Preliminary Study On Molecular Mechanism Of Functional Genes Relate To Ecdysone Signaling Pathway In Molting Of Litopenaeus Vannamei

Molting is an important physiological process in the whole life cycle of Litopenaeus vannamei,which is influenced by external environmental factors and regulated by the nervous system and endocrine system.Molting is a process of cascade regulation of up and down genes in the ecdysone signaling pathway with ecdysone synthesis as the core.In insects,the upstream neuropeptides of the CHH family inhibit the synthesis of ecdysone,while the downstream genes of the ecdysone signaling pathway,eclosion hormone（EH）and ecdysis-triggering hormone（ETH）can trigger the occurrence of molting behavior under the regulation of ecdysone.Compared with Arthropoda Insecta,the study of each gene of molting signaling pathway in crustaceans is not comprehensive.Most of them are borrowed from Insects,especially the functional genes related to ecdysone signaling pathway in the molting process of shrimp need to be further explored.In this study,Prokaryotic expression system was used to prepare recombinant proteins in vitro,RNAi technology and compared and analyzed the transcriptome data and studied CHH,EH and ETH of Litopenaeus vannamei.The effect of CHH on ecdysone synthesis was studied by in vivo injection and in vitro tissue incubation experiments,and the participation of EH and ETH in shrimp molting was regulated by ecdysone,and the synergistic participation of ETH in shrimp molting regulation through its receptor was confirmed.The results of the study revolve around the important element of ecdysone,to elucidate the molecular mechanism of key functional genes in the upstream and downstream of the ecdysone signaling pathway involved in the regulation of molting in Litopenaeus vannamei.The preliminary results of the study are as follows:1.LvCHH-L regulates molting of Litopenaeus vannamei by inhibiting ecdysone synthesis.Molecular cloning and bioinformatics analysis showed that LvCHH-L had the typical structural characteristics of CHH type I family neuropeptides.There was a CPRP structure between the mature peptide and the signal peptide,and there were six conserved cysteine residues in the mature peptide.The expression of LvCHH-L was mainly found in the eyestalk（P<0.05）,and the highest expression of LvCHH-L was found in the intermolt（P<0.05）,the transcription level gradually reduced along with the molting behavior coming.Short-term inhibition of LvCHH-L transcription significantly accelerated molting and increased molting rate（P<0.05）,while long-term inhibition of LvCHH-L transcription significantly increased body weight（P<0.05）and accelerated growth.Injection of ds LvCHH-L significantly increased the transcription levels of NVD,SAD and Ec R（P<0.05）.Interfering with the transcription of LvCHH-L increased the concentration of ecdysone in shrimp hemolymph and promoted the early molting of shrimp.The results showed that LvCHH-L inhibited the synthesis of ecdysone,which was involved in the regulation of molting of Litopenaeus vannamei.2.Comparative transcriptome analysis of Litopenaeus vannamei fed diets supplemented with ecdysoneComparative transcriptome analysis was performed on the hepatopancreas and intestine of Litopenaeus vannamei fed diets with different ecdysone levels（0,200,400,and 800mg/kg）for 2 months,and 24 high-quality databases ranging from 39361266 to 45214394were obtained.Through comparison of the Litopenaeus vannamei genome and prediction of new genes from the uncompared reads,32623 transcripts were obtained.The shortest one was 57 bp,and the longest one was 40511 bp.65140 transcripts were enriched in GO database,and 12394 transcripts were enriched in KEGG database.The results of differential gene analysis showed that the key genes EH,ETH,CHS,HR38,CHITINASE and RXR in the molting signaling pathway were significantly up-regulated in the 800 mg/kg hepatopancreas group（P<0.05）.Cytochrome P450 enzymes were significantly enriched in the KEGG differential pathway in the 800 mg/kg intestinal tissue experimental group.The expression of SREBP gene in INSIG-SREBP-SCAP signaling pathway was down-regulated with the increase of ecdysone content.From cholesterol regulatory protein binding element SREBP to cytochrome P450 enzymes and downstream genes of molting signaling pathway,there were significant differences in the expression of different ecdysone content feed groups.Through the analysis of transcriptome data in this study,the signaling pathways and genes that play a key role in molting growth of Litopenaeus vannamei regulated by ecdysone were excavated.3.LvEH involved in molting regulation of Litopenaeus vannamei.The LvEH gene was screened to be the key functional gene of ecdysone signaling pathway in Litopenaeus vannamei based on the transcriptome data.Bioinformatics analysis showed that the ORF of LvEH gene was 249 bp,encoding a signal peptide of 26 amino acids and a mature peptide of 56 amino acids,and the mature peptide contained six cysteine residues that could form intramolecular stable disulfide bonds.The expression level of LvEH gene was high in the gill,epidermis and brain in intermolt and during larval metamorphosis from nauplius to zoea,the expression level of LvEH gene was the lowest in the intermolt stage（P<0.05）,and gradually increased with the onset of molting.The recombinant protein r LvEH with a molecular weight of 26.8 k Da was successfully prepared by prokaryotic expression technology.Combined with RNAi technology,the regulation of LvEH on molting of shrimp was studied.In vitro incubation with ecdysone showed that increasing the concentration of ecdysone in a certain range significantly promoted the expression of LvEH（P<0.05）.Short-term interference of LvEH transcription significantly delayed the molting of shrimp（P<0.05）,but co-injection of ds LvEH and r LvEH had no significant effect on the molting of shrimp（P>0.05）.Injection of r LvEH accelerated the growth of shrimp,However,the growth of shrimp was significantly delayed by ds LvEH injection.LvEH significantly promoted the expression of LvETH,LvETHR,Lv Burs and LvCCAP（P<0.05）.The results showed that LvEH was regulated by ecdysone and affected the expression of downstream genes to regulate the molting of Litopenaeus vannamei.4.Involvement of LvETH and LvETHR in molting regulation of Litopenaeus vannameiLvETH,a functional gene of ecdysone signaling pathway,was screened from transcriptome data,and the ORF of LvETH gene was cloned,which was 405 bp in length and encoded 134 amino acids of LvETH.The first 19 amino acids formed a signal peptide.The LvETH gene was highly expressed in the muscle and eyestalk tissues during the intermolt period,and the LvETH transcript level was high in the N₆and N₅stages of nauplius,and was the lowest in the intermolt period（P<0.05）,gradually increase before molting.The results of molecular docking and yeast hybridization showed that LvETH and LvETHR could form a stable chemical bond,and yeast hybridization showed that they had a strong interaction,which interfered with the transcription of LvETH and significantly reduced the transcription level of LvETHR（P<0.05）.In vitro incubation experiments showed that LvETH was regulated by ecdysone,and combined injection experiments showed that injection of ds LvETH or ds LvETHR delayed molting,but synthetic peptide sys ETH could restore molting.Injection of ds LvETH delayed the growth of shrimp,while injection of sys ETH accelerated the growth of shrimp.The results showed that LvETH cooperated with LvETHR in molting regulation of Litopenaeus vannamei.