【摘要】 目的:本研究利用酵母三杂交系统从人脑海马回cDNA文库中筛选FXR1P的靶RNA,以进一步阐明FXR1基因的功能。方法:将表达FXR1P全长的质粒pYESTrp3/FXR1转化酵母菌株L40ura3/pHybLex/Zeo-MS2,检测毒性和自激活性;应用酵母三杂交技术从人脑海马回pRH3′-cDNA文库中筛选FXR1P的相互作用RNA;分离初步筛选的结果,再次转化含有诱饵质粒的融合菌株L40ura3/pHybLex/Zeo-MS2/pYESTrp3/FXR1,重新验证阳性结果;最后对阳性结果的外源插入片段进行测序和生物信息学分析。结果:酵母三杂交的筛选得到了3个阳性结果,经过测序和同源性分析,其中一个阳性结果中的插入片段为K-ALPHA-1 mRNA的部分序列。结论:K-ALPHA-1 mRNA可能是一种新的FXR1P的靶RNA。更多还原

【Abstract】 Objectiye:FXR1(Fragile X related gene 1)is a housekeeping gene that plays an important role in the development process of nervous system and muscular system.FXR1P,the translational product of FXR1,is a kind of RNA- binding protein,but the knowledge about FXR1P’s targeting-RNA is known little.Therefore,the aim of this study is to use yeast three hybrid system to screen FXR1P’s target RNA from pRH3’-cDNA library in gyrus hippocampi of human brain to further darify the function of FXR1 gene.Methods:The FXR1P whole long coding vector pYESTrp3/FXR1 was introduced into host strain L40 ura3/pHybLex/Zeo-MS2.Then the toxicity and self-activation of pYESTrp3/FXR1 were tested.The human brain pRH3’-cDNA library was screened by yeast transformation.The plasmids were extracted from the obtained positive yeast dones and reintroduced into the L40 ura3/pHybLex/Zeo-MS2/pYESTrp3/FXR1 one by one so as to validate the RNA-protein interactions.The inserted fragments of identified positive clones were sequenced and analyzed with BLAST in NCBI.Results: Three positive results were obtained through yeast three hybrid screening,in one of which,the inserted fragments was the partial sequence of K-ALPHA-1 mRNA by sequencing and BLAST analysis.Conclusions:K-ALPHA-1 mRNA is likely to be a new target RNA of FXR1P,which maybe plays a role in the genesis of Fragile X syndrome.更多还原