不同Ca2＋养殖条件下三角帆蚌外套膜和内脏团组织细胞的钙含量及其对碳酸酐酶的影响

Calcium content of mantle and visceral tissue cells from Hyriopsis cumingii and its effect on carbonic anhydrase in different Ca^(2+) culture conditions

为了探讨三角帆蚌(Hyriopsis cumingii)在不同钙离子浓度养殖时,内脏团和外套膜细胞对环境中钙离子的吸收,以及细胞内钙离子浓度对碳酸酐酶的影响,本实验设定了5种钙离子的环境浓度(0 m M、0.5m M、1.25 m M、2 m M、3 m M),采用流式细胞仪测定内脏团和外套膜组织细胞内钙离子含量,用荧光定量PCR检测α-碳酸酐酶基因(HcCA)表达,并用乙酸对硝基苯酯的水解间接测定碳酸酐酶活性,以期能阐明环境中钙浓度对组织细胞钙含量的影响,和组织细胞内不同钙含量与碳酸酐酶基因表达和酶活性之间的关系。研究结果表明,在相同环境的钙离子浓度下,活体三角帆蚌的内脏团细胞中钙含量显著高于外套膜细胞(P<0.05),并且表明从0 m M到2 m M浓度,内脏团和外套膜细胞内的钙含量显著上升(P<0.05),在3 m M浓度时出现下降(P<0.05)。同时,细胞内Ca^(2+)含量较低时,HcCA在内脏团和外套膜中的表达量均显著低于细胞内Ca^(2+)含量较高组(P<0.05),但细胞内Ca^(2+)荧光强度为8×104时达到饱和且开始下降,而HcCA的表达在细胞内Ca^(2+)荧光强度为6×104已经最高,并开始下降。环境钙离子浓度在0 m M、0.5 m M时碳酸酐酶活性显著高于1.25 m M、2 m M、3 m M浓度组(P<0.05),而且1.25 m M、2 m M、3 m M浓度组间无显著差异(P>0.05),由此发现碳酸酐酶在0.5 m M钙离子浓度中活性较高但表达量却较低,而在1.25 m M和2 m M浓度中表达量高但活性较低,并且碳酸酐酶外套膜细胞酶活性在各浓度均显著高于内脏团细胞(P<0.05)。本研究为三角帆蚌水体最适养殖钙浓度提供了重要依据。

To investigate visceral mass and mantle cell absorption of Ca^2＋ in Hyriopsis cumingii cultured in different Ca^2＋ concentrations, and the effect of intracellular Ca^2＋ concentrations on carbonic anhydrase, five kinds of Ca^2＋ concentrations were set for experiments （0 mM, 0.5 mM, 1.25 mM, 2 raM, 3 mM）. The paper used flow cytometry for the determination of content of calcium in the visceral mass and mantle tissue cells, expressed by fluorescence quantitative PCR detection of alpha-carbonic anhydrase gene HcCA, and with acetic acid hydrolysis of p-nitrophenyl for indirect determination of carbonic anhydrase activity to clarify the effect, between different calcium content and carbonic anhydrase gene expression and enzyme activity in cells and tissues. Results showed that in the same environment of Ca2＋ concentration, calcium content in the cells of visceral mass was significantly higher than that in mantle cells （ P 〈 0.05 ）, and from 0 mM to 2 mM concentrations intracellular calcium content increased significantly （P 〈 0.05 ）. In 3 mM concentration, calcium content decreased （ P 〈 0.05 ）. At the same time, the expression of HcCA was significantly lower in high Ca^2＋concentration group （ P 〈 0.05 ）. When the intracellular Ca2 ＋ fluorescence intensity reached 8×10^4,calcium content reached saturation and began to decline. The expression of Ca^2＋ in the cells with HcCA fluorescence intensity of 6 x l0t reached the highest and began to decrease. With Ca^2＋ concentration of 0 mM, 0.5 mM, carbonic anhydrase activity was significantly higher than that of 1.25 mM, 2 mM, 3 mM concentration groups （ P 〈 0.05 ） and there was no significant difference between 1.25 mM, 2 mM, 3 mM concentrations groups （P 〉 0.05 ）. Therefore, we found that carbonic anhydrase in Ca^2＋ concentration of 0.5 mM showed high activity but the expression was relatively low, while the expression in 1.25 mM and 2 mM concentration was in high volume but low activity and carbonic anhydrase mantle cell enzyme activities in each concentration were significantly higher than those of visceral mass cells （P 〈 0.05 ）. This study provides an important basis for the optimal cultured concentration of calcium in freshwater mussel.