【作者】 李莉；

【导师】 潘鲁青；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2011， 硕士

【摘要】 甲壳动物的渗透调节是维持甲壳动物正常生命活动的一项重要的生理功能,对甲壳动物渗透调节生理的研究也一直是国内外的热点。目前对甲壳动物渗透调节的研究也取得了一些阶段性的成果,但对甲壳动物渗透调节机制的深入研究还比较少,尤其是在神经内分泌系统调控下甲壳动物渗透调节中相关通路因子的变化的研究更少。而凡纳滨对虾（Litopenaeus vannamei）是当今世界养殖产量最高的三大虾类之一,又是我国对虾养殖产量中占总产量比例最高的品种。它具有很多养殖优势,主要有生长速度较快、能够适应高密度的养殖模式、适盐范围较广、能够在较低盐度的养殖水中生长、对饲料的蛋白质含量要求较低等。所以本文选择凡纳滨对虾作为研究对象,研究了凡纳滨对虾在甲壳动物高血糖激素（CHH）调控下相关渗透指标的变化,初步探讨了神经内分泌激素在凡纳滨对虾渗透调节中的作用以及通过通路中相关因子的变化来初步阐明这一机制。1凡纳滨对虾高血糖激素（CHH）基因的克隆与序列分析甲壳动物高血糖激素对于甲壳动物的生长发育生殖等生理生化过程具有重要作用。本实验通过相近物种CHH氨基酸序列的同源性比对,根据保守氨基酸序列设计兼并引物,利用RT-PCR技术成功克隆了凡纳滨对虾CHH的部分核心cDNA序列。将凡纳滨对虾的CHH与其它物种的CHH进行同源性比较发现,CHH基因具有高度保守性,同属于对虾属中的CHH基因比较相似,聚于进化树一个分支上。通过CHH的组织特异性研究发现,CHH基因在腹神经,眼柄,心脏、肝胰脏和肌肉中有表达,肌肉中的表达量相对较低,而在鳃中没有表达。2切除眼柄对凡纳滨对虾鳃丝离子调节信号转导通路的影响本实验研究了切除眼柄48h对凡纳滨对虾鳃丝离子调节信号转导通路中鳃丝三种生物胺、鳃丝cAMP和cGMP以及鳃丝三种离子转运酶活力的变化。结果显示,切除眼柄对凡纳滨对虾鳃丝3种生物胺含量、cGMP含量和三种离子转运酶活力的影响均显著（p<0.05）,而对cAMP含量影响不显著（p>0.05）。对照组和饥饿组鳃丝三种生物胺、cGMP和cAMP含量、Na⁺-K⁺-ATPase、V-ATPase和HCO₃^--ATPase活力在实验时间内无明显变化（p>0.05）。切眼组和交并组鳃丝DA、NE含量在48h内均呈峰值变化,在12h时达到最大值,然后呈逐渐下降趋势,于48h时显著低于对照组水平（p<0.05）;切眼组鳃丝5-HT含量在48h内呈峰值变化,在12h时达到最小值,随着时间的延长呈缓慢上升趋势然后趋于稳定,48h时显著低于对照组水平（p<0.05）;而交并组在实验时间内呈明显下降趋势,48时也显著低于对照组水平（p<0.05）。切眼组cAMP含量在实验时间内为先下降后升高的趋势,而交并组呈缓慢下降的趋势,而48h时均与对照组差异不显著（p>0.05）。切眼组和交并组鳃丝cGMP含量与处理时间呈负相关,在实验时间内呈逐渐下降趋势,48h时cGMP含量显著低于对照组水平（p<0.05）。切眼组鳃丝Na⁺-K⁺-ATPase、HCO₃^--ATPase活力在48h内均呈峰值变化,于12h时达到最小值,然后呈上升趋势,其中Na⁺-K⁺-ATPase活力在48h时恢复至对照组水平,HCO₃^--ATPase活力显著低于对照组（p<0.05）,而交并组均呈明显下降趋势,48h时显著低于对照组水平（p<0.05）;切眼组和交并组鳃丝V-ATPase活力在24h内明显下降,24h后趋于稳定并显著低于对照组水平（p<0.05）。同时在48h时3种生物胺含量、cGMP和cAMP含量、Na⁺-K⁺-ATPase、V-ATPase和HCO₃^--ATPase活力大小均表现为切眼组>交并组,由此可见,切除眼柄（CHH）对凡纳滨对虾鳃丝离子调节信号转导通路中的生物胺、第二信使以及离子转运酶活力有显著的影响,切眼组的数据大于交并组,说明在切除眼柄后,饥饿也会相应导致通路中这些指标的变化,并且通过第一信使生物胺、第二信使将信号传递到相应的基因表达调控原件,激活或者表达相应的靶器官来调节渗透平衡。3切除眼柄对凡纳滨对虾血糖代谢的影响本实验研究了切除眼柄48h对凡纳滨对虾血淋巴3种生物胺、肝胰脏cAMP和cGMP、血淋巴葡萄糖含量的影响。结果表明,切除眼柄对凡纳滨对虾血淋巴3种生物胺含量、肝胰脏cAMP和cGMP、血淋巴葡萄糖含量的影响显著（p<0.05）,而对照组无明显变化（p>0.05）。饥饿组血淋巴DA、NE含量在48h内均呈峰值变化,在12h时达到最大值,饥饿组5-HT含量在实验时间内变化较平稳,呈现先升高后下降的趋势,48h时3种生物胺含量低于对照组水平（p<0.05）;切眼组血淋巴DA含量呈下降趋势,48h时显著低于对照组（p<0.05）;切眼组NE含量在48h内呈峰值变化,于12h时达到最小值,48h时显著高于对照组（p<0.05）,切眼组5-HT含量在48h内呈平稳变化的趋势,48h时与对照组无显著差异（p>0.05）;交并组DA、NE含量在实验时间时内呈逐渐下降趋势,48h时显著低于对照组水平（p<0.05）;交并组5-HT含量呈现先升高后下降的趋势,48h时恢复至对照组水平。切眼组cAMP含量48h内呈现峰值变化,12h时达到最低值后逐渐上升,48h时恢复至对照组水平,切眼组cGMP含量呈下降趋势,24h时达到最低值后逐渐稳定,48h时显著低于对照组水平（p<0.05）;交并组cAMP、cGMP含量在实验时间内呈峰值变化,于12h时达到最低值,然后呈上升趋势,cAMP含量24h后趋于稳定,48h后显著低于对照组水平（p<0.05）,cGMP含量48h后恢复至对照组水平。饥饿组葡萄糖含量在实验时间内呈下降趋势,48h后与对照组差异显著（p<0.05）;切眼组葡萄糖含量在实验时间内呈峰值变化,于24h时达到最低值后升高,48h时显著高于对照组水平（p<0.05）;交并组葡萄糖含量在实验时间内呈下降趋势,48h时显著低于对照组水平（p<0.05）,并趋近于0。本实验说明,切除眼柄（CHH）后对凡纳滨对虾的血糖代谢有显著的影响,在不切除眼柄的处理组中,饥饿组的血淋巴生物胺含量和血糖含量都低于对照组,而cAMP和cGMP含量稍高于对照组,在切除眼柄的处理组中,切眼组的各项指标均高于交并组,这说明饥饿能够刺激细胞中的第二信使,通过传递相应的信号来调控相关的控制血淋巴葡萄糖含量的靶器官,而在切除眼柄的情况下,凡纳滨对虾可以通过食物来获得能量维持机体的生理平衡。更多还原

【Abstract】 There are lots of researchers focusing on the osmoregulation of Crustacean, which is a very important physiological function maintaining normal vital movement. But there were few correlative studies has reported, especially on the change of pathway factors of Crustacean osmoregulation modulated by the Neuroendocrine system. Litopenaeus vannamei is one of the three shrimps which have the largest production in the world, and have the largest production in China. It has many advantages, including fast growth rate, adaptability to high density and broad salinity and low demand to protein. The report studies the changes of osmoregulation factors modulated by CHH, the effect of Neuroendocrine hormone on the osmoregulation of Litopenaeus vannamei and the mechanism of the osmoregulation