本文選題：釀酒酵母 + 人類基因��； 參考：《江南大學(xué)》2017年碩士論文

【摘要】：釀酒酵母(Saccharomyces cerevisiae)二倍體細胞在缺乏氮源且存在非發(fā)酵型碳源時,營養(yǎng)細胞停止生長并進入產(chǎn)孢過程,通過減數(shù)分裂產(chǎn)生四個單倍體子囊孢子。產(chǎn)孢過程是釀酒酵母的一個特殊發(fā)育過程,該過程在哺乳動物細胞中并不存在,但是存在很多真核細胞中高度保守的細胞活動,例如減數(shù)分裂,膜的運輸與重組等。釀酒酵母孢子具有獨特的孢子壁結(jié)構(gòu),二酪氨酸層作為孢子壁最外層,其主要由二甲酰基-LL-二酪氨酸(Formyl-LL-dityrosine)組成。本研究利用二酪氨酸在紫外光下具有自熒光的特性,建立了一個篩選方法,通過相對熒光強度篩選特異性抑制釀酒酵母產(chǎn)孢過程的人類基因。在篩選過程中,使用本研究構(gòu)建的酵母表達質(zhì)粒在釀酒酵母細胞中單獨表達不同的人類基因,同時檢測該基因的表達對產(chǎn)孢過程的影響,篩選本研究所需的目的基因。在現(xiàn)階段的研究中,我們對3,128個人類基因進行篩選,共得到12個對釀酒酵母產(chǎn)孢過程具有明顯抑制作用的人類基因。在得到的目的基因中,大多數(shù)基因的表達會導(dǎo)致釀酒酵母無法進入減數(shù)分裂過程,導(dǎo)致產(chǎn)孢率下降。這些基因涉及多種細胞活動過程,其中一些基因的功能尚不清楚,利用釀酒酵母進行進一步的研究分析將有助于揭示這些基因產(chǎn)物的功能。此外,篩選得到的4-羥苯丙酮酸二加氧酶(4-Hydroxyphenylpyruvate dioxygenase,HPD)基因表現(xiàn)出了獨特的性質(zhì),該基因在釀酒酵母中表達會導(dǎo)致熒光強度下降但產(chǎn)孢率不變,推測可能導(dǎo)致了孢子壁缺陷。進一步研究發(fā)現(xiàn),HPD基因的表達會導(dǎo)致孢子的二酪氨酸層形成過程中出現(xiàn)缺陷,使其乙醚敏感性提升。同時,表達該基因的釀酒酵母在產(chǎn)孢過程中,培養(yǎng)基熒光強度上升,表明參與孢子壁組裝的二酪氨酸復(fù)合物大量泄漏至培養(yǎng)基中。此外,通過體外添加尿黑酸(Homogentisate,HGA)檢測發(fā)現(xiàn)酵母產(chǎn)孢過程中孢子具有氧化性。結(jié)果顯示,HPD蛋白的催化產(chǎn)物尿黑酸在產(chǎn)孢過程中被氧化并參與二酪氨酸層的組裝,導(dǎo)致二酪氨酸無法正確完成組裝。該發(fā)現(xiàn)為二酪氨酸層形成機制的研究提供了新的啟發(fā)與研究思路。本研究利用釀酒酵母的產(chǎn)孢過程對人類基因進行篩選,具有較強的創(chuàng)新性以及廣闊的研究前景。通過該研究不僅能夠更好地理解產(chǎn)孢過程,同時還對探索人類未發(fā)現(xiàn)基因以及揭示其基因產(chǎn)物功能有著重要的研究的意義與極高的研究價值。
[Abstract]:When the diploid cells of Saccharomyces cerevisiaeae were deficient in nitrogen and non-fermentative carbon sources, the vegetative cells stopped growing and entered the process of sporulation, and four haploid ascospores were produced by meiosis. Sporulation is a special developmental process of Saccharomyces cerevisiae, which does not exist in mammalian cells, but there are many highly conserved cell activities in eukaryotic cells, such as meiosis, membrane transport and recombination. The spore of Saccharomyces cerevisiae has a unique spore wall structure, and the dityrosine layer is the outermost layer of the spore wall, which is mainly composed of diformyl-L-L-tyrosine (Formyl-LL-dityrosine). Based on the autofluorescence of dityrosine in ultraviolet light, a screening method was established to screen human genes that specifically inhibit the sporulation process of Saccharomyces cerevisiae by relative fluorescence intensity. In the screening process, the yeast expression plasmid constructed in this study was used to express different human genes in Saccharomyces cerevisiae cells alone, and the effect of the expression of this gene on the sporulation process was detected. In the present study, we screened 3128 human genes and obtained 12 human genes which have obvious inhibitory effect on the sporulation process of Saccharomyces cerevisiae (Saccharomyces cerevisiae). Among the obtained target genes, the expression of most of the genes will lead to the inability of Saccharomyces cerevisiae to enter the meiosis process, resulting in a decrease in the sporulation rate. These genes are involved in a variety of cellular processes, and the functions of some of these genes are still unclear. Further research and analysis using Saccharomyces cerevisiae will help to reveal the function of these gene products. In addition, the 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate dioxygenase- HPD-HPD) gene, which was isolated from yeast, showed unique properties. Its expression in Saccharomyces cerevisiae led to a decrease in fluorescence intensity but a constant sporulation rate, which may lead to spore wall defects. It was further found that the expression of HPD gene may lead to defects in the formation of dityrosine layer of spores, and enhance the ether sensitivity of the spores. At the same time, the fluorescence intensity of the medium increased during the sporulation process of Saccharomyces cerevisiae which expressed the gene, indicating that the dityrosine complex involved in the assembly of spores wall was leaked into the medium. In addition, it was found that the spores were oxidized during sporulation by addition of Homogentisatein HGA in vitro. The results showed that the catalytic product of HPD was oxidized during sporulation and involved in the assembly of dityrosine layer, which resulted in the inability to complete the assembly of dityrosine correctly. The findings provide new insights and ideas for the study of the formation mechanism of dityrosine layer. In this study, the sporulation process of Saccharomyces cerevisiae was used to screen human genes, which had strong innovation and broad research prospect. This study not only can better understand the process of sporulation, but also has important significance and high research value in exploring human undiscovered genes and revealing the function of their gene products.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;TS261.11

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本文編號：1785597