筛选特异性抑制酿酒酵母产孢的人类基因

发布时间：2018-04-22 04:24

本文选题：酿酒酵母 + 人类基因　；参考：《江南大学》2017年硕士论文

【摘要】：酿酒酵母(Saccharomyces cerevisiae)二倍体细胞在缺乏氮源且存在非发酵型碳源时,营养细胞停止生长并进入产孢过程,通过减数分裂产生四个单倍体子囊孢子。产孢过程是酿酒酵母的一个特殊发育过程,该过程在哺乳动物细胞中并不存在,但是存在很多真核细胞中高度保守的细胞活动,例如减数分裂,膜的运输与重组等。酿酒酵母孢子具有独特的孢子壁结构,二酪氨酸层作为孢子壁最外层,其主要由二甲酰基-LL-二酪氨酸(Formyl-LL-dityrosine)组成。本研究利用二酪氨酸在紫外光下具有自荧光的特性,建立了一个筛选方法,通过相对荧光强度筛选特异性抑制酿酒酵母产孢过程的人类基因。在筛选过程中,使用本研究构建的酵母表达质粒在酿酒酵母细胞中单独表达不同的人类基因,同时检测该基因的表达对产孢过程的影响,筛选本研究所需的目的基因。在现阶段的研究中,我们对3,128个人类基因进行筛选,共得到12个对酿酒酵母产孢过程具有明显抑制作用的人类基因。在得到的目的基因中,大多数基因的表达会导致酿酒酵母无法进入减数分裂过程,导致产孢率下降。这些基因涉及多种细胞活动过程,其中一些基因的功能尚不清楚,利用酿酒酵母进行进一步的研究分析将有助于揭示这些基因产物的功能。此外,筛选得到的4-羟苯丙酮酸二加氧酶(4-Hydroxyphenylpyruvate dioxygenase,HPD)基因表现出了独特的性质,该基因在酿酒酵母中表达会导致荧光强度下降但产孢率不变,推测可能导致了孢子壁缺陷。进一步研究发现,HPD基因的表达会导致孢子的二酪氨酸层形成过程中出现缺陷,使其乙醚敏感性提升。同时,表达该基因的酿酒酵母在产孢过程中,培养基荧光强度上升,表明参与孢子壁组装的二酪氨酸复合物大量泄漏至培养基中。此外,通过体外添加尿黑酸(Homogentisate,HGA)检测发现酵母产孢过程中孢子具有氧化性。结果显示,HPD蛋白的催化产物尿黑酸在产孢过程中被氧化并参与二酪氨酸层的组装,导致二酪氨酸无法正确完成组装。该发现为二酪氨酸层形成机制的研究提供了新的启发与研究思路。本研究利用酿酒酵母的产孢过程对人类基因进行筛选,具有较强的创新性以及广阔的研究前景。通过该研究不仅能够更好地理解产孢过程,同时还对探索人类未发现基因以及揭示其基因产物功能有着重要的研究的意义与极高的研究价值。
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;TS261.11

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