DNA损伤结合蛋白1（Es-DDB1）在中华绒螯蟹精子发生过程中的功能研究

发布时间：2018-06-30 11:07

本文选题：中华绒螯蟹 + DDB1　；参考：《华东师范大学》2017年硕士论文

【摘要】：中华绒螯蟹(Eriocheir Sinensis)又称河蟹,隶属节肢动物门、甲壳纲、十足目,因具有较高的营养和经济价值而成为我国水产养殖主要品种之一。DNA损伤结合蛋白(DDB1)是一个多功能的蛋白,参与核酸剪切修复通路NER,通过识别紫外引起的DNA损伤从而激活核酸剪切修复进程,并且在泛素化过程中作为Culin4A的连接蛋白形成E3泛素连接酶调控细胞周期、基因转录、信号传导以及损伤修复等很多重要过程。近年来有研究发现DDB1参与胚胎发育和精子发生过程,然而甲壳类动物中DDB1的研究还较少。为了探究中华绒螯蟹DDB1是否参与调控生殖过程,本研究从中华绒螯蟹中克隆获得DDB1基因(Es-DDB1)cDNA全长,序列分析显示该基因由4871个核酸组成,ORF区为3414bp,编码1137个氨基酸,蛋白分子量为125KDa。生物信息学分析发现Es-DDB1包含一个MMS1_N和一个CPSF_A功能域,晶体结构包含三个BP结构域(BPA,BPB,BPC)和一个CTD结构域,通过对比发现Es-DDB1在进化中的功能域和结构都是高度保守的。实时定量PCR和蛋白免疫印迹实验发现Es-DDB1在中华绒螯蟹各组织均有表达,且无论是mRNA还是蛋白丰度都在精巢中较高,暗示Es-DDB1参与该蟹精巢发育过程。检测精子发生过程中Es-DDB1的表达量发现,Es-DDB1在精子发生的前期表达量最高,并随着发育的进行逐渐降低,暗示Es-DDB1可能参与调控了早期精子发生过程。免疫共沉淀实验发现Es-DDB1和Es-cu14在中华绒螯蟹中是以复合体的形式共同作用的。通过免疫荧光实验发现Es-DDB1主要分布在精原细胞期和精母细胞期的细胞质中,显示出非常强烈的阳性信号,这与实时定量PCR和蛋白免疫印迹实验结果一致,但随着发育的进行,Es-DDB1的阳性信号逐渐减弱直至精细胞期变得非常微弱,且发生了位置的转移,开始从细胞质中转移到细胞核中。以上实验结果表明中华绒螯蟹Es-DDB1参与调控生殖过程,与Es-Cul4结合成复合体的形式参与包括精子发生在内的重要的细胞过程。
[Abstract]:Eriocheir sinensis, also known as Eriocheir sinensis, belongs to Arthropoda, crustacea, Decapoda. Because of its high nutritional and economic value, Eriocheir sinensis has become one of the main aquaculture species in China. DNA damage binding protein (DDB1) is a multifunctional protein. Participate in the nucleic acid cleavage repair pathway ner, by recognizing the ultraviolet induced DNA damage, activate the nucleic acid shearing repair process, and in the course of Ubiquidization, form the E3 ubiquitin ligase to regulate cell cycle and gene transcription. Signal transduction and damage repair are many important processes. In recent years, it has been found that DDB1 is involved in embryonic development and spermatogenesis. However, there are few studies on DDB1 in crustaceans. In order to investigate whether DDB1 participates in the regulation of reproduction, the full-length cDNA of DDB1 gene (Es-DDB1) was cloned from Eriocheir sinensis. Sequence analysis showed that DDB1 gene was composed of 4871 nucleic acids in the ORF region of 3414 BP, encoding 1137 amino acids and the molecular weight of protein was 125 KDa. Bioinformatics analysis showed that Es-DDB1 contained a MMS1N and a CPSFA functional domain, and the crystal structure consisted of three BP domains (BPA-BPBPP-BPC) and a CTD domain. The functional domain and structure of Es-DDB1 were highly conserved in evolution. Real-time quantitative PCR and Western blot analysis showed that Es-DDB1 was expressed in all tissues of Eriocheir sinensis, and both mRNA and protein abundance were higher in spermary, suggesting that Es-DDB1 was involved in the development of spermary of Eriocheir sinensis. Detection of Es-DDB1 expression during spermatogenesis showed that Es-DDB1 expression was the highest in the early stage of spermatogenesis and decreased with the development of spermatogenesis, suggesting that Es-DDB1 may be involved in the regulation of early spermatogenesis. Immunoprecipitation assay showed that Es-DDB1 and Es-cu14 acted in the form of complex in Eriocheir sinensis (Eriocheir sinensis). Immunofluorescence assay showed that Es-DDB1 was mainly distributed in the cytoplasm of spermatogonia and spermatocytes, and showed very strong positive signals, which was consistent with the results of real-time quantitative PCR and Western blot. However, the positive signal of Es-DDB1 decreased gradually with the development until the spermatocyte phase became very weak, and the location of the transfer occurred, and began to transfer from the cytoplasm to the nucleus. These results suggest that Es-DDB1 is involved in the regulation of reproductive process, and the combination of Es-Cul4 and Es-Cul4 is involved in important cellular processes, including spermatogenesis.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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