發(fā)布時(shí)間：2018-07-20 14:15

【摘要】：母性效應(yīng)基因(maternal effect gene,MEG)通常被認(rèn)定是只在卵巢組織及卵母細(xì)胞中特異表達(dá),且目前已被證明在母型-合子型過(guò)渡(maternal zygotic transition,MZT),即母型調(diào)控向合子型調(diào)控的轉(zhuǎn)變過(guò)程中,以及哺乳動(dòng)物胚胎發(fā)育的初期和卵泡的發(fā)生、發(fā)育過(guò)程中發(fā)揮著重要的功能。合子阻滯因子1(zygote arrest 1,ZAR1)和生長(zhǎng)分化因子9(growth differentiation factor 9,GDF9)作為少數(shù)較早被挖掘到的母性效應(yīng)基因之一,在小鼠、大鼠、蛙、斑馬魚、牛、綿羊、豬等物種已被報(bào)道。ZAR1不但在卵母細(xì)胞的生長(zhǎng)和胚胎發(fā)育的初期過(guò)程中起著舉足輕重的作用,且被證明該基因某些位點(diǎn)的核苷酸多態(tài)性同其產(chǎn)仔數(shù)等繁殖性狀顯著相關(guān)。而GDF9基因作為TGF-β超家族的一員,已被證明是哺乳動(dòng)物卵巢的Qg源細(xì)胞因子,在卵泡的發(fā)生和卵母細(xì)胞的成熟過(guò)程中發(fā)揮著重要的調(diào)節(jié)作用,又因?yàn)槠湓谏尺^(guò)程中的重要作用,己成為動(dòng)物繁殖生物學(xué)研究的熱點(diǎn)。而目前國(guó)內(nèi)外關(guān)于這兩個(gè)基因在兔方面的研究仍然很少,對(duì)其作用機(jī)制的認(rèn)識(shí)還不深入。本研究基于實(shí)驗(yàn)室以前的工作,在新西蘭白兔克隆了ZAR1和GDF9基因,并進(jìn)行了相關(guān)生物信息學(xué)分析。為了檢測(cè)ZAR1和GDF9分別在高、低產(chǎn)新西蘭白兔不同組織的表達(dá)差異,試驗(yàn)采用實(shí)時(shí)熒光定量PCR技術(shù),檢測(cè)了兩基因分別在心臟、肝臟、脾臟、肺臟、腎臟、卵巢和子宮七種組織的相對(duì)表達(dá)情況,以期從ZAR1和GDF9基因的轉(zhuǎn)錄表達(dá)水平闡釋其在新西蘭白兔的作用機(jī)制和組織表達(dá)規(guī)律。通過(guò)上述方法得出如下試驗(yàn)結(jié)果:1、根據(jù)NCBI上已公布的ZAR1m RNA和基因組序列,設(shè)計(jì)特異性引物,從新西蘭白兔腎臟中擴(kuò)增出ZAR1基因跨1-2外顯子序列、跨2-4外顯子序列、跨外顯子4及其3’側(cè)翼序列,分別為141、357和338bp,拼接整合后共709bp。根據(jù)NCBI上已公布的GDF9m RNA和基因組序列,設(shè)計(jì)特異性引物,從新西蘭白兔肝臟中擴(kuò)增出跨5’側(cè)翼區(qū)和外顯子1序列、外顯子2的部分序列,分別為796bp和614bp。2、利用生物信息學(xué)相關(guān)軟件分析新西蘭白兔ZAR1基因核苷酸序列與其他物種的同源性,試驗(yàn)結(jié)果表明:ZAR1基因c DNA序列與已公布的兔、人、牛、綿羊和豬的一致性分別為100%,91%,88%,88%和87%,與小鼠、大鼠的一致性分別為86%、87%,與斑馬魚、非洲爪蟾的一致性分別為76%,82%。將所得ZAR1c DNA序列翻譯的氨基酸序列在Gen Bank數(shù)據(jù)庫(kù)中比對(duì),發(fā)現(xiàn)其與已公布兔、人、小鼠、大鼠的ZAR1氨基酸序列一致性分別為100%,84%,93%,94%,與牛和豬的一致性均為97%,與斑馬魚一致性為89%,與非洲爪蟾一致性為96%。3、利用生物信息學(xué)相關(guān)軟件分析新西蘭白兔GDF9基因與其他物種的核苷酸同源性,試驗(yàn)結(jié)果表明:GDF9基因c DNA序列與已公布兔、人、牛、猴子、綿羊和豬的一致性分別為99%,79%,79%,77%,76%和75%,與小鼠、大鼠的一致性均為72%,與斑馬魚一致性為68%,與非洲爪蟾一致性為73%。將所得GDF9c DNA序列翻譯的氨基酸序列在Gen Bank數(shù)據(jù)庫(kù)中比對(duì),發(fā)現(xiàn)GDF9氨基酸與已公布的兔、人、猴、小鼠、大鼠、豬的對(duì)應(yīng)序列的同源性分別為97%,71%,72%,66%、65%,69%,與牛和綿羊的一致性均為68%,與斑馬魚一致性為43%,與非洲爪蟾一致性為49%。4、使用半定量RT-PCR的方法,進(jìn)行了ZAR1和GDF9基因在心臟、肝臟、脾臟、肺臟、腎臟、卵巢和子宮的組織表達(dá)譜研究。結(jié)果發(fā)現(xiàn),在上述組織中均有ZAR1和GDF9基因的m RNA表達(dá),表明ZAR1和GDF9 m RNA在新西蘭白兔呈廣泛性表達(dá),不具卵巢特異性。5、在高、低產(chǎn)的新西蘭白兔中,ZAR1基因均在肺臟組織中表達(dá)含量最高,在脾臟和腎臟組織的表達(dá)量次之,而在心臟組織中表達(dá)最低;而GDF9基因均在卵巢和肝臟組織中表達(dá)含量最高,而在心臟和脾臟中表達(dá)最低。6、同一組織在高、低產(chǎn)組的差異比較顯示:ZAR1基因的相對(duì)表達(dá)量在肝臟、腎臟和卵巢(P0.01),心臟和子宮(P0.05)存在顯著性差異,在脾臟和肺臟,無(wú)顯著差異表達(dá)(P0.05);而GDF9基因在肝臟和子宮(P0.01),心臟、脾臟和卵巢組織(P0.05)中存在顯著性差異,而在肺臟和腎臟,無(wú)顯著差異表達(dá)(P0.05)。7、GDF9和ZAR1基因在高、低產(chǎn)新西蘭白兔相對(duì)表達(dá)差異比較顯示:GDF9基因在心臟、肝臟、腎臟、卵巢和子宮組織中的相對(duì)表達(dá)含量均極顯著高于ZAR1基因的表達(dá)量。
