Regulation Mechanism Of The Mirnas Targeting Key Genes Involved In Cuticle Chitin Hydrolysis In Macrobrachium Nipponense

The crustacean epidermis is the first barrier against the complex external environment.In order to adapt to changes in the environment and meet individual growth needs,crustaceans must periodically shed old skin and synthesize new skin at different stages of development.Macrobrachium nipponense N-acetyl-β-D-glucosaminidase（MnNAGase）and M.nipponense chitinase 3A（MnCHT3A）are povotal in the degradation process of chitin.However,its post-transcription level is less studied.Micro RNA（mi RNA）has been shown to suppress gene expression at the post-transcriptional level and plays an important role in the normal growth and development of animals.Previous studies have obtained the full-length gene and completed Small RNA deep sequencing.In order to explore the regulatory effect of mi RNA to MnNAGase and MnCHT3A,we first screen the mi RNA specifically expressed in the epidermis and use bioinformatics methods to predict the binding site of mi RNA.Then,q RT-PCR is used to analysis of the expression changes during the molting cycle.Thirdly,the dual luciferase assay analyses that mi RNA can directly interacted with MnNAGase and MnCHT3A in vitro.In vivo microinjection experiment analysis reciprocity of the candidate mi RNA with its target genes MnNAGase and MnCHT3A.The results are as follows:The bioinformatics prediction showed mi R-3049-5p and mi R-305-5p have binding sites in MnNAGase and MnCHT3A.The peak expression of MnNAGase is in D_0-2stage and the peak expression of mi R-3049-5p is in C stage.MnCHT3A has the highest expression level in A stage and the expression level of is highest in D_0-2stage.The expressions of mi R-3049-5p and mi R-305-5p are negatively correlated with the expression of MnNAGase and MnCHT3A.It is speculated that mi R-3049-5p and mi R-305-5p may negatively regulate the expression of Chitin degradation related genes MnNAGase and MnCHT3A.The dual luciferase assay show that mi R-3049-5p mimic binds to the 3’UTR of MnNAGase.Result in reducing the expression of luciferase gene.mi R-3049-5p inhibitor can inhibit mi RNA.It will result in increased the expression of luciferase gene.It indicates that in vitro experiments,mi R-3049-5p has an inhibitory effect on the expression of the corresponding target gene MnNAGase.After the binding site is mutated,the inhibitory effect is eliminated.Microinject mi RNA mimic or mi RNA inhibitor into M.nipponense to overexpress or inhibit the expression of corresponding mi RNA,and further verify the regulatory effect of mi R-3049-5p/mi R-305-5p on the target gene MnNAGase/MnCHT3A.The results show that mi R-3049-5p and mi R-305-5p have similar experimental results:after injection of mi RNA mimic,the expression of target genes is significantly or extremely significantly reduced,while injection of mi RNA inhibitor,the expression level of the target gene increased significantly.The change trend of related enzyme activities is basically consistent with the target gene.HE staining results show that the epidermis of the cephalothorax in C stage is divided into three layers:epicuticle,exocuticle,and endocuticle,from the outer to the inner layer.The epicuticle is the thinnest and darker stained upper epidermis.The exocuticle is more strongly basophilic and tightly arranged,while the endocuticle is weakly eosinophilic and loosely arranged.After injection of mi RNA mimic to overexpressed mi RNA,the thickness of exocuticle increased significantly,while the thickness of endocuticle remained relatively stable.After injection of mi RNA inhibitor to inhibit the expression of mi RNA,the epidermal structure was broken,but there was no obvious change in epidermal thickness.Fluorescence staining of chitin showed that the upper epidermis showed red because it did not contain chitin.The outer and inner epidermis showed blue and showed a vaguely lamellar structure,indicating that both were made of chitin-protein microfibers.The results has been constructed.But the inner skin is stained darker,indicating that the inner skin contains more chitin.After injection of mi RNA mimic or mi RNA inhibitor,the changes in epidermal structure are consistent with HE staining results.So we indicate that if the expression of mi R-3049-5p and mi R-305-5p have increases or inhibits,it will affect the degradation process of chitin,causing changes in the epidermal structure.Even the normal molting process of M.nipponense will be affected.In conclusion,mi R-3049-5p and mi R-305-5p and their target genes MnNAGase and MnCHT3A do play a key role in the hydrolysis of chitin in M.nipponense.To sum up,this study found that mi R-3049-5p and mi R-305-5p regulated MnNAGase and MnCHT3A by bioinformatics analysis,molting cycle expression analysis,in vitro dual luciferase experiment and in vivo injection experiment.The molting process of M.nipponense are regulated by controlling the degradation of chitin at the post-transcription level.This study also provides an important scientific basis for revealing the molecular mechanism of mi RNA-mediated post-transcriptional regulation of chitin hydrolysis mechanism.