Unraveling the mechanism of cryptic sexual cycle in Amoeba, Cochliopodium, using comparative transcriptome and immunocytochemistry

 描述(申请人提供)：利用比较转录组和免疫细胞化学，解开隐匿性周期发育的机制。这项拟议的工作旨在解开隐匿性生命周期的细胞学和遗传学基础。有性繁殖在真核生物中是非常有益的，因为它使它们能够增加：遗传多样性，在选择压力下的适应性和毒性，就像致病的真核生物一样。然而，大多数微生物真核生物的有性行为却知之甚少。这主要是因为它们的质量不同，生命周期复杂。与大多数其他阿米巴虫不同的是，包括溶组织阿米巴在内的大多数阿米巴虫被认为在休眠的包囊阶段(对实验研究来说是一个挑战)进行性行为，而Cochliopodium在活跃生长的营养阶段进行核融合。这使得它成为涉及现场实验的调查工作的理想候选者。这项拟议的工作将检验三个相互关联的假说，包括性发育的遗传机制、基因组缩减和遗传重组的机制以及Cochliopodium的交配系统。我们将结合多种方法，包括使用下一代测序获得的比较转录组，使用免疫细胞化学(ICC)获得的靶向性基因的细胞学证据，以及一系列培养实验来了解Cochlipodium的交配系统。我们已经建立了生物信息学管道和定制脚本来汇编和分析基因数据。新获得的从头数据统计工具将用于比较基因表达分析。解决提出的假说将有助于：(A)揭示Cochlipodium有性发育阶段的细胞学特征；(B)揭示Cochlipodium和其他生物中可能存在的隐蔽性生活周期的分子机制；(C)了解隐蔽生活周期中基因组减少和基因重组的机制；(D)探索可能用于对抗真核病原体的靶标；以及(E)深入了解微生物真核生物中性行为的起源和进化。

 DESCRIPTION (provided by applicant): Unraveling the Mechanism of Cryptic Sexual Cycle Development in Amoeba, Cochliopodium, using Comparative Transcriptome and Immunocytochemistry The proposed work aims to unravel the cytological and genetic underlying of the cryptic sexual life cycle of an amoeboid lineage, Cochliopodium. Sexual reproduction is highly beneficial in eukaryotes by enabling them to increase: genetic diversity, fitness under selective pressure and virulence as in pathogenic eukaryotes. However, sexuality in most microbial eukaryotes is poorly understood. This is mainly due to their diverse quality and complex life cycle. Unlike most other amoebae including E. histolytica that are assumed to engage in sex during the dormant, cyst stage (a challenge for experimental study), Cochliopodium undergoes nuclear fusion in the actively growing vegetative stage. This makes it an ideal candidate for investigative work involving live experimentation. The proposed work will test three interrelated hypotheses including genetic mechanisms of sexual development, mechanisms of genome reduction and genetic recombination and mating system in Cochliopodium. We will use a combination of methods including comparative transcriptome obtained using next generation sequencing, cytological evidence on targeted sex genes using immunocytochemistry (ICC), and a series of culturing experimentation to understand mating system in Cochliopodium. We have already built bioinformatics pipeline and custom scripts to assemble and analyze the genetic data. Newly available statistical tools for de novo data will be used for comparative gene expression analysis. Addressing the proposed hypotheses will enable: (a) uncover cytological signatures of the stages of sexual development in Cochliopodium, (b) shed light on the molecular mechanisms of a possible cryptic sexual life cycle in Cochliopodium and others, (c) understand mechanisms of genome reduction and genetic recombination in cryptic life cycles, (d) exploration of targets that might be used to combat eukaryotic pathogens, and (e) provide insights into the origin and evolution of sexuality in microbial eukaryotes.