【作者】 周舟；

【导师】 陈江野；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2002， 博士

【摘要】 白色念珠菌是一种重要的人体致病真菌，致病机制与其形态发生紧密相关。白色念珠菌在不同的生长条件下能发生显著的形态变化，这种变化由多种调控因子与信号转导途径所调控。酿酒酵母的G1 期细胞周期蛋白Cln1 和Cln2 参与了其形态发生，cln1/cln1 cln2/cln2 双缺失株不能形成菌丝。把白色念珠菌基因组文库导入cln1/cln1 cln2/cln2 缺失株，筛选能校正菌丝形成缺陷的基因，分离得到白色念珠菌中的CaBEM1 基因。从核苷酸序列推导，CaBEM1 编码一个632个氨基酸的蛋白，氨基酸序列分析表明在其N 端有两个SH3 结构域，中部有一个PX结构域，C端有一个PB1结构域；CaBEM1的氨基酸序列与酿酒酵母的Bem1同源性达38%， 与裂殖酵母的Scd2同源性达32％。 在酿酒酵母的缺失株中异源表达CaBEM1， 能够部分校正它们在氮源缺乏条件下的菌丝形成缺陷。这种菌丝形成的校正作用绕过MAPK途径和cAMP/PKA途径，表明CaBem1在菌丝形成中的作用可能位于这两条信号传导途径的下游。酿酒酵母Flo8 因子在其形态发生中起重要作用，我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中，筛选能够互补flo8侵入生长缺陷的基因，分离到了14个新基因，包括CaSRB9， CZF2和CaFLO11等基因。CaSRB9 基因编码区全长4998 bp， 编码一个1665 个氨基酸的蛋白质。该基因产物与酿酒酵母SRB9基因编码的产物具有最高的同源性，因此命名为CaSRB9（C. albicans SRB9）。 在双倍体酿酒酵母中异源表达CaSRB9可以部分校正MAPK途径基因缺失株以及flo8 缺失株的菌丝生长缺陷；在单倍体酿酒酵母中表达能够互补flo8 缺失株的侵入生长缺陷，但在MAPK 途径基因缺失株中不能形成侵入生长。CZF2 Candida albicans zinc finger protein 基因的编码区为1875bp ，由序列<WP=4>推断编码一种624个氨基酸的蛋白Czf2， 属于GAL4真菌转录调控蛋白家族。Czf2的N端有一个保守的Zn2Cys6锌指结构域，这一结构域中有六个半胱氨酸残基，结合两个锌离子形成结合DNA 的锌簇（zinc cluster）。 在双倍体酿酒酵母中异源表达CZF2 可以部分校正MAPK 途径基因缺失株以及flo8 缺失株的菌丝生长缺陷；在单倍体酿酒酵母中异源表达Czf2能够校正MAPK途径基因缺失株以及flo8缺失株的侵入生长缺陷。用同源重组法敲除了白色念珠菌CZF2基因。czf2/czf2缺失株在一些固体培养基中有轻微的菌丝形成缺陷。在YPD 中培养时形成伸长的细胞。表明CZF2 基因的缺失对其形态发生有一定影响。以小鼠系统感染模型研究发现，czf2/czf2缺失株的毒性比野生型略有下降。CaFLO11 （C. albicans FLO11） 基因编码区为1962bp， 由序列推断编码一个653 个氨基酸的蛋白质。从氨基酸序列分析预测CaFlo11 是一个细胞壁蛋白，其N端有信号肽序列，C末端具有GPI结构域，中部含有多个由丝氨酸和苏氨酸组成的串联重复序列。在双倍体酿酒酵母中异源表达CaFLO11 可以强烈地激活菌丝生长，这种激活绕过MAPK 途径以及cAMP/PKA 途径。在单倍体酿酒酵母中表达能够使MAPK 途径基因缺失株以及flo8 缺失株形成很强的侵入生长。用同源重组法敲除白色念珠菌CaFLO11 基因，caflo11/caflo11 缺失株没有明显的菌丝形成缺陷，但在某些液体培养基中形成的菌丝较为粗短。以小鼠系统感染模型研究发现，CaFLO11的缺失对白色念珠菌的毒性几乎没有影响。酿酒酵母MAPK 途径成员缺失株biofilm 形成能力降低。外源表达CZF2 和CaFLO11 能不同程度地校正这些缺失株的biofilm 形成缺陷，但不能校正flo8 缺失株的biofilm 形成缺陷。CaFLO11 能轻微地校正flo11 缺失株的biofilm 形成缺陷。更多还原

