基于RIP技术对SRSF1蛋白相互作用的性发育相关mRNA的研究

发布时间：2018-02-21 14:56

本文关键词： RIP SRSF1蛋白 RNA测序性发育　出处：《东华大学》2017年硕士论文　论文类型：学位论文

【摘要】：在哺乳动物中,性发育启动受到下丘脑-垂体-性腺轴的调控,其分泌的各种激素直接或间接调控着性发育的启动。本实验室利用杂交F2代和同类系,进行全基因组的STR扫描和ISCSs精细定位,筛选出miR-505-3P作为可靠的候选基因。利用表达谱和Targetscan预测并验证得到SRSF1是miR-505-3p的靶基因。通过在过表达miR-505-3p的GT1-7中过表达SRSF1编码区,发现miR-505-3p所抑制的性发育相关基因的表达水平有不同程度的恢复,说明SRSF1可以促进性发育相关基因的表达。而SRSF1是如何通过下游基因来影响性发育亟待研究。本研究首先以Hela细胞为模型,构建甲醛交联RNA免疫共沉淀(RNA Immunoprecipitation,RIP)方案。第一步,针对细胞固定条件、裂解液强度及超声条件进行摸索,结果显示以0.1%甲醛固定细胞,选择1%NP-40的低强度裂解液,结合低超声条件处理固定细胞,成功捕获目的蛋白及其相互作用的RNA。第二步,针对RIP捕获的SRSF1蛋白相互作用的RNA样本,建立RNA富集效率检测方案。以酿酒酵母RNA的β-actin作为内参,采用qPCR技术精准定量检测目标RNA的富集效率。结果显示,0.1%甲醛交联RIP技术特异性捕获得到SRSF1蛋白-RNA复合物;qPCR技术检测阳性基因PABP、SRSF1在SRSF1抗体捕获的产物和IgG抗体的产物中相应RNA的浓度差异均在60倍以上。利用上述建立的RIP技术,在GT1-7细胞中特异性捕获SRSF1蛋白相互作用的RNA。同时结合western blot和考马斯染色的结果,确保适当的抗体量高效富集细胞内源性目的RNA,并将捕获的目的RNA进行测序。之后,首先将测序获得的原始数据用NGStookit软件进行质控处理,然后用tophat软件将过滤之后的reads比对到小鼠基因组上,再用cufflink软件将比对结果进行拼接及表达量的计算。根据实验组和对照组表达量的差异筛选出与SRSF1蛋白相互作用的基因,筛选标准是差异倍数大于2倍的基因作为候选基因。之后对筛选出的候选基因进行GO注释和KEGG注释,获得候选基因的功能信息,最终找出并验证与性发育相关的13个基因。这些工作为性发育的分子机制的研究奠定了基础,从而为性早熟,生殖障碍的预防、鉴别诊断和治疗提供分子靶点和理论依据,同时对人类正常生殖功能的维持及生殖障碍的预防将有更全面的了解。
[Abstract]:In mammals, the initiation of sexual development is regulated by the hypothalamus-pituitary-gonadal axis, and the hormones secreted by it directly or indirectly regulate the initiation of sexual development. STR scanning and ISCSs fine mapping of the whole genome were performed to screen miR-505-3P as a reliable candidate gene. Expression profiles and Targetscan were used to predict and verify that SRSF1 was the target gene of miR-505-3p. It was found that the expression level of sex-development-related genes inhibited by miR-505-3p recovered to varying degrees. It is suggested that SRSF1 can promote the expression of genes associated with sexual development. However, it is urgent to study how SRSF1 affects sexual development through downstream genes. The first step of this study was to construct a formaldehyde-crosslinked RNA immunoprecipitation scheme based on Hela cells. The results showed that the fixed cells were fixed with 0.1% formaldehyde, and the cells were treated with NP-40 low intensity lytic solution, combined with low ultrasound condition, according to the condition of cell immobilization, the intensity of lysate and the ultrasonic condition, the results showed that the cells were fixed with 0.1% formaldehyde. The second step of successfully capturing the target protein and its interaction, the RNA sample of SRSF1 protein interaction captured by RIP was used to establish a RNA enrichment efficiency detection scheme. 尾 -actin of Saccharomyces cerevisiae RNA was used as the internal control, and 尾 -actin from Saccharomyces cerevisiae was used as internal control. The enrichment efficiency of target RNA was accurately and quantitatively detected by qPCR technique. The results showed that 0.1% formaldehyde crosslinked RIP technique was specific to capture SRSF1 protein-RNA complex qPCR to detect the captured products of the positive gene PABPP-SRSF1 in SRSF1 antibody and the IgG antibody. The concentration difference of corresponding RNA in the product is more than 60 times. Using the established RIP technology, The specific SRSF1 protein interaction was captured in GT1-7 cells. Combined with the results of western blot and Cormas staining, it was ensured that appropriate antibody levels were highly efficient in enriching cellular endogenous RNAs, and the captured target RNA was sequenced. The original data obtained by sequencing was first processed with NGStookit software for quality control, and then filtered reads was compared to the mouse genome by tophat software. The results were spliced and expressed by cufflink software. According to the difference of expression between experimental group and control group, the genes interacting with SRSF1 protein were screened out. The screening criteria are that the genes whose difference ratio is more than 2 times are used as candidate genes. Then go annotation and KEGG annotation are carried out to obtain the functional information of the candidate genes. Finally, 13 genes related to sexual development were identified and verified. These work laid the foundation for the study of the molecular mechanism of sexual development, thus providing molecular targets and theoretical basis for precocious puberty, prevention of reproductive disorders, differential diagnosis and treatment. At the same time, the maintenance of human normal reproductive function and the prevention of reproductive disorders will have a more comprehensive understanding.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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