小鼠GV期卵母细胞核质比、ATP8表达的研究及其RNAi载体构建

发布时间：2018-01-09 00:02

本文关键词：小鼠GV期卵母细胞核质比、ATP8表达的研究及其RNAi载体构建　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:1、分析小鼠卵巢生发泡(germinal vesicle,GV)期卵母细胞的核质比,探讨核质比在卵母细胞成熟发育中的作用;2、分析GV期卵母细胞线粒体ATP合酶亚基8(ATP8)的表达及其生物信息学数据,研究卵母细胞成熟与线粒体功能的关系;3、构建ATP8基因的RNA干扰载体,为研究ATP8基因在卵母细胞成熟发育过程中的作用及其对卵母细胞发育参数—核质比的调控奠定基础。方法:1、用挤压法从卵巢中分离获得GV期卵母细胞并置leica Mps 60倒置显微镜下拍照,测量系统经标准图片校正后,测量GV期卵母细胞面积和半径,按核质比(nucleus-plasma ratio,NP)公式:NP=V_N/(V_c-V_N),式中V_N为核的体积,V_c为细胞的体积,由V=4/3pr~3分别求得,计算出GV期卵母细胞的核质比;2、以Genbank(NC_005089)公布的线粒体ATP8序列为参考,设计两对引物,用RT-PCR检测GV期单个卵母细胞中ATP8基因的表达:其中,cDNA的合成分两种方法进行:一是将GV期单个卵母细胞直接进行RT合成cDNA,二是先用DNA酶加EcoRⅠ酶祛除mtDNA和核DNA后再进行RT;回收产物构建克隆质粒并测序;3、用Blastn、VECTOR NIT 9.0、RNAdraw、Treeview等工具对测序结果进行生物信息学分析,在此基础上用RNA聚合酶Ⅲ启动子表达siRNA法构建ATP8的RNA干扰载体。结果1、分离获得GV期卵母细胞51枚,测得卵径为40.940±3.341μm,核径为15.020±2.236μm,其核质比为0.054±0.019;2、RT-PCR结果显示ATP8基因在GV期卵母细胞中表达。克隆测序显示所测昆明小鼠ATP8序列与Genbank序列完全一致;3、blastp序列比对和mRNA二级结构图均显示12种动物间的ATP8氨基酸序列和ATP8核酸序列有较大差异,其中不同品种小鼠间也有差异;4、不同物种ATP8核苷酸序列构建出的分子进化树中,小鼠与棕熊进化距离较远,与田鼠、家猫进化距离相对较近。5、设计ATP8基因干扰序列并且重组质粒经过限制性内切酶和测序鉴定,表明成功构建了pGPH1/GFP/Neo-mouse-shATP8 RNA干扰质粒。结论1、本文测得小鼠GV期卵母细胞的核质比为0.054±0.019,与文献报道其它物种同时相核质比在相同范围内,提示核质比是卵母细胞发育成熟的重要指标;2、线粒体ATP8基因在GV期卵母细胞中有表达,提示GV期卵母细胞发育成熟需要线粒体的参与;3、氨基酸序列比对和mRNA二级结构分析均显示,ATP8序列在多种哺乳动物间、不同品种小鼠间有较大差异;4、成功设计并构建了小鼠线粒体ATP8基因RNA干扰载体,为进一步研究ATP8在小鼠卵母细胞发育过程中的作用奠定了基础。
[Abstract]:Objective: 1. Analysis of mouse ovarian germinal vesicle (germinal vesicle, GV) oocytes of nuclear to cytoplasmic ratio, cytoplasmic ratio in oocyte maturation in the development; 2, analysis of phase GV oocyte mitochondrial ATP synthase subunit 8 (ATP8) expression and bioinformatics data, research on the relationship between oocyte maturation and mitochondrial function; 3, construction of RNA interference vector of ATP8 gene, and lay the foundation for the effect of ATP8 gene on oocyte maturation during the development of oocyte development parameters - nucleocytoplasmic ratio regulation.
Methods: 1 GV oocytes Leica Mps 60 inverted microscope with juxtaposed extrusion method isolated from the ovary under the camera, measuring system by standard image correction, area and radius measurement of GV stage oocytes by cytoplasmic ratio (nucleus-plasma ratio NP) formula: NP=V_N/ (V_c-V_N), nuclear volume in V_N, V_c cell volume, were obtained by V=4/3pr~3, calculated GV oocytes with Genbank 2, karyoplasmic ratio; (NC_005089) mitochondrial ATP8 sequences published for reference, two pairs of primers were designed for detection of GV expression of ATP8 RT-PCR during the period of single genes in oocytes which were: the synthesis of cDNA in two ways: one is the single stage GV oocytes directly RT synthesis of cDNA, two is the first with the DNA enzyme enzyme EcoR I get rid of mtDNA and nuclear DNA after RT; the product of RT-PCR was cloned and sequenced by Blastn; 3, NIT 9, VECTOR, RNAdraw, Treeview etc. work On the basis of the bioinformatics analysis of the sequencing results, the RNA interference vector of ATP8 was constructed by using the RNA polymerase III promoter to express the siRNA method.
The results of 1 isolated GV oocytes 51, measured egg diameter was 40.940 + 3.341 m, nuclear size is 15.020 + 2.236 m, the nuclear cytoplasmic ratio was 0.054 + 0.019; 2, RT-PCR showed that the expression of ATP8 gene in GV oocytes. Cloning and sequencing of display exactly the same Kunming mouse ATP8 sequence and Genbank sequence; 3, blastp sequences and mRNA two level structure showed 12 kinds of animal between the amino acid sequences of ATP8 and ATP8 nucleic acid sequences have great differences, which differ between different varieties of mice; 4, molecular phylogenetic tree of different species ATP8 nucleotide sequences constructed in mice and the brown bear the evolutionary distance, and voles, cat evolutionary distance is relatively close to.5, design ATP8 gene interference sequence and recombinant plasmid by restriction endonuclease and sequencing revealed the successful construction of pGPH1/GFP/Neo-mouse-shATP8 RNA plasmid.
Conclusion 1 to mouse GV oocytes measured the nucleo cytoplasmic ratio was 0.054 + 0.019, and reported in the literature of other species and nucleocytoplasmic ratio in the same range, that is an important indicator of development of the nuclear cytoplasmic ratio of mature oocytes; 2, mitochondrial ATP8 gene expression in GV oocytes, suggesting that GV oocytes maturation involve mitochondria; 3, analysis showed that the amino acid sequence of mRNA and two grade structure, ATP8 sequence in a variety of mammals, there are differences between different varieties of mice; 4, successfully designed and constructed gene RNA interference vector of mouse mitochondrial ATP8, lays a foundation for further research in ATP8 the process of mouse oocyte development in vitro.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321-33

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