【Abstract】 Candida albicans is the most frequently isolated fungal pathogen in humans. Thepathogenic fungus Candida albicans has a dimorphic transition in various environmentalconditions. Many regulatory factors and several transduction pathways have beenidentified in controlling filamentous growth. G1 cyclins Cln1 and Cln2 have been reportedas involved in the control of morphogenesis in Saccharomyces cerevisiae. Diploidcln1/cln1 cln2/cln2 strains completely lost the ability to form pseudohyphae. A C.albicans genomic DNA library was introduced into cln1/cln1 cln2/cln2 mutant to screengenes which could complement its filamentous growth defect. In this screening, a BEM1homolog, CaBEM1, was identified. The CaBEM1 gene has an ORF of 1899 bp, encodinga putative protein of 632 amino acids. The CaBem1 protein shares highest homology inamino acids with Bem1(38%) of S. cerevisiae and Scd2(32%) of Schizosaccharomycespombe. Sequence analysis showed that the CaBem1 contains two N-terminal SH3domains, a PX domain and a C-terminal PB1 domain. It is believed these domains arerequired for binding to proteins involved in polarized growth in S. cerevisiae and S. pombe.Ectopic expression of the CaBEM1 gene in diploid S. cerevisiae suppressed defects infilamentous growth of some mutants under nitrogen starvation conditions. Thissuppression bypassed MAPK pathway and cAMP/PKA pathway in filamentous growth.These results suggest that the CaBem1 protein may be a downstream component of thesetwo signal transduction pathways for filaments formation.Flo8 plays an important role in morphogenesis of Saccharomyces cerevisiae. In this<WP=6>work, a C. albicans genomic DNA library was introduced into an S. cerevisiae flo8/flo8mutant to screen genes which could complement its invasive growth defect. In thisscreening, 14 novel genes were isolated. One gene shares highest similarity in amino acids(38%) with Srb9 of S. cerevisiae and designated CaSRB9 (Candida albicans SRB9 gene).The deduced translation production of another gene is belong to a GAL4 fugaltranscription regulatory protein family, which was named CZF2 ( Candida albicans zincfinger protein). The third gene encoding a cell wall protein was designated CaFLO11.The CaSRB9 gene has an ORF of 4998 bp, encoding a predicted protein of 1665amino acids. Ectopic expression of the CaSRB9 gene in diploid S. cerevisiae suppresseddefect in filamentous growth of some mutants in filamentation MAPK pathway (ste7/ste7,ste12/ste12, and tec1/tec1) and flo8/flo8 mutant under nitrogen starvation conditions. Inhaploid S. cerevisiae, ectopic expressed CaSrb9 complemented the invasive growth defectof flo8 mutant but failed to complement the invasive growth defects of the mutants infilamentation MAPK pathway.The CZF2 gene has an ORF of 1875 bp, encoding a deduced protein of 624 aminoacids. Czf2 has a N-terminal conserved Zn2Cys6 zinc cluster motif. The region forms abinuclear zinc cluster, in which two Zn atoms are bound by six Cys residues . Ectopicexpression of the CZF2 gene in diploid S. cerevisiae partially suppressed defects infilamentous growth of the mutants in filamentation MAPK pathway and flo8/flo8 mutantunder nitrogen starvation conditions. In haploid S. cerevisiae, ectopic expressed Czf2complemented the invasive growth defects of the mutants in filamentation MAPKpathway and flo8 mutant. The deletion of CZF2 gene in C. albicans caused slightly defectin hyphal formation. czf2/czf2 mutant strain grown in YPD medium formed elongatedcells. These results suggested CZF2 contributed to C. albicans morphogenesis. In asystemic mouse model, czf2/czf2 mutant strains showed reduced virulence comparingwith wild type strains..The CaFLO11 gene has an ORF of 1962 bp, encoding a hypothetic protein of 653amino acids. CaFlo11 is similar in overall structure to the class of yeast serine/threoninerich GPI-anchored cell wall proteins. An N-terminal domain containing a signal sequence<WP=更多还原