为了探索miRNA对日本沼虾几丁质酶3A(Macrobrachium nipponense chitinase 3A, MnCHT3A)基因的调控作用，实验采用生物信息学方法预测并筛选与MnCHT3A靶向结合的miRNA—miR-305-5p；利用qRT-PCR、生物化学以及组织学方法，研究了在体条件下，miR-305-5p对靶基因的调控作用。结果显示，一个蜕皮周期中miR-305-5p与MnCHT3A的表达量呈负相关，前者C期最高、A期最低；而MnCHT3A的表达趋势则相反。注射miR-305-5p mimics和miR-305-5p inhibitor后，与对照组相比，MnCHT3A转录水平分别降低60%和升高166%；MnCHT酶活性分别降低39.53%和升高133%。组织学观察发现，C期的头胸甲表皮为3层结构，由外向内依次为上表皮、外表皮和内表皮(H.E染色)；几丁质荧光染色结果显示，几丁质分布在内、外表皮；扫描电镜结果清晰显示了内、外表皮的板层结构。与对照组相比，miR-305-5p mimics组内表皮的板层厚度增加，呈蓝色荧光的几丁质条带有增厚趋势；而miR-305-5p inhibitor组表皮结构紊乱，几丁质荧光分布不均且部分区域明显减弱。研究表明，miR-305-5p的靶基因为MnCHT3A，在体条件下，miR-305-5p能够靶向抑制MnCHT3A的转录。

In order to explore the regulatory effect of microRNA (miRNA) on Macrobrachium nipponense chitinase 3A(MnCHT3A) gene, bioinformatics approach was firstly used to predict and screen the miRNA--miR-305-5p bound specifically to MnCHT3A. Using qRT-PCR, biochemical and histological methods, the regulation of miR-305-5p on target gene MnCHT3A was studied in vivo. The results showed that the expression change of miR-305-5p was negatively correlated with MnCHT3A during the molting cycle of M. nipponense. The level of miR-305-5p peaked at stage C and was the lowest at stage A, while the expression trend of MnCHT3A mRNA was opposite. After injection of miR-305-5p mimics or miR-305-5p inhibitor, the transcription level of MnCHT3A was decreased by 60% or increased by 166%, and the activity of MnCHT enzyme, meanwhile, was decreased by 39.53% or increased by 133%, respectively compared with the control group. Histological results showed that a three-layer cuticle structure of carapace in stage C was observed by means of H-E staining, namely, epicuticle, exocuticle and endocuticle from outside to inside. The images of chitin fluorescence staining showed the presence of chitin in the exocuticle and endocuticle. Results from scanning electron microscopy clearly showed the lamellar structure of the exocuticle and endocuticle. Compared with the control group, a thickening trend in the endocuticle with the lamellae as well as the blue fluorescence chitin stripe was observed in miR-305-5p mimics group. But in miR-305-5p inhibitor group, the culticular structure was disordered. Correspondingly, the blue fluorescence chitin stripe was not uniform and weakened in some areas. The results obtained above indicate that the target gene of miR-305-5p is MnCHT3A, and miR-305-5p can specifically inhibit the transcription of MnCHT3A in vivo.

Q786；S917.4

河南省自然科学基金(182300410033)