tRNA来源的小RNA（tsRNA）因其独特的结构和功能而广受关注。然而，关于tsRNA的大多数研究集中在动物细胞和植物细胞中，在细菌、酵母细胞中也有少量涉及，唯独缺少在丝状真菌中的探索。本实验室前期研究发现产紫杉醇真菌小孢拟盘多毛孢Pestalotiopsis microspora NK17中大量存在tsRNA，且来源于tRNAAlaCGC1的tsRNA具有调控菌株孢子形成和次级代谢产物合成的功能，但其形成机制尚未可知。为了深入探究丝状真菌tsRNA形成机制，本研究参考动物、植物细胞中tsRNA形成机制研究成果，针对NK17中Dicer蛋白及RNase T2蛋白进行功能探索，并得出以下结论：
1) 与拟南芥中报道结果一致，Dicer蛋白缺失对NK17中tsRNA产生无影响，说明Dicer蛋白不参与tsRNA的形成；
2) NK17中一共存在3个与拟南芥RNSs蛋白同源性较高的蛋白PmRNS1，PmRNS2和PmRNS3，且PmRNS蛋白对维持NK17的生长是必须的，但是激活任意一个Pmrns基因的表达均为维持菌体的生长；
3) 与拟南芥中RNS蛋白不同的是，PmRNS1剪切tRNA关键活性位点不是CAS I和CAS II的组氨酸位点，该位点的突变不能导致酶活性失活，因此PmRNSs剪切tRNA机制还有待更进一步的研究；
4) 模式真菌构巢曲霉中存在唯一RNase T2蛋白AnRNS，该蛋白能够在NK17中异源表达完全替代PmRNSs的功能；
5) 构巢曲霉中也存在大量tsRNA，且AnRNS同样也参与了这些tsRNA的形成；
6) NK17中激活不同的RNase T2蛋白时，黑色素形成和次级代谢产物形成通路基因表达有明显的差异，主要次级代谢产物dibenzodioxocinone类化合物产量也有显著变化，说明NK17中tsRNA具有调控功能的。

tRNA-derived small RNA (tsRNA) is well known for its specific strunture and function in cells. However, the majority of research on tsRNA was in mammalian or plant cells, while the biogenesis of tsRNAs in filamentous fungi has rarely been studied. Previously studies showed that tsRNA derived from tRNAAlaCGC1 regulates the sporulation and biosynthesis of metabolites in taxol producing fungus Pestalotiopsis microspora NK17. Nevertheless, the biogenesis of tsRNA in NK17 was unknown. Thus, to investigate the biogenesis of tsRNA in filamentous fungi, in this study, we focused on the Dicer or RNase T2 proteins which were reported to be the main player in biogenesis of tsRNA in mammalian or plant cells. As a result, we got the following conclusions:
1) The deletion of Dicer proteins had no effect on the production of tsRNA in NK17, indicating that Dicer proteins were not involved in the formation of tsRNA, thich is consisted with the results in Arabidopsis;
2) There are three RNase T2 proteins, PmRNS1, PmRNS2 and PmRNS3, sharing high homology with RNS proteins in Arabidopsis. Moreover, activating the expression of any PmRNSs could maintain the growth of NK17;
3) Different from the RNSs in Arabidopsis, Cas I and Cas II are not the key sites to catalytic the reaction of tRNA cleavage. Therefore, the mechanism of tRNA cleavage by RNase T2 in NK17 remains unknown;
4) There is only one RNase T2 protein, AnRNS, in Aspergillus nidulans RJMP1.59. And AnRNS could completely replace the function of PmRNSs in NK17;
5) sRNA-sequencing results showed there are a large number of tsRNA in A. nidulans. And AnRNS is the major player of tsRNA biogenesis in A. nidulans;
6) When different RNase T2 proteins are activated in NK17, there are significant differences in the expression levels of genes in melanin formation and secondary metabolic pathway. The yield of dibenzodioxocinone compounds also changed, indicating that tsRNA acts as metabolic regulator in NK17.