according to the changes of relating factors.1 Cloning and sequence analysis of CHH in Litopenaeus vannameiCHH is very important in the growth and reproduction of Crustacean. The degenerate primers were designed from CHH protein multiple sequence alignments from related species. The partial CHH cDNA sequence was obtained through the technology of RT-PCR. The results show that CHH is highly conserved when compared among species, especially the CHH cDNA of shrimps, Together on a branch in the evolutionary tree. And it can be drawn from the experiment that CHH mRNA expresses in abdominal nerve, eyestalk, heart,hepatopancreas and muscle. It is lowly expressed in muscle and isn’t expressed in gill.2 Effect of Eyestalk Ablation on the pathway of signal transport ion of gill of Litopenaeus vannameiThe studies shows that the changes of contents of three kinds of biogenic amines, cAMP and cGMP and three kinds of ion transportation enzyme activity in gills. The results show that eyestalk ablation can affect significantly contents of three kinds of biogenic amines and cGMP and three kinds of ion transportation enzyme activities in gills （p<0.05）, and can not affect significantly contents of cAMP （p>0.05）. The contents of three kinds of biogenic amines, cAMP and cGMP and three kinds of ion transportation enzyme activities in the gills of control group and hunger group don’t change significantly （p>0.05）. The concentration of DA and NE in the gill of eyestalk ablation group and interactive group showed peak changes within 48h, reached the peak value at 12h, then decreased and were finally lower than the control group at 48h （P<0.05）. The contents of 5-HT in the gill of excising eyestalk group showed peak changes within 48h, reached the minimum value at 12h, then increased slowly, became stable and was finally lower than the control group at 48h （P<0.05）. But the contents of 5-HT in the gill of interactive group decreased significantly and was finally lower than the control group at 48h （P<0.05）. The concentration of cAMP in the gill of eyestalk ablation group decreased at first and then increased. The contents of cAMP in the gill of interactive group decreased slowly and had no significant differences from the control group at 48h. The concentration of cGMP in the gill of eyestalk ablation group and interactive group had negative correlation with treat time, decreased and was finally lower than the control group at 48h （P<0.05）. Na⁺-K⁺-ATPase and HCO₃^--ATPase activity in the gill of eyestalk ablation group showed peak changes within 48h, reached the minimum value at 12h, and then increased. Na⁺-K⁺-ATPase recovered to control level. But HCO₃^--ATPase activity was finally lower than the control group at 48h （P<0.05）. Na⁺-K⁺-ATPase and HCO₃^--ATPase activity in the gill of interactive group decreased significantly and was finally lower than the control group at 48h （P<0.05）. V-ATPase activity in the gill of excising eyestalk group and interactive group decreased significantly, became stable at 24h and was finally lower than the control group （P<0.05）. The concentration of three kinds of biogenic amines, cAMP and cGMP, Na⁺-K⁺-ATPase, V-ATPase and HCO₃--ATPase activity in the gill of eyestalk ablation group were higher than those of interactive group. Eyestalk removal affected significantly biogenic amines, second messengers and ion transportation enzyme activity. And the data of eyestalk ablation group was higher than that of interactive group. The result indicated that huger induced the changes of signal channel and activated