Molecular cloning and expression analysis of StARgene from oriental river pawn(Macrobrachium nipponense)in response to hypoxia
Steroid acute regulatory protein(StAR)is an important enzyme in the early stage of steroid hormone synthesis.Recent studies have found that aquatic hypoxia can influence synthesis pathway of steroid hormones in fish as an endocrine disruptor,however,little information was observed in crustaceans.In order to investigate the roles of StAR in the oriental river prawn Macrobrachium nipponense under hypoxia,StAR gene has been cloned and characterized.A full-length cDNA sequence of StAR from M.nipponense was cloned by RACE(rapid ampli-fication of cDNA ends)PCR.We examined the expression levels of StAR gene in different tissues by semi-quantit-ative RT-PCR,at the same time,the expression profile of StAR gene transcription level and protein expression abundance in testis of M.nipponense under hypoxia were also examined by quantitative real-time PCR(qRT-PCR)and Western blot,respectively.The results showed that the cDNA of StAR gene was 3 361 bp in length(NCBI:MW263908),including 323 bp 5′-untranslated regions(UTR),2 129 bp 3′-UTR,909 bp open reading frame(ORF),which encodes 302 amino acids.Based on bioinformatics analysis,phylogenetic tree analysis showed that the StAR gene of M.nipponense was closely related to the StAR gene of Portunus trituberculatus.The semi-quantitative RT-PCR results showed that StAR was highly expressed in testis tissues,but the lowest expressed in hepatopancreas and heart tissues.qRT-PCR results showed that the mRNA expression of StAR in the testis from hypoxia 1-48 h was significantly higher than those in control group,but there was a significant lower mRNA expression of StAR in hypoxia 96 h group than those in control group.In addition,the recombinant plasmid was constructed and the StAR polyclonal antibody was obtained by immunizing rabbit.The expression abundance of StAR protein was detected by Western blot,which was basically similar to the gene expression pattern.In addition,immunohistochemical results showed that StAR protein was expressed in sperm cells and Leydig cell in testis tis-sue.Acute hypoxic stress significantly inhibited expression level of StAR,which may play an important role in spermatogenesis.The results of this study lay a foundation for further analysis of the molecular mechanism of ster-oid hormone synthesis in M.nipponense.
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