WDR5对SOX9基因的初步表观调控

发布时间：2018-05-10 19:45

本文选题：WDR5 + 真核表达　；参考：《大理大学》2017年硕士论文

【摘要】：WD重复蛋白5(WD repeat-containing protein 5,WDR5)是WD-40蛋白家族的成员,由333个氨基酸组成,其肽链含有7个WD重复结构形成的β-折叠,是WDR5的主要功能结构域。作为WD40家族的成员,它广泛参与了生物体的基因表达调控,在混合谱系白血病的激活、肿瘤形成、VISA相关的信号复合体的装配及IRF3和NF-κB的激活等方面都发挥了巨大作用。但迄今为止,WDR5还有许多生物学作用机理尚待阐明。本课题第二部分利用基因重组技术构建人源WDR5全长结构基因真核表达载体,建立稳定表达WDR5的LNCaP细胞株。实验中通过PCR扩增WDR5基因后,将WDR5插入pCI-neo载体中,酶切及测序鉴定重组质粒。采用脂质体转染法将重组质粒转染LNCaP细胞,Real Time-PCR检测转染细胞WDR5的mRNA表达水平。经酶切及测序证实成功构建了真核表达载体pCI-neo-WDR5构建正确,重组质粒转染到LNCaP细胞后,经G418筛选获得稳定表达的细胞株,Real Time-PCR方法检测到WDR5基因在转录水平的表达,初步表明稳定表达WDR5的LNCaP细胞株的建立,为下一步WDR5的深入研究及应用奠定了基础。在哺乳动物个体发育过程中,DNA甲基化对性别决定和分化具有重要调节作用。近来在我们实验研究中证实了WDR5和SRY能共定位在SOX9的启动子上,并且通过两者协调作用上调SOX9的表达,同时抑制beta-Catenin的表达。因此,WDR5在性别决定中起着重要的调节作用。但是WDR5如何参与及具体调节SOX9基因表达机制却不清楚。故本课题的第三部分,我们应用与WDR5直接紧密相关的组蛋白标记H3K4me1/2/3抗体进行染色体免疫沉降(ChIP)实验,以验证WDR5分子对SOX9基因启动子的初步表观调节。
[Abstract]:WD repeat protein 5 (WD repeat-containing protein 5, WDR5) is a member of the WD-40 protein family and consists of 333 amino acids. Its peptide chain contains 7 WD duplicated beta folds. It is the main functional domain of WDR5. As a member of the WD40 family, it is widely involved in the regulation of gene expression in the biological body and the activation of the mixed lineage leukemia. The formation of tumor, the assembly of VISA related signal complex and the activation of IRF3 and NF- kappa B have played a great role. But so far, many biological mechanisms of WDR5 have yet to be elucidated. The second part of this subject uses gene recombination technology to construct eukaryotic expression vector of human WDR5 fully long structure gene and establish a stable expression of WDR5. In the experiment, the WDR5 gene was amplified by PCR, and WDR5 was inserted into the pCI-neo vector, and the recombinant plasmid was identified by enzyme digestion and sequencing. The recombinant plasmid was transfected into LNCaP cells by liposome transfection, and Real Time-PCR was used to detect the mRNA expression level of WDR5 in the transfected cells. The eukaryotic expression carrier was successfully constructed by enzyme digestion and sequencing. After the construction was correct, the recombinant plasmid was transfected into LNCaP cells and screened by G418 to obtain the stable expression of the cell line. The Real Time-PCR method was used to detect the expression of WDR5 gene at the transcriptional level. It preliminarily indicated the establishment of the LNCaP cell line that stably expressed WDR5, which laid the foundation for the further study and application of the next step of WDR5. DNA methylation plays an important regulatory role in gender determination and differentiation. In recent experimental studies, we have demonstrated that WDR5 and SRY can be Co located on the promoter of SOX9, and the expression of SOX9 is up-regulated through the coordination of both, and the expression of beta-Catenin is inhibited. Therefore, WDR5 plays an important role in the regulation of sex determination. It is not clear how WDR5 participates in and specifically regulates the mechanism of SOX9 gene expression. Therefore, in the third part of this topic, we use the histone labeled H3K4me1/2/3 antibody, which is closely related to WDR5, to carry out the chromosome immunization (ChIP) experiment to verify the preliminary apparent regulation of the WDR5 molecule on the SOX9 gene promoter.

【学位授予单位】：大理大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416

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