三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化  

MOLT-INHIBITING HORMONE LEVELS AND ECDYSTEROID TITER DURING A MOLT CYCLE OF PORTUNUS TRITUBERCULATUS

作　　者：汪春建[1] 朱冬发[1] 亓一舟[1] 胡则辉[1] 谢熙[1] 沈洁[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,宁波315211

出　　处：《水生生物学报》2013年第1期22-28,共7页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金项目(40976098);国家科技富民强县专项行动计划项目(2011F012);宁波市科技局科研项目(2012A610137;2011B81003)资助

摘　　要：甲壳动物的蜕皮过程被认为是由位于眼柄的X器-窦腺复合体(XO-SG)分泌蜕皮抑制激素(MIH)通过调节Y器(YO)合成蜕皮激素而调控的。通过实时荧光定量PCR(qRT-PCR)发现MIH基因在三疣梭子蟹眼柄X器-窦腺复合体中表达最强。采用qRT-PCR分析了MIH基因在三疣梭子蟹蜕皮周期中的表达变化,结果表明;A期为(0.42±0.08)倍,B期为(1.09±0.09)倍,C期为(1.35±0.16)倍,D0亚期为(1.00±0.10)倍,D1亚期(0.78±0.07)倍,D2亚期为(0.27±0.08)倍,D3/4亚期为(0.20±0.04)倍。采用高效液相色谱-电喷雾串联质谱(LC-MS/MS)法完成了三疣梭子蟹蜕皮周期中蜕皮激素(20E)浓度变化的测定。A/B期蜕皮激素的浓度较低,低于仪器检测限0.33 pg,C期为(1.666±0.762)ng/mL,D0亚期为(4.047±1.5133)ng/mL,D1亚期为(6.756±4.928)ng/mL,D2亚期为(8.609±3.827)ng/mL,D3亚期为(19.534±4.799)ng/mL,D4亚期为11.616 ng/mL。在三疣梭子蟹蜕皮周期中,MIH基因表达量与血淋巴中蜕皮激素浓度呈现一定拮抗性,揭示MIH抑制Y器合成蜕皮激素而调控着三疣梭子蟹蜕皮的发生和进行。Portunus trituberculatus as a popular table delicacy is one of the most important fishery and aquaculture species of crab around the coast of China.In crustaceans,molt-inhibiting hormone（MIH）,a polypeptide secreted by the X-organ–sinus gland（XO-SG） of the eyestalks,had been proposed to regulate molting by inhibiting the synthesis of ecdysteroids from Y-organs（YO）.The method for determining the levels of MIH mRNA in the swimming crab had been developed using relative quantification of quantitative real-time PCR（qRT-PCR）.We found the expression level of MIH mRNA was the highest in the XO-SG.By taking surstage D0as the control group,the levels of MIH mRNA were analyzed by 2 ΔΔCt in a molt cycle,and the results showed that MIH transcripts down-regulated 0.42±0.08,increased（1.09±0.09,increased 1.35±0.16 fold in stage A,B,C,respectively,and down-regulated 0.78±0.07,down-regulated 0.27±0.08,down-regulated 0.20±0.04 fold in surstage D1,D2,D3/4,respectively.In addition,we used the method of high performance liquid chromatography-e1ectrospray ionization tandem mass spectrometry（LC-MS/MS） to complete the process of measuring the consistency of portunus molting ecdysteroid（20-hydroxyecdysone,20E） in hemolymph.The results showed that the consistency of ecdysone was below the instrument detection limit of 0.33 pg in the post molt stage（A/B）.In the inter-molt period（C）,the consistency of ecdysone gradually returned to（1.666±0.762） ng/mL.In the pre-molt ecdysteroid titer increased gradually to（4.047±1.5133）,（6.756±4.928） and（8.609±3.827） ng/mL in surstage D0,D1and D2,respectively.The ecdysteroid titer increased steadily to a peak of（19.534±4.799） ng/mL in the surstage D3,then dropped to 11.616 ng/mL in surstage D4.These stage-specific expression changes in MIH mRNA levels were accompanied by significant fluctuations in hemolymph ecdysteroid titer.During a molt cycle of the swimming crab,the expression of MIH exhibited a negative correlation with ecdysone in hem

关 键 词：三疣梭子蟹 蜕皮周期 蜕皮抑制激素 实时荧光定量PCR 高效液相色谱-电喷雾串联质谱 

分 类 号：Q344[生物学—遗传学]