长牡蛎AMPK基因在三倍体性腺发育中的表达特性及调控机制

Expression Characteristics and Regulation Mechanism of AMPK Gene in Gonad Development of Triploid Pacific Oyster(Crassostrea gigas)

为探究三倍体长牡蛎(Crassostrea gigas)性腺发育的分子调控机制,对AMPK基因及其靶基因在长牡蛎性腺发育过程中的表达特性进行研究。长牡蛎AMPK包含AMPKα、AMPKβ和AMPKγ三个亚基。荧光定量PCR结果显示,这三个亚基的mRNA表达量在二倍体长牡蛎性腺发育过程中均呈下降趋势,在三倍体长牡蛎性腺发育过程中均呈上升趋势。二倍体长牡蛎性腺中AMPKα亚基Thr172位点磷酸化(p-AMPKα)水平在增殖期高,到成熟期几乎完全消失,这说明在长牡蛎性腺发育早期AMPK基因可能起到重要调控作用;在三倍体长牡蛎中,不育型三倍体(3nβ)的p-AMPKα水平显著高于可育型三倍体(3nα),推测AMPKα亚基磷酸化增强可能与三倍体不育密切相关。AMPKα亚基活性蛋白主要定位在滤泡细胞、精原/精母细胞和卵原/卵母细胞,说明该蛋白可能参与调控长牡蛎配子发生的早期过程。对AMPK潜在靶基因(GCS、CREB、SREBP1、ACC和Raptor)进行表达量分析,结果显示长牡蛎中p-AMPKα可能参与了CREB和SREBP1基因表达的调控。推测在可育型三倍体长牡蛎性腺发育早期AMPK基因可能调控促进糖原和脂质的分解,为生殖细胞分裂和分化提供能量;而在不可育型三倍体长牡蛎中由于生殖细胞分裂不旺盛,AMPK基因表达未被抑制,AMPK发挥维持体内能量稳态的调控作用。

To explore the molecular regulation mechanism of gonadal development in triploid Crassostrea gigas,the expression characteristics of AMPK gene and its target genes during gonadal development in C.gigas were investigated.AMPK contained three subunits,AMPKα,AMPKβand AMPKγ.The results of real time PCR showed that the three AMPK subunits mRNA all showed a decreasing trend during the gonadal development in diploid C.gigas and an increasing trend in triploid C.gigas.The phosphorylation level of AMPKαat Thr172(p-AMPKα)of gonadal development in diploid C.gigas was high at the proliferative stage and almost completely disappeared by the mature stage,indicating that AMPK may play an important regulatory role in the early stage of gonadal development in C.gigas,while in triploid C.gigas,the level of p-AMPKαwas significantly higher in sterile triploids(3nβ)than in fertile triploids(3nα),speculating that p-AMPKαenhancement might be closely related to triploid infertility.AMPKαactive protein was mainly located in follicular cells,spermatogonia/spermatocytes and oogonia/oocytes,suggesting that this protein may be involved in the regulation of the early gametogenesis of C.gigas.The expression of potential AMPK targeting genes(GCS,CREB,SREBP1,ACC and Raptor)were analyzed,and the results showed that p-AMPKαmay be involved in the regulation of CREB and SREBP1 expression.It is speculated that AMPK may promote glycogen and lipid breakdown and provide energy for germ cell division and differentiation at the early stage of gonadal development in fertile triploid C.gigas.In the sterile triploid C.gigas,the expression of AMPK was not inhibited due to the lack of vigorous germ cell division,which maintained the energy homeostasis in the body.