氮源等环境因子对皮状丝孢酵母形态转换的调控及其分子机制

The dimorphism fungi Trichosporon cutaneum was an important candidate for microbial oil production for its high oil content and can hydrolyze lignocellulose. Our previous study showed that nitrogen source can efficiently regulate the morphologic transition of T. cutaneum and it has become a key factor to affect the fermentation level. Therefore, probe the environmental factors and its regulatory mechanism for morphologic transition of T. cutaneum is significant no only to discover its morphologic transition pattern and enrich the morphologic transition theory of dimorphism fungi but also to improve the fermentation level. However, study on morphologic transition of T. cutaneum is rather insufficiency. In present study, we explored the environmental factors especially the nitrogen resource (amino acid) to regulate the morphologic transition of T. cutaneum in plate culture and chemostat culture, and on the basis of that we applied the technology of suppression subtractive hybridization (SSH) and expressed sequence tags analysis to compare the gene expression difference between the yeast and pseudohyphal forms of T. cutaneum cell. Additionally, the real-time quantitative PCR method was also used to check the differentially expressed gene so as to elucidate the molecular mechanism of morphologic transition of T. cutaneum under different environments.

皮状丝孢酵母（Trichosporon cutaneum）为二型态真菌，其含油量高并可利用木质纤维素，是微生物油脂生产的重要候选菌种。在前期研究中，我们发现氮源可有效调控T. cutaneum的形态，成为影响发酵水平的关键。因此开展T. cutaneum细胞形态转换的环境调控因子及其分子机制研究不仅能揭示其形态转换规律，丰富二型态真菌形态转换理论，而且对于提高二型态真菌的发酵水平具有重要意义。但迄今有关T. cutaneum形态转换未见研究。本项目以T. cutaneum为对象，采用平板结合反应器恒化培养，探寻影响其形态转换的环境因子，并着重探讨氮源（氨基酸）对其形态的调控过程，在此基础上，利用抑制消减杂交（SSH）技术并结合表达序列标签（ESTs）分析，比较其酵母型和菌丝型间的基因表达差异，同时利用实时定量PCR技术进行验证，以揭示不同条件下T. cutaneum形态转换的分子机制。

本课题以皮状丝孢酵母为对象，探寻影响其形态转换的环境因子，并着重开展氮源（氨基酸）对皮状丝孢酵母形态调控过程，在此基础上，利用转录组测序技术，比较两种细胞形态（酵母型和菌丝型）间的基因表达差异并用实时定量PCR加以验证，以揭示不同条件下皮状丝孢酵母形态转换的分子机制。课题组首先分析了不同环境因子对皮状丝孢酵母二型态转换的影响，发现氮源、碳源浓度对皮状丝孢酵母 B3形态转换至关重要，低浓度的氮源（≤1 g/L）、低碳源浓度（≤3 g/L）有利于菌体呈酵母型生长，高浓度氮源（≥5 g/L）或碳源（≥40 g/L）有利于诱导其菌丝形成；此外，pH、温度和溶氧等环境因子对皮状丝孢酵母 B3形态转换也有重要影响。在此基础上，课题组利用转录组测序技术，构建了全面而高质量的皮状丝孢酵母转录组数据库，筛选出与皮状丝孢酵母二型态转换相关的关键功能基因。结果表明，在低氮条件下，酵母细胞cAMP-PKA, MAPK途径的下游转录因子RGT1，NRG1，MIG1，TUP1以及SSN6基因显著上调，TOR途径中的TOR同源物（c14858_g1）和Tap42（c9323_g1）都显著提高，Rim13（c8226_g1）与Rim101（c1563_g1）在低氮条件下也是显著性地转录上调，这表明皮状丝孢酵母能通过cAMP-PKA, MAPK，TOR和Rim101等途径控制菌体成酵母型生长及油脂合成。研究结果不仅丰富了二型态真菌形态转换理论，而且为提高皮状丝孢酵母的微生物油脂生产水平奠定基础，同时也为其他二型态真菌的工业发酵调控提供有利的借鉴。