拟盘多毛孢属（Pestalotiopsis）真菌是一类在热带和亚热带地区广泛分布的内生植物真菌，由于植物与内生菌群之间长期的相互协作进化，导致植物内生菌在代谢途径的多样性和复杂性方面具有独特的优势，因此它们能够产生一系列的结构新颖、功能独特的有机化合物。越来越多的证据表明，拟盘多毛孢属（Pestalotiopsis）是一个巨大的、大量未被开发的天然产物资源库，其化学结构经过进化优化，具有生物和生态相关性。到目前为止，该属已发现约300种次级代谢产物。

我们实验室分离得到一株真菌：小孢拟盘多毛孢P.microspora NK17，它能够产生紫杉醇（taxol）。此外，我们还发现了一种CETP家族蛋白抑制剂dibenzodioxocinone类化合物pestalotiollide B，它具有治疗心血管疾病的潜力。此前的研究中，我们发现当菌株NK17缺失一个编码丙氨酸tRNA的基因（tRNA^Ala_CGC1）时，突变株分生孢子及次级代谢产物的生成都受到了极大地抑制，并且对外界压力变得极为敏感。经深入研究发现，tRNA^Ala_CGC1在菌体的产孢和次级代谢过程中，通过裂解产生小RNA（tsRNAs）起到调节作用，此小RNA经测序鉴定为：5-tRF^Ala_CGC1。为了进一步研究其作用机制，分析RNA pull down实验数据，发现了一类能与5-tRF^Ala_CGC1结合的下游功能蛋白。

这类蛋白经过保守结构域预测和生物信息学分析确认为Septin蛋白，并与其它10个模式真菌的septin蛋白家族核心组分对比，鉴定出菌株NK17中四个Septin蛋白家族核心组分PmSep3（gme10087）、PmSep4（gme11531）、PmSep5（gme13112）和PmSep6（gme15866），这类蛋白结构在进化上具有保守性。借助基因功能缺陷突变体的构建，通过系统性地研究其与野生型的表型差异，我们期望从遗传学的角度阐明Septin蛋白家族对细胞的生命活动的调控机制。这类蛋白基因的缺失会减慢甚至限制NK17的营养生长，但是最终并不影响菌丝的生物量，但是突变株孢子数量和野生型孢子数量有显著性差异，次级代谢产物合成能力也明显下降，说明这类蛋白基因正向调控菌体的生长繁殖和次级代谢。除此之外，Septin蛋白在菌体生长中应对环境胁迫中也起到了重要的作用。

本研究首次在丝状真菌中发现了tRNA^Ala_CGC1来源的5-tRF通过与Septin蛋白特异性结合来调节丝状真菌的产孢和次级代谢产物的合成，推测可能是通过影响细胞的分裂导致一系列表型变化。这一研究结果进一步扩展了对于NK17真菌产孢和次级代谢调控机制的探究，尤其是tRNA^Ala_CGC1所产生的5-tRF对于该过程的调节作用。这些发现在一定程度上也为寻找新型药物提供了有益的启示和方向。

Pestalotiopsis is a kind of widely distributed endophytic fungi, particularly in regions with tropical and subtropical climates. Due to the long-term co-evolution between plant endophytes and their hosts, plant endophytes can produce a series of organic compounds with novel structures and unique functions due to the diversity and complexity of their metabolic pathways. More and more evidences show that Pestalotiopsis is a huge and largely untapped resource pool of natural products. Its chemical structure has been optimized by evolution and has biological and ecological relevance. So far, about 300 secondary metabolites have been found in this genus.

In our laboratory, we successfully isolated a fungus : P.microspora NK17, which can produce taxol. In addition, we also found a molecule pestalotiollide B similar to the CETP family protein inhibitor dibenzodioxocinone, which has the potential to treat cardiovascular diseases. In previous studies, we found that when the strain NK17 lacked a gene encoding alanine tRNA ( tRNA^Ala_CGC1 ), the production of conidia and secondary metabolites of the mutant strain was greatly inhibited and became extremely sensitive to external pressure.Further investigation revealed that tRNA^Ala_CGC1 plays a regulatory role in sporulation and secondary metabolism through the production of small RNAs ( tsRNAs ) by division. The tRNA-derived small RNA was identified by sequencing as 5-tRF^Ala_CGC1.In order to further explore its mechanism of action, we used RNA pull down experiments to find downstream functional proteins that can bind to 5-tRF^Ala_CGC1.

These proteins were identified as Septin proteins by conservative domain prediction and bioinformatics analysis. Compared with the core components of the septin protein family of other 10 model fungi, four core components of the septin protein family in strain NK17 were identified: PmSep3 ( gme10087 ), PmSep4 ( gme11531 ), PmSep5 ( gme13112 ) and PmSep6 ( gme15866 ). The structure of these proteins is evolutionarily conserved. At the same time, the gene function defect mutants were constructed, and the phenotypic differences between the mutants and the wild type were compared and analyzed to determine the biological function of the Septin protein family genes. The deletion of these protein genes will slow down or even limit the vegetative growth of NK17, but ultimately does not affect the mycelial biomass, but the number of spores of the mutant strain is significantly different from that of the wild type, and the synthesis ability of secondary metabolites is also significantly reduced, indicating that these protein genes positively regulate the growth and reproduction of bacteria and secondary metabolism. In addition, Septin protein also plays an important role in the growth of bacteria in response to environmental stress.

For the first time, it was found that 5-tRF derived from tRNA^Ala_CGC1 regulates the sporulation and secondary metabolite synthesis of filamentous fungi by specifically binding to Septin protein, which may lead to a series of phenotypic changes by affecting cell division. The results of this study further expanded the exploration of the regulation mechanism of sporulation and secondary metabolism of NK17 fungi, especially the regulation of 5-tRF produced by tRNA^Ala_CGC1 on this process.These findings also provide useful inspiration and direction for finding new drugs to a certain extent.