鸡miR-2954超表达载体的构建及其性腺发育相关靶基因的研究

发布时间：2018-06-07 09:07

本文选题：鸡 + miR-2954　；参考：《华中农业大学》2017年硕士论文

【摘要】：禽类与哺乳动物均属于遗传性别决定,但与哺乳动物不同,禽类的性别决定机制还不确定,上游性别决定因子还有待于进一步探寻。研究表明在转录后起重要调控作用的miRNAs在动物性腺发育过程有重要作用,本课题组拟从miRNA水平寻找与鸡性别决定及早期性腺发育相关的上游调控因子。miR-2954是位于禽类Z染色体上的miRNA,本课题组前期研究发现其在雄性性腺及其他组织中的表达量显著高于雌性,与先前报道的在其他禽类中的研究结果相似,这提示了miR-2954可能作为一个细胞自主决定因子在鸡早期性别决定及性腺发育中起调控作用。本研究首先构建了miR-2954超表达载体,并结合基因的双荧光素酶报告系统验证了miR-2954与性腺发育及功能相关靶基因的调控关系,为进一步确定及研究miR-2954在鸡早期性腺发育中的功能奠定基础。本研究的实验结果如下:1.制备了不同性别原代鸡胚成纤维细胞(CEF),通过定量PCR检测miR-2954在不同CEF细胞及商品化DF1细胞中的表达量,结果显示miR-2954在雄性CEF细胞中的表达量最高,并极显著地高于雌性和混样(雌雄等量混合)CEF及商品化DF1细胞,这与miR-2954雄性高表达特性相符;且结果表明本室的商业化DF1细胞适用于后续的miR-2954超表达载体效果检测及靶基因验证研究。2.构建了PSIN-2954、PSIN-eGFP-2954和PSIN-eGFP三种表达载体,通过序列测定和转染细胞后荧光观察初步确定其效果。定量PCR检测结果显示miR-2954在转染了PSIN-2954和PSIN-eGFP-2954载体的细胞中表达量均显著提高,这表明miR-2954超表达载体构建成功。3.转染PSIN-2954载体后通过PI染色法检测mi R-2954对DF1细胞周期的影响。结果显示:转染miR-2954组G1期的细胞比例呈极显著降低,而S期和G2期的细胞则极显著升高,这表明miR-2954的超表达可促进细胞增殖。4.根据鸡转录组的mRNA(包括5’UTR、CDS和3’UTR)与miR-2954序列的匹配情况与功能分析,筛选出四个与鸡性腺发育及功能相关的基因AMH、DMRT1、SF1及VLDL作为miR-2954的候选靶基因,并成功的构建了靶序列的荧光素酶报告重组载体。5.候选靶基因(AMH、DMRT1、SF1和VLDL)的双荧光素酶报告系统检测结果显示,miR-2954可显著或极显著地降低SF1和VLDL-CDS双荧光素酶的活性,而AMH和DMRT1-CDS双荧光素酶活性无明显变化。结果表明SF1和VLDL是与miR-2954存在负调控关系的靶基因。综上所述,本研究结果初步确定了miR-2954与SF1等早期性腺发育有关因子的调控关系,并成功构建了miR-2954的超表达载体,为接下来在鸡胚中研究miR-2954对早期性腺分化及发育的功能奠定了重要基础。
[Abstract]:Both birds and mammals are genetic determinants, but different from mammals, the sex determination mechanism of birds is uncertain, and the upstream sex determinants need to be further explored. Studies have shown that miRNAs, which plays an important role in posttranscriptional regulation, plays an important role in the development of gonad in animals. Our team intends to look for the upstream regulatory factor .miR-2954, which is related to sex determination and early gonadal development of chicken, at the miRNA level, which is located on the Z chromosome of poultry. Our previous study found that it is a surface in male gonads and other tissues. The amount reached was significantly higher than that in females. Similar to previous studies in other birds, this suggests that miR-2954 may play a regulatory role as a cellular autonomic determinant in early sex determination and gonadal development in chickens. In this study, miR-2954 superexpression vector was constructed, and the regulatory relationship between miR-2954 and gonadal development and function related target genes was verified by double luciferase report system. To further determine and study the role of miR-2954 in chicken early gonadal development. The experimental results of this study are as follows: 1. The expression of miR-2954 in different CEF cells and commercial DF1 cells was detected by quantitative PCR. The results showed that miR-2954 expression was the highest in male CEF cells. It was significantly higher than that of female and mixed (female and male) mixed CEF and commercial DF1 cells, which was consistent with the high expression of miR-2954 male. The results showed that the commercial DF1 cells were suitable for the subsequent miR-2954 superexpression vector detection and target gene validation. 2. Three kinds of expression vectors PSIN-2954 PSIN-eGFP-2954 and PSIN-eGFP were constructed and their effects were preliminarily confirmed by sequencing and fluorescence observation after transfection of PSIN-2954 and PSIN-eGFP-2954. The results of quantitative PCR analysis showed that the expression of miR-2954 in the cells transfected with PSIN-2954 and PSIN-eGFP-2954 vectors was significantly increased, which indicated that the miR-2954 superexpression vector was successfully constructed. 3. After transfection of PSIN-2954 vector, the effect of miR-2954 on DF1 cell cycle was detected by Pi staining. The results showed that the percentage of cells transfected with miR-2954 in G1 phase was significantly decreased, while that in S phase and G2 phase was significantly increased, which indicated that the overexpression of miR-2954 could promote cell proliferation. According to the matching and functional analysis of miR-2954 sequences in chicken transcriptome (including 5UTRCDS and 3UTRs), four genes related to gonadal development and function, AMHN DMRT1SF1 and VLDL, were selected as candidate target genes for miR-2954. And successfully constructed the target sequence of luciferase report recombinant vector. The results of double luciferase reporting system for candidate target gene AMHG DMRT1 SF1 and VLDL showed that mmiR-2954 could significantly or extremely reduce the activity of SF1 and VLDL-CDS double luciferase, but AMH and DMRT1-CDS double luciferase activities had no significant change. The results showed that SF1 and VLDL were the target genes of negative regulation with miR-2954. In conclusion, this study preliminarily confirmed the regulatory relationship between miR-2954 and early gonadal development related factors such as SF1, and successfully constructed the superexpression vector of miR-2954. It lays an important foundation for studying the function of miR-2954 in early gonad differentiation and development in chicken embryo.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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