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

  • 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。

     

    Abstract: 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 D0 as the control group, the levels of MIH mRNA were analyzed by 2-DDCt in a molt cycle, and the results showed that MIH transcripts down-regulated 0.42?.08, increased (1.09?.09, increased 1.35?.16 fold in stage A, B, C, respectively, and down-regulated 0.78?.07, down-regulated 0.27?.08, down-regulated 0.20?.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?.762) ng/mL. In the pre-molt ecdysteroid titer increased gradually to (4.047?.5133), (6.756?.928) and (8.609?.827) ng/mL in surstage D0, D1 and D2, respectively. The ecdysteroid titer increased steadily to a peak of (19.534?.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 hemolymph, and this correlationindicated that MIH regulated the occurrence and advance of molt by inhibiting the ecdysteroid synthesis from YO.

     

/

返回文章
返回