[Abstract]:Maternal effect gene (MEG) is usually identified only in ovarian tissue and oocyte, and is now proved to be in the transition process of the parent type transition (maternal zygotic transition, MZT), that is, the transformation of the parent type to the zygotic type, as well as the early development of the mammalian embryo and the follicle. It plays an important role in the process of development. The zygote block factor 1 (zygote arrest 1, ZAR1) and growth differentiation factor 9 (growth differentiation factor 9, GDF9) are one of the few maternal effects genes that have been excavated earlier. In mice, rats, frogs, zebrafish, cattle, sheep, pigs, and other species have been reported that.ZAR1 is not only in oocyte. The GDF9 gene, as a member of the TGF- beta superfamily, has been proved to be the Qg source factor of the mammalian ovary, in the follicular and oocyte, as a member of the TGF- beta superfamily, and is proved to play an important role in the early stages of growth and embryonic development. In the process of maturation, it plays an important regulatory role, and because of its important role in the reproductive process, it has become a hot spot in the research of animal reproductive biology. At present, the research on these two genes in rabbits is still very few, and the understanding of its mechanism is not deep. This study is based on the previous work in the laboratory. The rabbits cloned the ZAR1 and GDF9 genes and analyzed the related bioinformatics. In order to detect the difference in the expression of different tissues between the high and low yield New Zealand white rabbits, ZAR1 and GDF9 were detected by real-time fluorescence quantitative PCR, and the two genes were detected in the heart, liver, spleen, lung, kidney, ovary and uterus, respectively. The mechanism of action and tissue expression in New Zealand white rabbits were explained from the transcriptional expression level of ZAR1 and GDF9 genes. The following experimental results were obtained through the above methods: 1, specific primers were designed according to the ZAR1m RNA and genome sequence published on NCBI, and the ZAR1 gene was amplified from New Zealand white rabbit kidney by 1-2. Exon sequence, cross 2-4 exon sequence, exon 4 and 3 'flanking sequence, 141357 and 338bp respectively. After splicing and integration, 709bp. based on the published GDF9m RNA and genome sequence on NCBI, designed specific primers, and amplified the 5' side wing and exon 1 sequence from the New Zealand white rabbit liver, and the partial sequence of exon 2. 796bp and 614bp.2, using bioinformatics related software to analyze the homology of New Zealand white rabbit ZAR1 nucleotide sequence and other species. The results showed that the consistency of C DNA sequence of ZAR1 gene was 100%, 91%, 88%, 88% and 87% respectively, and the consistency with mice and rats was 86%, 87%, respectively. The consistency between the zebrafish and the Xenopus Xenopus was 76%, respectively. 