选择性白腐真菌Phlebia tremellosa降解木质纤维素的机制研究

As the most efficient and important degraders of lignin, white rot fungi play an increasingly important role in the carbon recycle in nature. Two main patterns of lignin biodegradation have been distinguished: selective degradation and simultaneous degradation. Compared with simultaneous white rot fungi, selective white rot fungi are exemplified by selectively and early degradation of lignin followed by less cellulose and hemicellulose degradation. Therefore, it’s of great value to carry out the research on selective white rot fungi. However, most of the researches have been carried out on the non-selective model white rot fungi Phanerochaete chrysosporium. Very limited data have been reported on selective white rot fungi. In this project, a selective white rot fungus Phlebia tremellosa is chosen as the research subject. Firstly, protoplast monokaryogenesis technique will be applied to obtain the monokaryon. Using the genomic DNA from high purity monokaryon, the whole genomic sequencing will be developed with high-throughput technology and then de novo assembled, and annotated. Comparative genomics study of the annotated genome of Ph. tremellosa, Ph. centrifuga, Ceriporiopsis subvermispora, and P. chrysosporium will be conducted to search the homologous proteins with the known functions, especially lignocellulolytic enzymes. And then, the transcriptome of Ph. tremellosa under different induction will be sequenced, and simultaneously, the lignocellulose degradation will be analyzed based on the protocols of NREL. The transcriptome analysis contains of genetic structure optimization, expression annotation, differential expression analysis, new transcript prediction and alternative splicing analysis. New genes related to induction and lignocellulose degradation are expected based on transcriptome analysis and the gene function will be verified via qRT-PCR. Finally, the molecular basis of lignocellulose degradation by the selective white rot fungi Ph. tremellosa will be comprehensively elaborated based on all the results. Results of the project will provide theoretical guidance and technical support for the scientific application of selective white rot fungi in practice.

选择性白腐真菌能较好地降解木质素和保留纤维素，应用价值大，但目前多以非选择性降解菌为研究对象，关于选择性白腐真菌的研究相对较少。本项目拟以一株选择性株菌Phlebia tremellosa为研究材料，在采用原生质体单核化技术获得其单核体的基础上，基于高通量测序技术对该菌株的基因组进行de novo测序，并对所得序列进行组装和基因组注释，进一步通过同属菌株及典型选择性菌株和非选择性菌株的比较基因组学研究，发现与已知功能（尤其是木质纤维素降解酶）的基因序列同源的蛋白；在此基础上，采用RNA-seq技术获得不同诱导条件下的转录组数据并测定其木质纤维素降解情况，分析转录组对不同诱导物的响应，比较诱导物引起的基因响应差异，以期找到已注释蛋白之外表达受诱导的“全新”基因并验证其功能；最终，综合阐述Ph. tremellosa降解木质素纤维素的分子基础，为选择性白腐真菌的科学应用提供理论指导和技术支撑。

本项目以一株选择性降解木质素的胶质射脉革菌Phlebia tremellosa BBEL0901为对象，在对其全基因组进行测序和解析的基础上，采用生物信息学方法挖掘和分析其降解木质纤维素的相关基因，并通过比较转录组学探究其降解生物质的差异表达基因。项目首先基于单因素实验研究了菌龄、酶解时间、溶壁酶浓度、转速、稳渗剂种类和浓度对原生质体产量的影响，结合正交试验设计优化了原生质体制备条件。在此基础上进行原生质体再生，采用细胞DAPI核荧光染色、ITS序列分析和Kmer曲线杂合度测定从多角度筛选获得了单核体M73。提取单核体M73的全基因组DNA，基于二代、三代相结合的测序技术进行de novo测序、组装与分析。全基因组最终产生了8.23G的原始数据，组装得到43个Scaffolds，测序深度达到133×。全基因组大小为40.7 Mb，GC碱基含量约为51.41%，共有12,362个编码基因、272个tRNA和18个rRNA。菌株BBEL0901虽然拥有较完整的纤维素、半纤维素和果胶的降解酶系，但纤维素酶基因的拷贝数相对偏低；具备丰富的木质素降解酶系，包括86个与木质素降解相关的基因，13个参与木质素降解的代谢通路，4个以上木质素及其中间体的代谢途径；拥有丰富的P450蛋白基因。在明确漆酶、锰过氧化物酶和木质素过氧化物酶基因的保守结构域、进化关系、启动子顺式元件和蛋白质结构的基础上，分析了各木质素降解酶基因的顺式作用元件结合位点。基于比较转录组学揭示了菌株BBEL0901对甘草渣的利用与碳水化合物代谢、戊糖与葡萄糖醛酸的相互转化、乙醛酸和二羧酸酯代谢、异生物质降解、芳香族氨基酸降解等通路密切相关，并挖掘出12个与木质纤维素降解相关的重要差异表达基因。.综上，胶质射脉革菌BBEL0901拥有完整的木质纤维素降解酶系，拥有较强的木质素降解酶系统和丰富的木质素代谢通路，选择性降解木质素的物质基础较好，生物质能够诱导木质纤维素降解及其中间体代谢的相关基因表达，并显著富集与能量代谢和木质纤维素降解有关的通路。该研究明确了胶质射脉革菌选择性降解木质素的物质基础，为解析木质素纤维素降解的分子机制奠定了基础。