本文關(guān)鍵詞：小鼠GV期卵母細胞核質(zhì)比、ATP8表達的研究及其RNAi載體構(gòu)建　出處：《山西醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小鼠 生發(fā)泡 卵母細胞 ATP合酶亞基8 RNA干擾

【摘要】： 目的:1、分析小鼠卵巢生發(fā)泡(germinal vesicle,GV)期卵母細胞的核質(zhì)比,探討核質(zhì)比在卵母細胞成熟發(fā)育中的作用;2、分析GV期卵母細胞線粒體ATP合酶亞基8(ATP8)的表達及其生物信息學(xué)數(shù)據(jù),研究卵母細胞成熟與線粒體功能的關(guān)系;3、構(gòu)建ATP8基因的RNA干擾載體,為研究ATP8基因在卵母細胞成熟發(fā)育過程中的作用及其對卵母細胞發(fā)育參數(shù)—核質(zhì)比的調(diào)控奠定基礎(chǔ)。 方法:1、用擠壓法從卵巢中分離獲得GV期卵母細胞并置leica Mps 60倒置顯微鏡下拍照,測量系統(tǒng)經(jīng)標準圖片校正后,測量GV期卵母細胞面積和半徑,按核質(zhì)比(nucleus-plasma ratio,NP)公式:NP=V_N/(V_c-V_N),式中V_N為核的體積,V_c為細胞的體積,由V=4/3pr~3分別求得,計算出GV期卵母細胞的核質(zhì)比;2、以Genbank(NC_005089)公布的線粒體ATP8序列為參考,設(shè)計兩對引物,用RT-PCR檢測GV期單個卵母細胞中ATP8基因的表達:其中,cDNA的合成分兩種方法進行:一是將GV期單個卵母細胞直接進行RT合成cDNA,二是先用DNA酶加EcoRⅠ酶祛除mtDNA和核DNA后再進行RT;回收產(chǎn)物構(gòu)建克隆質(zhì)粒并測序;3、用Blastn、VECTOR NIT 9.0、RNAdraw、Treeview等工具對測序結(jié)果進行生物信息學(xué)分析,在此基礎(chǔ)上用RNA聚合酶Ⅲ啟動子表達siRNA法構(gòu)建ATP8的RNA干擾載體。 結(jié)果1、分離獲得GV期卵母細胞51枚,測得卵徑為40.940±3.341μm,核徑為15.020±2.236μm,其核質(zhì)比為0.054±0.019;2、RT-PCR結(jié)果顯示ATP8基因在GV期卵母細胞中表達�？寺y序顯示所測昆明小鼠ATP8序列與Genbank序列完全一致;3、blastp序列比對和mRNA二級結(jié)構(gòu)圖均顯示12種動物間的ATP8氨基酸序列和ATP8核酸序列有較大差異,其中不同品種小鼠間也有差異;4、不同物種ATP8核苷酸序列構(gòu)建出的分子進化樹中,小鼠與棕熊進化距離較遠,與田鼠、家貓進化距離相對較近。5、設(shè)計ATP8基因干擾序列并且重組質(zhì)粒經(jīng)過限制性內(nèi)切酶和測序鑒定,表明成功構(gòu)建了pGPH1/GFP/Neo-mouse-shATP8 RNA干擾質(zhì)粒。 結(jié)論1、本文測得小鼠GV期卵母細胞的核質(zhì)比為0.054±0.019,與文獻報道其它物種同時相核質(zhì)比在相同范圍內(nèi),提示核質(zhì)比是卵母細胞發(fā)育成熟的重要指標;2、線粒體ATP8基因在GV期卵母細胞中有表達,提示GV期卵母細胞發(fā)育成熟需要線粒體的參與;3、氨基酸序列比對和mRNA二級結(jié)構(gòu)分析均顯示,ATP8序列在多種哺乳動物間、不同品種小鼠間有較大差異;4、成功設(shè)計并構(gòu)建了小鼠線粒體ATP8基因RNA干擾載體,為進一步研究ATP8在小鼠卵母細胞發(fā)育過程中的作用奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R321-33

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本文編號：1399181