82%. compared the amino acid sequence translated by the ZAR1c DNA sequence in the Gen Bank database, and found that the consistency of the ZAR1 amino acid sequences of the published rabbits, people, mice and rats were 100%, 84%, 93%, 94% respectively, and the consistency with the cattle and pigs were 97%, and the consistency with zebrafish was 89%, and that of the zebrafish was 89%. The conformance of Xenopus laevis was 96%.3, and the nucleotide homology of New Zealand white rabbit GDF9 gene and other species was analyzed by bioinformatics software. The results showed that the consistency of C DNA sequence of GDF9 gene was 99%, 79%, 79%, 77%, 76% and 75%, respectively, and 72% in mice and rats. The conformance of zebrafish was 68%. The homology of the amino acid sequence translated from the GDF9c DNA sequence was compared with the African Xenopus 73%., and the homology of the GDF9 amino acid to the corresponding sequence of the published rabbit, human, monkey, mouse, rat, and pig was 97%, 71%, 72%, 66%, 65%, 69%, respectively, and was 68% with the cattle and sheep. The conformance of zebrafish was 43%. The expression of ZAR1 and GDF9 genes in the heart, liver, spleen, lungs, kidneys, ovary and uterus were studied with 49%.4 and semi quantitative RT-PCR. The results showed that there were m RNA expressions of ZAR1 and GDF9 genes in the above-mentioned tissues, indicating that ZAR1 and GDF9 m RNA are in New Zealand. The white rabbit is widely expressed and does not have ovarian specific.5. In high and low yield New Zealand white rabbits, the ZAR1 gene expresses the highest content in the lung tissue, the expression of the spleen and kidney and the lowest expression in the heart tissue, while the GDF9 gene is the highest in the ovarian and liver tissues, but in the heart and spleen. The difference in the expression of the lowest.6 and the same tissue in the high and low yield groups showed that the relative expression of the ZAR1 gene was significantly different in the liver, the kidneys and the ovary (P0.01), the heart and the uterus (P0.05), and there was no significant difference in the spleen and lungs (P0.05), while the GDF9 gene was stored in the liver and uterus (P0.01), the heart, spleen, and ovarian tissue (P0.05). In the significant difference, in the lungs and kidneys, there was no significant difference in expression (P0.05).7, the GDF9 and ZAR1 genes were high, and the relative expression of the low yield New Zealand white rabbits showed that the relative expression of GDF9 gene in the heart, liver, kidney, ovary and uterus was significantly higher than that of the ZAR1 gene.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S829.1

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