小鼠Prestin基因表达及启动子转录活性分析

发布时间：2019-02-28 17:42

【摘要】：本研究通过实时荧光定量PCR、基因克隆、双荧光素酶报告实验等方法,研究了prestin基因在各组织中的表达和差异,对该基因不同转录变异体对应的不同启动子转录活性进行了深入分析,并结合生物信息学的方法预测了相关顺式调控元件和调控因子,获得以下结果：(1)通过实时荧光定量对prestin基因在大脑、睾丸和耳蜗中总体表达量进行检测,发现prestin除了在耳蜗中特异性高表达外,在小鼠睾丸和大脑中也有表达。随后检测了prestin基因转录变异体1表达量,发现除了耳蜗外,大脑和睾丸中同样存在着不同的转录变异体表达,转录变异体1在耳蜗、大脑和睾丸组织中分别占总表达量的58.08%、43.77%、62.91%。(2)通过分析prestin基因已知的3种：mRNA序列,针对不同转录变异体设计了特异性片段扩增,定性的研究该基因不同转录变异体的表达,发现耳蜗、大脑、睾丸、GC-1spg细胞中prestin基因均有转录本1的表达,在耳蜗中也扩增出转录变异体2或3。结合5'RACE实验扩增出睾丸中prestin基因mRNA的5’端序列,发现prestin基因在睾丸中表达转录变异体2或3,但没有发现其他未知的转录变异体。(3)通过克隆prestin基因5’端DNA序列,构建了启动子1和启动子2缺失片段载体,运用双萤光素酶报告系统,检测启动子区转录活性变化规律,发现启动子1在GC-1spg小鼠精原细胞中转录活性较低,而启动子2的转录活性较高。第一外显子上游-280bp到-765bp以内存在转录激活的转录调控元件,-765bp到-1096bp内存在转录抑制的调控元件,第二外显子上游-952bp内存在转录激活的调控元件,-952bp到-1954bp内存在转录抑制的调控元件。(4)结合生物信息学分析预测了上述调控区潜在的大量转录因子结合位点,发现转录调控因子GATA-3,-2,-1潜在的结合位点,结合已有报道,推测其可能在小鼠精原细胞中调控prestin基因的表达。本研究分析了prestin基因在耳蜗、睾丸和大脑中的表达,检测了启动子各区域的转录活性,为下一步阐明该基因的特异性表达调控模式奠定了基础。
[Abstract]:In this study, real-time quantitative PCR, gene cloning and double-luciferase reporting assay were used to study the expression and difference of prestin gene in various tissues. The transcriptional activities of different promoters corresponding to different transcriptional variants of the gene were analyzed, and the related cis-regulatory elements and regulatory factors were predicted by bioinformatics. The following results were obtained: (1) the total expression of prestin gene in the brain, testis and cochlea was detected by real-time fluorescence quantitative analysis. It was found that prestin was expressed not only in the cochlea but also in the testis and brain of mice. Then we detected the expression of transcription variant 1 of prestin gene, and found that in addition to cochlea, there were different expression of transcription variant 1 in the brain and testis, and transcription variant 1 was in the cochlea. Brain and testicular tissue accounted for 58.08%, 43.77%, 62.91% of the total expression respectively. (2) by analyzing three known prestin genes: mRNA sequences, specific fragments were designed for different transcription variants. Qualitative study on the expression of prestin gene in cochlea, brain, testis and GC-1spg cells showed that transcript 1 was expressed in cochlea, brain, testis and transcription variant 2 or 3. The 5'- terminal sequence of prestin gene mRNA was amplified by 5'RACE assay. It was found that prestin gene expressed transcription variant 2 or 3 in testis, but no other unknown transcription variants were found. (3) the 5'- terminal DNA sequence of prestin gene was cloned. Promoter 1 and promoter 2 deletion vectors were constructed. The transcriptional activity of promoter 1 was detected by double luciferase reporting system. It was found that promoter 1 had low transcriptional activity in spermatogonia of GC-1spg mice. The transcriptional activity of promoter 2 was higher. There were transcriptional regulatory elements in-280bp to-765bp in the upstream of exon 1, regulatory elements in-765bp to-1096bp and regulatory elements in-952bp in the upstream of exon 2, and transcription-activated elements in-952bp in the upstream of exon 2, and transcription-activated elements in-765bp to-1096bp in the upstream of exon 1. There are regulatory elements of transcription inhibition in-952bp to-1954bp. (4) binding bioinformatics analysis predicted a large number of potential transcription factor binding sites in these regulatory regions, and found the potential binding sites of transcriptional regulatory factor GATA-3,-2,-1. It has been reported that it may regulate the expression of prestin gene in mouse spermatogonia. In this study, the expression of prestin gene in cochlea, testis and brain was analyzed, and the transcriptional activity of the promoter region was detected, which laid a foundation for further elucidating the specific expression and regulation pattern of the gene.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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