- 學位論文
隱球菌 CRK1與 GAT1 共同調控有性生殖過程
Sexual differentiation is coordinately regulated by Cryptococcus neoformans CRK1 and GAT1
摘要
隱球菌（Cryptococcus neoformans），屬於擔子菌門，為異宗交配型真菌具有MATa與MAT兩種不同交配型。隱球菌存在兩種不同細胞形態，在營養充足環境下，以酵母細胞無性繁殖，而當氮素源缺乏誘導有性生殖時，兩性細胞融合可產生菌絲形態。隱球菌有性生殖過程中，包括酵母菌絲形態轉換、菌絲延長、擔子柄形成、減數分裂，及產生擔孢子等過程。隱球菌 CRK1 基因，為啤酒酵母菌（Saccharomyces cerevisiae）IME2 與玉米黑穗病菌（Ustilago maydis）crk1之同源基因，具有負向調控隱球菌雙性有性生殖之角色。本研究探討CRK1對隱球菌有性生殖及菌絲發育之影響、Crk1在細胞中的分布，及其在訊息網絡的交互作用。crk1突變株的有性生殖過程，其細胞融合效率及雙核生殖菌絲發育時程，皆顯著提高及加速，並且相較於野生株，其擔子柄（basidium）與擔孢子（basidiospore）的形成皆提早18小時；雖然突變株雙核菌絲的長度明顯的短於野生株，但相關構造皆完整存在。基因表現分析顯示，CRK1調控有性生殖各階段，如 ZNF2, DMC1與CSA1等基因的表現。而有性生殖表型分析結果顯示，NAT2與ZNF2基因的突變，皆造成crk1突變株無法形成雙核生殖菌絲。藉由基因體生物資訊的分析，預測轉錄因子Gat1與 Rgm1可能為 Crk1 下游的標的蛋白質。經由表型與遺傳分析結果顯示，Crk1與Gat1在有性生殖的過程具有重疊功能，形成一迴路調控Mat2，參與生殖菌絲發育進程及形態轉換的調控。總結，隱球菌Crk1為雙核生殖菌絲分化的調控因子，透過調控Gat1負向調控隱球菌的有性生殖。
並列摘要
Cryptococcus neoformans is a heterothallic basidiomycete which contains two mating types, MATa and MATα. C. neoformans grows vegetatively as yeast and filamentous hyphae are produced in the sexual state. Morphological progression of bisexual reproduction in C. neoformans is as follows: yeast to hyphal transition, filament extension, basidium formation, meiosis, and sporulation. C. neoformans Cdk-related kinase 1 (CRK1) is a negative regulator of bisexual mating, also a homologue of Saccharomyces cerevisiae IME2 and Ustilago maydis crk1. In this study, we characterized the effect of CRK1 on mating process and the morphology of dikaryotic filamentation, the localization of Crk1 in yeast cells and hyphal cells, and the genetic interaction in mating regulatory pathways. In the bilateral crk1 mutant cross, the timing of basidium formation was approximately 18 hrs earlier than wild-type cross. Despite shorter filament length than the wild-type, sexual filaments still retained intact structures in the bilateral crk1 mutant cross. Furthermore, gene expression analyses revealed that CRK1 modulated the genes involved in the progression of hyphal elongation, basidium formation, karyogamy and meiosis. Phenotypic results showed that although deletion of C. neoformans CRK1 gene increased the efficiency of bisexual mating, filamentation in the crk1 mutant was blocked the mutation of MAT2 or ZNF2. Moreover, a bioinformatic survey predicted the C. neoformans GATA type transcriptional factor Gat1 and zinc finger transcriptional factor Rgm1 as potential substrates of Crk1 kinase. Our phenotypic and genetic findings suggest that C. neoformans Crk1 and Gat1 shared overlapping functions to form a regulatory circuit which negatively regulated Mat2 to control filamentation progression and transition during bisexual mating. C. neoformans CRK1 functions as a regulator to maintain dikaryotic filament differentiation and coordinates with GAT1, as a negative regulator, to control bisexual mating.
參考文獻
延伸閱讀
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