斑马鱼rsph3基因在早期发育中的研究及斑马鱼prdx突变体的制备

发布时间：2018-02-01 17:26

本文关键词： 基因敲除技术 rsph3基因左右不对称性 prdx基因氧胁迫　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：基因敲除技术是反向遗传学研究的高效手段之一。CRISPR/Cas9和TALENs已经被证实为高效的敲除工具。斑马鱼是一种可用于分析功能基因组学、研究人类疾病发病机制的优良动物模型。纤毛病是细胞上纤毛结构发生功能障碍所导致的疾病,是一种遗传异质性的疾病。临床表现为新生儿呼吸窘迫症、慢性呼吸道感染、支气管扩张、多囊肾、耳聋、内脏倒位以及肥胖、糖尿病等。在纤毛病患者中发现径向轮辐蛋白3基因(radial spoke 3 homolog,rsph3)突变类型,其纤毛9+2结构受到破坏。RSPH3是组成径向轮辐复合体的二十多种蛋白之一,对纤毛轴丝的滑动是必须的。本实验利用TALENs技术敲除斑马鱼rsph3基因,得到-8、-10、-15 bp三种F1代杂合突变体和rsph3--(-8,-15 bp)纯合突变体,这些突变均产生截短的蛋白质。原位杂交结果显示rsph3是母源表达的基因,并在斑马鱼早期胚胎发育中广泛表达。rsph3纯合突变体互交,约48%的受精卵不卵裂。在存活的突变体中,通过原位杂交检测发现其肝脏右移、心脏右移、心管不弯曲,显示身体左右不对称轴被破坏。显微注射rsph3 mRNA可以拯救突变体肝脏右移的表型。突变体中kupffer'svesicle(KV)的标记基因Dand5的表达模式和野生型相同,在6体节期都呈现不完全包围的C形,显示纤毛结构的破坏未影响到KV的形态。由此推测左右对称轴的破坏可能不是由于KV的结构异常,而是其中纤毛结构和功能的异常导致的。斑马鱼rsph3-/-突变体可以作为了解人类纤毛病的一种动物模型。过氧化物氧还蛋白(Peroxiredoxins,prdx)是细胞在清除活性氧过程中起重要作用的酶。利用CRISPR/Cas9技术对斑马鱼prdx家族进行敲除,获得了prdx1,prdx5,prdx6三个亚型的突变体。其中prdx1、prdx6获得F0代突变体;prdx5得到+2、-2、-5、-8 bp四种类型F1代突变杂合体和-5 bp类型的F2代突变纯合体,这些突变均产生截短的蛋白质。原位杂交的结果显示斑马鱼prdx1、prdx5、prdx6是母源表达的基因,并且在早期胚胎中广泛表达。RT-PCR的结果显示prdx1、prdx5、prdx6在成体的卵巢和精巢中有很高的表达量。其中prdx5在精巢中表达量最高。用15 mmol/L的H202处理,发现斑马鱼prdx5+/-胚胎耐受氧胁迫能力降低。
[Abstract]:Gene knockout is one of the most efficient methods for reverse genetic research. CRISPRR / Cas9 and TALENs have been proved to be efficient knockout tools. Zebrafish is a useful tool for analyzing functional genomes. Learn. The fine animal model to study the pathogenesis of human disease. Fibrinopathy is a disease caused by ciliated structure dysfunction on the cell, it is a disease of genetic heterogeneity. The clinical manifestation is neonatal respiratory distress. Chronic respiratory infections bronchiectasis polycystic kidney deafness visceral inversion and obesity. Diabetes mellitus and so on. The radial spoke 3 homologue rsph3) mutation type was found in patients with ciliatosis. Its ciliated 9.2 structure was destroyed. RSPH3 is one of the more than 20 proteins that make up the radial spoke complex. The TALENs technique was used to knockout the rsph3 gene of zebrafish. The heterozygous mutants of F _ 1 generation and rsph _ 3 ~ (-8) -15 BP) were homozygous. All these mutations produced truncated proteins. In situ hybridization results showed that rsph3 was a maternal expression gene and was widely expressed in zebrafish early embryonic development, and the. Rsph3 homozygous mutants intercrossed. About 48% of the fertilized eggs were not cleavage. In the surviving mutants, the liver shifted to the right, the heart shifted to the right and the cardiac tube did not bend by in situ hybridization. It shows that the dissymmetry axis of the body is broken. Microinjection of rsph3 mRNA can save the phenotype of the right shift of the mutant liver. The expression pattern of Dand5 was the same as that of wild type. At the 6 body node, the shape of C is not completely surrounded, which shows that the destruction of ciliated structure does not affect the shape of KV. It is inferred that the destruction of the left and right symmetry axis may not be due to the abnormal structure of KV. It is the abnormal structure and function of ciliates. The rsph3-r-rsph-mutants of zebrafish can be used as an animal model to understand human fiber problems. Peroxiredoxins. PRDX) is an enzyme that plays an important role in the scavenging of reactive oxygen species (Ros). The prdx family of zebrafish was knocked out by CRISPR/Cas9 technique and prdx1 / prdx5 was obtained. Among the three subtypes of prdx6, PRDX1 / prdx6 obtained F 0 generation mutants. The heterozygotes of F _ 1 generation and F _ 2 generation of -5 BP type were obtained by prdx5. All these mutations produced truncated proteins. In situ hybridization showed that the zebrafish prdx1, prdx5, prdx6 were maternal expressed genes. The results of RT-PCR showed that prdx1 and prdx5 were widely expressed in early embryos. The expression of prdx6 was high in adult ovary and testis. The highest expression of prdx5 was found in the testis and treated with H202 of 15 mmol/L. It was found that the ability of zebrafish embryos to withstand oxygen stress was decreased.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596;Q78

【相似文献】