斑马鱼SDC4基因的克隆与遗传进化分析

Cloning and Genetic Evolution Analysis of Zebrafish SDC4 Gene

目的:克隆斑马鱼多配体聚糖(SDC)4基因并分析其遗传进化关系,为探究斑马鱼SDC4基因的功能奠定分子生物学基础。方法:将分子克隆得到的斑马鱼SDC4基因全长cDNA利用Gibson组装方法构建至pCS2+载体并对其进行序列分析;利用qRT-PCR检测受精后1 d和3 d的AB、Tübingen(TU)野生型斑马鱼SDC4 mRNA表达量和胚胎发育前10 d的TU野生型斑马鱼SDC4 mRNA的表达差异;根据NCBI和ensemble网络信息,对斑马鱼SDC4、人SDC4和小鼠SDC4基因在染色体上的基因连锁信息作出分析,并利用MegAlign软件对斑马鱼SDC4、人SDC4和小鼠SDC4蛋白进行氨基酸序列比对分析其同源性,再应用MEGAX软件对不同物种SDC4蛋白构建系统发生树。结果:测序结果显示克隆得到的斑马鱼SDC4基因与ensemble公布的序列一致;AB、TU野生型斑马鱼在受精后前3 d的SDC4 mRNA表达差异无统计学意义(P>0.05),TU野生型斑马鱼SDC4 mRNA表达在胚胎发育前期呈现一定程度的母系遗传控制;染色体连锁基因座位图发现斑马鱼SDC4与人SDC4、小鼠SDC4的连锁基因不完全相同,蛋白序列比对分析斑马鱼SDC4蛋白与人SDC4、小鼠SDC4蛋白同源性分别为38.3%、44.2%,根据不同物种的SDC4氨基酸序列构建的系统发生树反映了生物进化的关系。结论:成功克隆了斑马鱼SDC4基因并对其进行了遗传进化分析,补充了斑马鱼SDC4的分子生物学信息。

Objective:To clone the zebrafish syndecan(SDC)4 gene,analyze the evolutionary relationship,and lay a molecular biological basis for exploring the function of the zebrafish SDC4 gene.Methods:The full-length cDNA of SDC4 was constructed into a pCS2+vector by the Gibson assembly method;qRT-PCR was used to detect the SDC4 mRNA expression level in AB and TU wild-type zebrafish at 1 and 3 days post-fertilization,and in TU wild-type zebrafish during the first ten days of embryonic development;According to NCBI and ensemble online information,the gene linkage information of zebrafish SDC4,human SDC4 and murine SDC4 genes on chromosomes was analyzed.Zebrafish SDC4,human SDC4,and murine SDC4 proteins were compared to analyze their amino acid sequence homology by MegAlign software,Then MEGAX software was used to construct a phylogenetic tree of SDC4 protein molecules from different species.Results:The sequencing result showed that the cloned zebrafish SDC4 gene was consistent with the sequence published in the ensemble;There was no statistical difference in the expression level of SDC4 mRNA between AB and TU wild-type zebrafish of the first 3 days post fertilization(P>0.05).SDC4 mRNA expression of TU wild-type zebrafish exhibited a degree of maternal genetic control during early embryonic development.The chromosomally syntenic analysis found that the linkage genes of zebrafish SDC4 were not identical with human SDC4 and murine SDC4.The protein sequence alignment found that the zebrafish SDC4 protein shared 38.3%and 44.2%identity with human and murine SDC4 proteins,respectively.The phylogenetic tree constructed according to the SDC4 amino acid sequences of different species reflected the relationship of biological evolution.Conclusion:The zebrafish SDC4 gene was successfully cloned and bioinformatics analysis was carried out,which supplemented the molecular biological information of zebrafish SDC4.