ZAR1和GDF9基因的克隆及其在新西兰白兔的差异表达研究

发布时间：2018-07-20 14:15

【摘要】：母性效应基因(maternal effect gene,MEG)通常被认定是只在卵巢组织及卵母细胞中特异表达,且目前已被证明在母型-合子型过渡(maternal zygotic transition,MZT),即母型调控向合子型调控的转变过程中,以及哺乳动物胚胎发育的初期和卵泡的发生、发育过程中发挥着重要的功能。合子阻滞因子1(zygote arrest 1,ZAR1)和生长分化因子9(growth differentiation factor 9,GDF9)作为少数较早被挖掘到的母性效应基因之一,在小鼠、大鼠、蛙、斑马鱼、牛、绵羊、猪等物种已被报道。ZAR1不但在卵母细胞的生长和胚胎发育的初期过程中起着举足轻重的作用,且被证明该基因某些位点的核苷酸多态性同其产仔数等繁殖性状显著相关。而GDF9基因作为TGF-β超家族的一员,已被证明是哺乳动物卵巢的Qg源细胞因子,在卵泡的发生和卵母细胞的成熟过程中发挥着重要的调节作用,又因为其在生殖过程中的重要作用,己成为动物繁殖生物学研究的热点。而目前国内外关于这两个基因在兔方面的研究仍然很少,对其作用机制的认识还不深入。本研究基于实验室以前的工作,在新西兰白兔克隆了ZAR1和GDF9基因,并进行了相关生物信息学分析。为了检测ZAR1和GDF9分别在高、低产新西兰白兔不同组织的表达差异,试验采用实时荧光定量PCR技术,检测了两基因分别在心脏、肝脏、脾脏、肺脏、肾脏、卵巢和子宫七种组织的相对表达情况,以期从ZAR1和GDF9基因的转录表达水平阐释其在新西兰白兔的作用机制和组织表达规律。通过上述方法得出如下试验结果:1、根据NCBI上已公布的ZAR1m RNA和基因组序列,设计特异性引物,从新西兰白兔肾脏中扩增出ZAR1基因跨1-2外显子序列、跨2-4外显子序列、跨外显子4及其3’侧翼序列,分别为141、357和338bp,拼接整合后共709bp。根据NCBI上已公布的GDF9m RNA和基因组序列,设计特异性引物,从新西兰白兔肝脏中扩增出跨5’侧翼区和外显子1序列、外显子2的部分序列,分别为796bp和614bp。2、利用生物信息学相关软件分析新西兰白兔ZAR1基因核苷酸序列与其他物种的同源性,试验结果表明:ZAR1基因c DNA序列与已公布的兔、人、牛、绵羊和猪的一致性分别为100%,91%,88%,88%和87%,与小鼠、大鼠的一致性分别为86%、87%,与斑马鱼、非洲爪蟾的一致性分别为76%,82%。将所得ZAR1c DNA序列翻译的氨基酸序列在Gen Bank数据库中比对,发现其与已公布兔、人、小鼠、大鼠的ZAR1氨基酸序列一致性分别为100%,84%,93%,94%,与牛和猪的一致性均为97%,与斑马鱼一致性为89%,与非洲爪蟾一致性为96%。3、利用生物信息学相关软件分析新西兰白兔GDF9基因与其他物种的核苷酸同源性,试验结果表明:GDF9基因c DNA序列与已公布兔、人、牛、猴子、绵羊和猪的一致性分别为99%,79%,79%,77%,76%和75%,与小鼠、大鼠的一致性均为72%,与斑马鱼一致性为68%,与非洲爪蟾一致性为73%。将所得GDF9c DNA序列翻译的氨基酸序列在Gen Bank数据库中比对,发现GDF9氨基酸与已公布的兔、人、猴、小鼠、大鼠、猪的对应序列的同源性分别为97%,71%,72%,66%、65%,69%,与牛和绵羊的一致性均为68%,与斑马鱼一致性为43%,与非洲爪蟾一致性为49%。4、使用半定量RT-PCR的方法,进行了ZAR1和GDF9基因在心脏、肝脏、脾脏、肺脏、肾脏、卵巢和子宫的组织表达谱研究。结果发现,在上述组织中均有ZAR1和GDF9基因的m RNA表达,表明ZAR1和GDF9 m RNA在新西兰白兔呈广泛性表达,不具卵巢特异性。5、在高、低产的新西兰白兔中,ZAR1基因均在肺脏组织中表达含量最高,在脾脏和肾脏组织的表达量次之,而在心脏组织中表达最低;而GDF9基因均在卵巢和肝脏组织中表达含量最高,而在心脏和脾脏中表达最低。6、同一组织在高、低产组的差异比较显示:ZAR1基因的相对表达量在肝脏、肾脏和卵巢(P0.01),心脏和子宫(P0.05)存在显著性差异,在脾脏和肺脏,无显著差异表达(P0.05);而GDF9基因在肝脏和子宫(P0.01),心脏、脾脏和卵巢组织(P0.05)中存在显著性差异,而在肺脏和肾脏,无显著差异表达(P0.05)。7、GDF9和ZAR1基因在高、低产新西兰白兔相对表达差异比较显示:GDF9基因在心脏、肝脏、肾脏、卵巢和子宫组织中的相对表达含量均极显著高于ZAR1基因的表达量。
[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.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S829.1

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