Identification And Physiological Function Of Molting Cascade Signaling Pathway Genes In Neocaridina Denticulata Sinensis

During the growth and development of crustaceans,molting is the hallmark feature,which is regulated by both the nervous system and endocrine system.The molting cascade signaling pathway is inextricably linked to the growth,metamorphosis,molting and reproduction of crustaceans.Methylenefarnesoate(MF),an essential endocrine hormone,plays a critical role in the crustacean molting cascade signaling pathway,regulating molting,growth,development,and reproduction.In this study,the key genes of MF biosynthesis were screened and cloned based on the genomic and transcriptomic information of Neocaridina denticulata sinensis.The experiments and analyses were carried out at the gene,protein,and individual phenotype.RNA interference(RNAi)technology was used to investigate further the role and molecular regulation of the molting cascade pathway and its impact on the metabolic network by combining differential expression profiling and metabolic pathway analysis.This study is vital for elucidating molecular mechanisms in the complex molting cascade signaling pathway.It provides new ideas to solve the problems of excessive molting frequency and early sexual maturation in crustacean breeding.1.Cloning and identification of key genes of MF biosynthesis pathwayFour key genes in the MF synthesis pathway were screened based on the transcriptomic and genomic information from the N.denticulata sinensis,and the candidate sequences were initially confirmed in the NCBI Nr database.3-hydroxy-3-methyl glutaryl coenzyme A reductase(HMGR),farnesyl pyrophosphate synthase(FPPS),farnesoic acid Omethyltransferase(FAMe T),Juvenile hormone acid O-methyltransferase(JHAMT)were cloned from N.denticulata sinensis for the first time,and named Nd HMGR,Nd FPPS,Nd FAMe T,Nd JHAMT,respectively.Sequence analysis and tissue expression identification of these four key genes were also performed.2.Protein analysis and RNA interference of farnesoic acid O-methyltransferase in N.denticulata sinensisFarnesoic acid O-methyltransferase(FAMeT)catalyzes the generation of FA into MF and is the key rate-limiting enzyme in the MF biosynthesis process.Nd FAMe T recombinant expression plasmid was constructed,and the recombinant protein was obtained by expressing it in the prokaryotic expression system.The protein was purified,detected by SDS-PAGE,and the enzyme activity was verified.To further investigate the function of Nd FAMe T,the knockdown efficiency and enzyme activity were examined after injection of ds Nd FAMe T using RNA interference technology,and the results showed that RNA interference reduced the relative expression of Nd FAMe T by more than 90% and enzyme activity by 13%.After staging the molting cycle of N.denticulata sinensis,microscopic observation of the shrimp after RNA interference showed that the knockdown of Nd FAMe T did not prevent the molting of N.denticulata sinensis.3.Ovarian transcriptome and metabolic responses of RNAi-mediated farnesyl pyrophosphate synthase knockdown in N.denticulata sinensisTo further investigate the function of Nd FPPS,its knockdown efficiency was examined after injection of ds Nd FPPS using RNA interference technology.Ovarian tissues with the highest Nd FPPS expression were selected for transcriptome sequencing analysis.The differential expression genes(DEGs)of the experimental group(E1,E2,E3)and the control group(C1,C2,C3)injected with ds Nd FPPS were compared,annotated and enriched by ovarian transcriptome analysis.Six libraries were constructed from DNA,and their sizes were E1: 7.23 G,E2: 6.41 G,E3: 6.89 G,C1: 6.95 G,C2: 6.47 G,C3: 6.99 G.RNA-seq generated 281,427,802 raw reads,and after quality control,272,902,124 clean reads and 40.94 G clean bases were left,with base Q30(value of Phred >30)values >92%.A total of 9,230 differential genes was identified in the experimental and control groups based on FPKM values,of which 5,082 were up-regulated genes and 4,148 were down-regulated genes.A total of 761 GO terms were enriched for the differential genes.The number of down-regulated genes was significantly higher in the 30 GO terms significantly enriched for the differential genes.A total of 102 KEGG pathways were enriched,including four molting-related pathways and four ovarian development-related pathways.These pathways were included in the 20 most significantly enriched KEGG pathways,demonstrating that Nd FPPS knockdown affected the molting,ovarian development,and reproduction processes in N.denticulata sinensis.