Identification and expression regulation analysis of E93 gene from Scylla paramamosian
Ecdysteroids(Ecds)and juvenile hormones(JHs)coordinate to regulate arthropods growth and development.In insects,E93 is a primary response gene to 20-hydroxyecdysone(20E),which mediates the 20E-induced autophagy and apoptotic signals to trigger metamorphosis during development,while JH inhibits E93 expression through JH signal to prevent precious metamorphosis.Crustacean has different development patterns and life cycles,especially during metamorphosis,whether E93 still plays a pivotal role in crustacean development still needs to be elucidated.Currently,rare information is known about crustacean E93.In this study,we identified E93 gene from Scylla paramamosian,named Sp-E93.Sp-E93 was located at the position 15 396 399 in the positive chain of pseudo-chromosome LG26,and its genomic structure contained six exons and five introns,with a total length of 100 679 bp.The mRNA length of Sp-E93 was 3 756 bp,containing a 2 982 bp open reading frame(ORF)encoding 993 amino acids with a predicted protein molecular mass of 106.34 ku.Two conserved helix-turn-helix(HTH)domains were found in Sp-E93,while LLQHLL was lost.Results from multiple sequence alignment revealed that E93 amino acids sequences were less consistent across species except two HTH domain regions.The phylogenetic tree indicated that the evolution of E93 was basically the same with species evolution,but many clades had a low node support,indicating that E93 might undergo evolution after species differentiation.Furthermore,the expression of Sp-E93 in different tissues of male and female adult crabs revealed that Sp-E93 had the highest expression in Y organ(YO)and mandibular organ(MO),and relatively low expression in midgut and muscle in both females and males,indicating that the expression of Sp-E93 might have some relationship with biosynthesis and secretion of Ecd and methyl farnesoate(MF),which were synthesized in YO and MO respectively.Significantly higher expression of Sp-E93 was found in males than females,this might be due to the different levels of hormone titers in different genders.In addition,we analyzed the regulation role of 20E,MF,and MF's analog methoprene on the expression of Sp-E93 using hepatopancreas culture.The results showed that MF and methoprene could significantly up-regulate E93 gene expression in hepatopancreas,and the regulation effect was higher with the increase of hormone concentration,and 10 μmol·L-1 MF could upregulate Sp-E93 expression 56 folds compared with the control.However,MF's analog methoprene could not significantly regulate Sp-E93 expression in the ovary of female crabs;similarly,20E was also able to regulate the expression of E93 gene in hepatopancreas,and 0.1 μmol·L-1 20E had the most remarkable regulatory effect,while 20E could also not significantly regulate Sp-E93 expression in the ovary,alone or with methoprene.In summary,these results indicate that the regulation of Sp-E93 in Scylla paramamosian might still function in the hormone signals,but the regulatory mechanism might have changed compared with insects.This study will promote our understanding of Ecd and JH regulatory pathways and provide further direction for the function studies of E93.
万方数据
维普
