果显示,Slitrk3氨基酸序列在鱼类中具有较高的保守性;大黄鱼slitrk3基因启动子区域预测存在2个潜在的转录起始位点、2个CpG岛以及Sp1、GR、C/EBPα和C/EBPβ等多种转录因子结合位点。双荧光素酶报告基因系统结果显示,大黄鱼slitrk3基因启动子区域的−1970—−1614 bp、−1210—−667 bp存在正调控元件,−1614—−1210 bp、−667—−376 bp、−376—−147 bp存在负调控元件,推测−147—+16 bp为核心启动子。

Slitrk3,a neurosynaptic-related protein,can regulate the development of inhibitory synapses.To explore the tran-scriptional regulation mechanism of slitrk3 gene of the large yellow croaker(Larimichthys crocea)can provide a new idea for solving the problems of growth,stress and anti-stress in the L.crocea culture.We conducted a multiple sequence alignment and a phylogenetic tree analysis of amino acids for Slitrk3 in L.crocea and other species to investigate the transcriptional regulation mechanism of the neural cell adhesion molecule for slitrk3 gene.Besides,we predicted the potential core promoter regions,CpG islands and transcription factor binding sites of slitrk3 gene by bioinformatics methods,and detected the Luciferase activity of promoter of slitrk3 gene by the Dual-Luciferase Reporter System.Bioinformatics analysis shows that the amino acid sequences of Slitrk3 were highly conserved in fish.There were two transcription start sites,two CpG islands,and multiple transcription factor binding sites such as Sp1,GR,C/EBPαand C/EBPβin promoter of slitrk3 gene.Dual-Luciferase Reporter System shows that the regions from-1970 to-1614 bp and from-1210 to-667 bp contained positive regulatory elements;while the regions from-1614 to-1210 bp,from-667 to-376 bp and from-376 to-147 contained negative regulatory elements;and the regions from-147 to+16 bp might be core promoter of slitrk3 gene.The results lay a theoretical foundation for the further study of transcriptional regulation mechanism of slitrk3 gene in L.crocea.