斑节对虾cyclin G2基因克隆及不同刺激下的表达分析

细胞周期蛋白G2(cyclin G2)是细胞周期中的重要调控因子。该研究初步探讨斑节对虾(Penaeus monodon)细胞周期蛋白G2(Pmcyclin G2)在卵巢发育中的作用,利用RACE技术克隆获得4075bp Pmcyclin G2 c DNA全长,其开放阅读框(ORF)1161bp,编码386个氨基酸。利用荧光定量PCR技术研究了Pmcyclin G2组织表达模式,结果显示,Pmcyclin G2在斑节对虾的心脏、血淋巴、肝胰腺、卵巢等组织中均有表达,其中肌肉中表达量最高;卵巢发育不同阶段Pmcyclin G2的表达分析则表明,Pmcyclin G2在III期表达量最高;注射5-羟色胺(5-HT)能诱导卵巢Pmcyclin G2基因表达升高,多巴胺(DA)则抑制卵巢中Pmcyclin G2基因表达。利用原核表达技术获得了cyclin G2的体外重组蛋白,Western Blot分析证实重组蛋白为cyclin G2蛋白。此结果为进一步研究该蛋白的功能提供了条件。以上研究结果表明,Pmcyclin G2基因可能与斑节对虾卵母细胞发育有密切关系,该结果可为进一步探究斑节对虾卵巢的发育机理提供理论依据。

Molecular cloning and expression analysis of cyclin G2 gene from black tiger shrimps (Penaeus monodon) under different stimulation

Penaeus monodon is the largest species of shrimp, with its high economic and natural nutritional value. However, during its breeding process, broodstock breeding technology has been a very important limiting factor. With the development of molecular biology techniques, there are growing concerns about the function of cell cycle genes of crustaceans, especially shrimp. Recently, molecular mechanisms of cell cycle regulatory proteins have been researched. In the study, cell cycle-related protein family genes relating to ovarian development also become a hot topic. Cyclin G2 is a newly discovered cell cycle-related protein that plays an important role during embry-onic development, cell cycle progression and development, and disease. Cyclin G2 in some species have been cloned out, but the study of the gene has not been reported in the black tiger shrimp (Penaeus monodon). Gonads of the black tiger shrimp,Penaeus monodon, mature if the eye stalk is removed. Thus, the full-length cyclin G2 cDNA sequence fromPenaeus monodon (denoted asPmcyclin G2) was cloned by means of rapid amplification of cDNA ends (RACE) approaches to better understand the potential function of cyclin G2 in the regulation of shrimp reproduction. The full-length cDNA sequence was 4075 bp and contained 189 bp 5′untranslated region (UTR) and a 2725 bp 3′UTR. The open reading frame was 1161 bp and coded 386 amino acids (aa) which was highly homology to other species cyclin G2. Bioinformatics analysis showed that the amino acid coding sequence had a conserved cyclin box and the homologous protein box structure domain was 50–150 aa. The predicted molecular weight was about 43.4 kD, and the theoretical isoelectric point was 8.25. The deduced amino acid of cyclin G2 con-tained 29 phosphorylation sites including 17 Ser, 7 Thr, and 5 Tyr residues. SignalP 4.0 analysis revealed that cyclin G2 did not contain a typical signal peptide sequence. The temporal expression ofPmcyclin G2 in different tissues (ovary, heart, intestine, hepatopancreas, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by Real-time quantitative polymerase chain reaction (QRT-PCR). The lowest expression level ofPmcyclin G2 was observed in the hepatopancreas, and the highest in the muscle. During the ovary development stages,Pmcyclin G2 was significantly high expressed in stage III ovaries than the other stages. The relative expression ofPmcyclin G2 in the ovary was up-regulation after 5-hydroxytryptamine (5-HT) injection, while down-regulation after dopamine (DA) challenge. The study obtained recombinant expression Pmcyclin G2 in prokaryotes and offered theoretical basis for further research on Pmcyclin G2 protein function. Those results offered additional information to understand the devel-opment mechanisms ofP. monodon ovaries.