3＇RACE验证Long-SAGE文库构建后白念珠菌不同菌相的表达基因
发布时间：2018-11-08 20:41
【摘要】：白念珠菌菌相的转换与白念珠菌的感染和致病密切相关,但目前对白念珠菌菌相转换的分子机制尚不清楚;尽管有文献报道与白念珠菌菌相转换相关的一些基因,如VPS11、CaCLA4、INT1、CaRSR1等的表达,及以MAPK、cAMP为代表的白念珠菌菌相转换的信号传导网络有关,但这些研究主要局限于某个基因或信号传导通路的某一成分上,而对调控其菌相转换的基因网络缺乏全面、系统的研究。 白念珠菌基因组测序工作几近完成,但大量的未知基因未得到充分注释,与其菌相转换的关键或全部基因尚未得到全面阐述,其功能研究更是远远滞后;近几年来,本课题组通过采用基因表达系列分析(serial analysis of gene expression, SAGE),构建了白念珠菌不同菌相的LongSAGE 文库,获得了数以千计的序列表达标签,可以在较短时间内检测细胞内几乎所有表达基因的mRNA,并且能对这些mRNA 的表达进行定量与定性分析,不仅可用于白念珠菌表型差异的基因分析,同时也可能发现潜在的功能未知基因。 为验证LongSAGE文库构建后,白念珠菌菌相转换时菌相间的部分差异表达基因,本研究在上述工作的基础上,通过运用3′RACE 技术,经过三部分实验:(1) 白念珠菌ATCC90028 菌丝相的诱导;(2) 白念珠菌ATCC-90028 不同菌相总RNA 的制备和分析;(3)运用3′RACE 验证白念珠菌不同菌相LongSAGE 文库标签的差异表达基因,对Long-SAGE 文库中的多重匹配标签进行了3′端的全长扩增,获得了从标签至mRNA-3′端的基因序列,证实了表达序列代表的表达基因,本研究结果为进一步验证白念珠菌LongSAGE 文库的阳性库容、以及为能发现新基因以及对菌相转换起关键作用的调控基因打下分子生物学基础。 本研究主要结果和结论: 1. 孢子以密度为5×106/L 分别接种于1640 培养基和DMEM 培养基(pH7. 35, 37℃),振荡培养下可诱导出芽管形成率大于95%的可以满足分子生物学需要的白念珠
[Abstract]:The phase transition of Candida albicans is closely related to the infection and pathogenicity of Candida albicans, but the molecular mechanism of phase transition of Candida albicans remains unclear. Although it has been reported that some genes related to phase transformation of Candida albicans, such as VPS11,CaCLA4,INT1,CaRSR1, and signal transduction networks of Candida albicans represented by MAPK,cAMP, have been reported. However, these studies are mainly limited to a gene or a component of the signal transduction pathway, but there is no comprehensive and systematic study on the gene network that regulates the transformation of the bacteria. The genome sequencing of Candida albicans was nearly completed, but a large number of unknown genes were not fully annotated, and the key or all genes involved in the phase transformation of Candida albicans were not fully described, and the study of their function was far behind. In recent years, the LongSAGE library of Candida albicans was constructed by using gene expression series (serial analysis of gene expression, SAGE), and thousands of sequence expression tags were obtained. The mRNA, of almost all the expressed genes in the cell can be detected in a short time, and the expression of these mRNA can be quantitatively and qualitatively analyzed, which can not only be used for the genetic analysis of phenotypic differences in Candida albicans. Potential functional unknown genes may also be found. In order to verify the partial differentially expressed genes of Candida albicans during phase transformation after the construction of LongSAGE library, based on the above work, 3'RACE technique was used in this study. After three parts of the experiment: (1) the induction of ATCC90028 hyphal phase of Candida albicans; (2) preparation and analysis of total RNA in different phases of Candida albicans ATCC-90028; (3) 3'RACE was used to verify the differentially expressed genes in the LongSAGE library tags of Candida albicans in different phases. The full length of the multiple matching tags in the Long-SAGE library was amplified by 3'-terminal amplification, and the gene sequence from the label to the mRNA-3' terminal was obtained. The results of this study provide a molecular biological basis for further verification of the positive library capacity of Candida albicans LongSAGE library and for the discovery of new genes and the regulatory genes that play a key role in the phase transition of Candida albicans. The main results and conclusions of this study are as follows: 1. The spores were inoculated in 1640 and DMEM medium (pH7.) with a density of 5 脳 106 / L. 35 鈩，
本文编号：2319629
[Abstract]:The phase transition of Candida albicans is closely related to the infection and pathogenicity of Candida albicans, but the molecular mechanism of phase transition of Candida albicans remains unclear. Although it has been reported that some genes related to phase transformation of Candida albicans, such as VPS11,CaCLA4,INT1,CaRSR1, and signal transduction networks of Candida albicans represented by MAPK,cAMP, have been reported. However, these studies are mainly limited to a gene or a component of the signal transduction pathway, but there is no comprehensive and systematic study on the gene network that regulates the transformation of the bacteria. The genome sequencing of Candida albicans was nearly completed, but a large number of unknown genes were not fully annotated, and the key or all genes involved in the phase transformation of Candida albicans were not fully described, and the study of their function was far behind. In recent years, the LongSAGE library of Candida albicans was constructed by using gene expression series (serial analysis of gene expression, SAGE), and thousands of sequence expression tags were obtained. The mRNA, of almost all the expressed genes in the cell can be detected in a short time, and the expression of these mRNA can be quantitatively and qualitatively analyzed, which can not only be used for the genetic analysis of phenotypic differences in Candida albicans. Potential functional unknown genes may also be found. In order to verify the partial differentially expressed genes of Candida albicans during phase transformation after the construction of LongSAGE library, based on the above work, 3'RACE technique was used in this study. After three parts of the experiment: (1) the induction of ATCC90028 hyphal phase of Candida albicans; (2) preparation and analysis of total RNA in different phases of Candida albicans ATCC-90028; (3) 3'RACE was used to verify the differentially expressed genes in the LongSAGE library tags of Candida albicans in different phases. The full length of the multiple matching tags in the Long-SAGE library was amplified by 3'-terminal amplification, and the gene sequence from the label to the mRNA-3' terminal was obtained. The results of this study provide a molecular biological basis for further verification of the positive library capacity of Candida albicans LongSAGE library and for the discovery of new genes and the regulatory genes that play a key role in the phase transition of Candida albicans. The main results and conclusions of this study are as follows: 1. The spores were inoculated in 1640 and DMEM medium (pH7.) with a density of 5 脳 106 / L. 35 鈩，
本文编号：2319629
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