【摘要】：白念珠菌菌相的轉(zhuǎn)換與白念珠菌的感染和致病密切相關(guān),但目前對(duì)白念珠菌菌相轉(zhuǎn)換的分子機(jī)制尚不清楚;盡管有文獻(xiàn)報(bào)道與白念珠菌菌相轉(zhuǎn)換相關(guān)的一些基因,如VPS11、CaCLA4、INT1、CaRSR1等的表達(dá),及以MAPK、cAMP為代表的白念珠菌菌相轉(zhuǎn)換的信號(hào)傳導(dǎo)網(wǎng)絡(luò)有關(guān),但這些研究主要局限于某個(gè)基因或信號(hào)傳導(dǎo)通路的某一成分上,而對(duì)調(diào)控其菌相轉(zhuǎn)換的基因網(wǎng)絡(luò)缺乏全面、系統(tǒng)的研究。 白念珠菌基因組測(cè)序工作幾近完成,但大量的未知基因未得到充分注釋,與其菌相轉(zhuǎn)換的關(guān)鍵或全部基因尚未得到全面闡述,其功能研究更是遠(yuǎn)遠(yuǎn)滯后;近幾年來(lái),本課題組通過(guò)采用基因表達(dá)系列分析(serial analysis of gene expression, SAGE),構(gòu)建了白念珠菌不同菌相的LongSAGE 文庫(kù),獲得了數(shù)以千計(jì)的序列表達(dá)標(biāo)簽,可以在較短時(shí)間內(nèi)檢測(cè)細(xì)胞內(nèi)幾乎所有表達(dá)基因的mRNA,并且能對(duì)這些mRNA 的表達(dá)進(jìn)行定量與定性分析,不僅可用于白念珠菌表型差異的基因分析,同時(shí)也可能發(fā)現(xiàn)潛在的功能未知基因。 為驗(yàn)證LongSAGE文庫(kù)構(gòu)建后,白念珠菌菌相轉(zhuǎn)換時(shí)菌相間的部分差異表達(dá)基因,本研究在上述工作的基礎(chǔ)上,通過(guò)運(yùn)用3′RACE 技術(shù),經(jīng)過(guò)三部分實(shí)驗(yàn):(1) 白念珠菌ATCC90028 菌絲相的誘導(dǎo);(2) 白念珠菌ATCC-90028 不同菌相總RNA 的制備和分析;(3)運(yùn)用3′RACE 驗(yàn)證白念珠菌不同菌相LongSAGE 文庫(kù)標(biāo)簽的差異表達(dá)基因,對(duì)Long-SAGE 文庫(kù)中的多重匹配標(biāo)簽進(jìn)行了3′端的全長(zhǎng)擴(kuò)增,獲得了從標(biāo)簽至mRNA-3′端的基因序列,證實(shí)了表達(dá)序列代表的表達(dá)基因,本研究結(jié)果為進(jìn)一步驗(yàn)證白念珠菌LongSAGE 文庫(kù)的陽(yáng)性庫(kù)容、以及為能發(fā)現(xiàn)新基因以及對(duì)菌相轉(zhuǎn)換起關(guān)鍵作用的調(diào)控基因打下分子生物學(xué)基礎(chǔ)。 本研究主要結(jié)果和結(jié)論: 1. 孢子以密度為5×106/L 分別接種于1640 培養(yǎng)基和DMEM 培養(yǎng)基(pH7. 35, 37℃),振蕩培養(yǎng)下可誘導(dǎo)出芽管形成率大于95%的可以滿足分子生物學(xué)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，

本文編號(hào)：2319629