摘要
目的在我们的前期研究工作中,通过Tol2转座子介导的插入突变,筛选到了一批组织特异性表达绿色荧光蛋白GFP的斑马鱼品系。其中一个品系Tol2:20141221t的GFP在神经系统中表达,但还没有鉴定到Tol2转座子插入到基因组什么位置,造成了哪个基因的突变。本文的主要研究目标就是对这一由Tol2转座子插入诱导的斑马鱼突变品系进行鉴定。方法使用交错式热不对称PCR(thermal asymmetric interlaced PCR,TAIL-PCR)鉴定Tol2转座子插入的基因组位置;利用原位杂交检测被突变的基因的时空表达是否与该品系GFP表达具有一致性;筛选鉴定纯合体突变体,并进一步分析该基因突变造成的发育缺陷。结果在该品系中,Tol2转座子插入到了亚精胺/精胺N1-乙酰基转移酶1a(spermidine/spermine N1-acetyltransferase 1a,sat1.a)的第八个内含子区域,致使sat1.a基因转录提前终止。我们筛选到了sat1.a纯合突变体,但是没有检测到明显的发育缺陷。结论筛选鉴定的Tol2转座子介导的斑马鱼sat1.a突变体没有明显的发育缺陷,但可以作为研究神经系统发育的有力工具。
Objective In our previous study,we had generated various zebrafish mutant lines with tissue-specific GFP expression by Tol2 transposon-mediated insertional mutagenesis. Among these mutants,the Tol2： 20141221 t line expresses GFP in nervous system,while the position within zebrafish genome where transposon inserted has not yet been identified. The aim of this study was to identify and analyze this genetically modified mutant. Methods The transgenic insertion loci in the genome of Tol2： 20141221 t line was identified by TAIL-PCR and the spatial and temporal expression profile of the affected gene was examined by in situ hybridization. Homozygous mutant of Tol2： 20141221 t was generated for exploring related developmental defects. Results Tol2 transposon was inserted into the 8th intron region of sat1.a gene,and induced premature transcription termination. The maternal and zygotic mutants of Tol2： 20141221 t was generated,while without apparent developmental defects. Conclusions We have generated and identified the zebrafishsat1.a mutant mediated by Tol2 transposon. This gene insertion mutant exhibits no obvious developmental abnormalities,but may serve as a powerful tool to study the development of nervous system.
作者
万传璐
闫一芳
王鹏
林宇
曹羽
王强
WAN Chuan-lu YAN Yi-fang WANG Peng LIN Yu CAO Yu WANG Qiang(School of Life Science, Anhui University, Hefei, 230601 ,China State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101)
出处
《中国实验动物学报》
CAS
CSCD
北大核心
2016年第6期551-557,共7页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金面上项目(No.31271532)资助
