本文選題：雞 + miR-2954�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：禽類(lèi)與哺乳動(dòng)物均屬于遺傳性別決定,但與哺乳動(dòng)物不同,禽類(lèi)的性別決定機(jī)制還不確定,上游性別決定因子還有待于進(jìn)一步探尋。研究表明在轉(zhuǎn)錄后起重要調(diào)控作用的miRNAs在動(dòng)物性腺發(fā)育過(guò)程有重要作用,本課題組擬從miRNA水平尋找與雞性別決定及早期性腺發(fā)育相關(guān)的上游調(diào)控因子。miR-2954是位于禽類(lèi)Z染色體上的miRNA,本課題組前期研究發(fā)現(xiàn)其在雄性性腺及其他組織中的表達(dá)量顯著高于雌性,與先前報(bào)道的在其他禽類(lèi)中的研究結(jié)果相似,這提示了miR-2954可能作為一個(gè)細(xì)胞自主決定因子在雞早期性別決定及性腺發(fā)育中起調(diào)控作用。本研究首先構(gòu)建了miR-2954超表達(dá)載體,并結(jié)合基因的雙熒光素酶報(bào)告系統(tǒng)驗(yàn)證了miR-2954與性腺發(fā)育及功能相關(guān)靶基因的調(diào)控關(guān)系,為進(jìn)一步確定及研究miR-2954在雞早期性腺發(fā)育中的功能奠定基礎(chǔ)。本研究的實(shí)驗(yàn)結(jié)果如下:1.制備了不同性別原代雞胚成纖維細(xì)胞(CEF),通過(guò)定量PCR檢測(cè)miR-2954在不同CEF細(xì)胞及商品化DF1細(xì)胞中的表達(dá)量,結(jié)果顯示miR-2954在雄性CEF細(xì)胞中的表達(dá)量最高,并極顯著地高于雌性和混樣(雌雄等量混合)CEF及商品化DF1細(xì)胞,這與miR-2954雄性高表達(dá)特性相符;且結(jié)果表明本室的商業(yè)化DF1細(xì)胞適用于后續(xù)的miR-2954超表達(dá)載體效果檢測(cè)及靶基因驗(yàn)證研究。2.構(gòu)建了PSIN-2954、PSIN-eGFP-2954和PSIN-eGFP三種表達(dá)載體,通過(guò)序列測(cè)定和轉(zhuǎn)染細(xì)胞后熒光觀察初步確定其效果。定量PCR檢測(cè)結(jié)果顯示miR-2954在轉(zhuǎn)染了PSIN-2954和PSIN-eGFP-2954載體的細(xì)胞中表達(dá)量均顯著提高,這表明miR-2954超表達(dá)載體構(gòu)建成功。3.轉(zhuǎn)染PSIN-2954載體后通過(guò)PI染色法檢測(cè)mi R-2954對(duì)DF1細(xì)胞周期的影響。結(jié)果顯示:轉(zhuǎn)染miR-2954組G1期的細(xì)胞比例呈極顯著降低,而S期和G2期的細(xì)胞則極顯著升高,這表明miR-2954的超表達(dá)可促進(jìn)細(xì)胞增殖。4.根據(jù)雞轉(zhuǎn)錄組的mRNA(包括5’UTR、CDS和3’UTR)與miR-2954序列的匹配情況與功能分析,篩選出四個(gè)與雞性腺發(fā)育及功能相關(guān)的基因AMH、DMRT1、SF1及VLDL作為miR-2954的候選靶基因,并成功的構(gòu)建了靶序列的熒光素酶報(bào)告重組載體。5.候選靶基因(AMH、DMRT1、SF1和VLDL)的雙熒光素酶報(bào)告系統(tǒng)檢測(cè)結(jié)果顯示,miR-2954可顯著或極顯著地降低SF1和VLDL-CDS雙熒光素酶的活性,而AMH和DMRT1-CDS雙熒光素酶活性無(wú)明顯變化。結(jié)果表明SF1和VLDL是與miR-2954存在負(fù)調(diào)控關(guān)系的靶基因。綜上所述,本研究結(jié)果初步確定了miR-2954與SF1等早期性腺發(fā)育有關(guān)因子的調(diào)控關(guān)系,并成功構(gòu)建了miR-2954的超表達(dá)載體,為接下來(lái)在雞胚中研究miR-2954對(duì)早期性腺分化及發(fā)育的功能奠定了重要基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S831

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本文編號(hào)：1990634