the target organ to regulate osmotic equilibrium according to biogenic amines and second messengers.3 Effect of eyestalk ablation on the hemolymph glucose metabolism of Litopenaeus vannameiThe report studies the effects of eyestalk ablation on the contents of three kinds of biogenic amines in hemolymph, cAMP, cGMP in the hepatopancreas and the hemolymph glucose. The result indicated that eyestalk removal affected significantly the contents of three kinds of biogenic amines in hemolymph, cAMP, cGMP in the hepatopancreas and the hemolymph glucose （P<0.05）. But there were no significant differences in those of control group（p>0.05）. The contents of DA and NE in the hemolymph of huger group showed peak changes within 48h, reached the peak value at 12h. The content of 5-HT in huger group increased and then decreased, but didn’t change significantly. The contents of three kinds of biogenic amines was finally lower than the control group at 48h （P<0.05）. The concentration of DA in the hemolymph of eyestalk ablation group decreased and was finally lower than the control group at 48h （P<0.05）. The content of NE in the hemolymph of eyestalk ablation group showed peak changes within 48h, reached the minimum value at 12h and was finally higher than the control group at 48h （P<0.05）. The content of 5-HT in the hemolymph of eyestalk ablation group didn’t change significantly, and had no significant difference with the control group at 48h （p>0.05）. The content of DA and NE in the hemolymph of interactive group decreased during the experiment and was finally lower than the control group at 48h （P<0.05）. The content of 5-HT in the hemolymph of interactive group increased, then decreased and recovered to control level. The content of cAMP in the eyestalk ablation group showed peak changes within 48h, reached the minimum value at 12h, then increased and recovered to control level at 48h. The content of cGMP in the eyestalk ablation group decreased, reached the minimum value at 24h, then became stable, and was finally lower than the control group at 48h （P<0.05）. The contents of cAMP and cGMP in the interactive group showed peak changes during the experiment, reached the minimum value at 12h, and then increased. The content of cAMP became stable at 24h and was finally lower than the control group at 48h （P<0.05）. But the content of cGMP recovered to control level at 48h. The content of hemolymph glucose in huger group decreased during the experiment and had significant difference with the control level （P<0.05）. The content of hemolymph glucose in eyestalk ablation group showed peak changes during the experiment, reached the minimum value at 24h, then increased and was finally higher than the control group at 48h （P<0.05）. The content of hemolymph glucose in the interactive group decreased to about 0 during the experiment and was finally lower than the control group at 48h （P<0.05）. The results indicated that eyestalk ablation affected significantly the hemolymph glucose metabolism of Litopenaeus vannamei. The contents of biogenic amines in hemolymph and the hemolymph glucose in no eyestalk ablation groups was lower than the control level. The contents of cAMP and cGMP was higher than the control level. The data of eyestalk ablation group were all higher than interactive group. The results indicated that huger activated second massagers in the cell and then regulated target organ related to hemolymph glucose. Litopenaeus vannamei can maintain physiological equilibrium according to obtain energy from food in the absent of CHH.更多还原