Cloning and expression of UBE2H gene in Penaeus monodon

ObjectiveThe present study was conducted to clone ubiquitin-conjugating enzyme E2 H gene(UBE2H) of Penaeus monodon and explore its tissue specific expression, in order to provide references for revealing ovary develop-ment of P. monodon. MethodOpen reading frame of UBE2H gene in P. monodon was amplified by PCR, and UBE2H gene expressions in different tissues, and in hepatopancreas and ovary of P. monodon at different ovary developmental stages were detected using real-time quantitative PCR. pET32a-UBE2H prokaryotic expression recombinant plasmid was con-structed, and induction expression and protein purification was carried out. ResultThe cloned UBE2H gene(GenBank accession No. KU870456)was1010bpinlength,including5'UTRof77bpand3'UTRof378bpandanopenreading frame(ORF) of 555 bp(encoding 184 amino acids). Isoelectric point(pI) of UBE2H protein was 4.88, molecular weight was 20.85 kD. Amino acid sequence of UBE2H protein was highly similar to those of other species, the similarity reached 78.00%-83.00%. Real-time quantitative PCR results indicated that expression quantity of UBE2H gene was the highest in lymphoid tissue in P. monodon, and followed by gill. The expression quantities of UBE2H gene in hepatopan-creas and ovary at different ovary developmental stages both went through an up-down variation, but peak times were dif-ferent. The largest expression quantity of UBE2H gene in hepatopancreas occurred at ovary developmental stageⅢ, whereas the largest expression quantity in ovary occurred at ovary developmental stageⅡ. Fusion protein UBE2H obtained through prokaryotic expression was 39.00 kD, protein concentration after purification was 2.92 μg/μL. ConclusionUBE2H gene of P. monodon participate in oocyte development and transport of yolk protein in hepatopancreas, and is closely related to ovary development of P. monodon.