蜕壳周期内中华绒螯蟹钙含量、组织结构及相关基因表达变化

运用生化分析、组织切片和分子生物学等技术,测定蜕壳期(蜕下旧壳后0.5 h内)和蜕壳后期(蜕下旧壳0.5 h后至新壳未完全硬化之前)平均体质量(5.63±2.35)g中华绒螯蟹肝胰腺和鳃中的钙含量,观察肝胰腺和鳃组织结构变化,用荧光定量PCR技术分析肝胰腺和鳃中的钙网蛋白(CRT)基因的表达情况。试验结果表明,蜕壳后期肝胰腺中钙含量较高;蜕壳期和蜕壳后期鳃中钙含量较低。组织学观察发现,整个蜕壳周期内,肝胰腺中的细胞内容物含量变化明显;在鳃中,鳃叶末端边缘管处的空腔结构尚未恢复正常形态。荧光定量分析结果表明,肝胰腺和鳃的钙网蛋白基因的表达量先降后升,且均在蜕壳前期最小。蜕壳前期钙网蛋白基因在肝胰腺中的表达量最低,蜕壳期最高(P<0.05)。与蜕壳期相比,蜕壳后期的表达量略有降低,但变化不显著(P>0.05)。蜕壳前期鳃中钙网蛋白基因的表达量最低,在蜕壳后期最高(P<0.05)。本试验结果将为中华绒螯蟹蜕壳和钙转运研究提供一定理论基础。

Changes in Calcium Content,Histological Structure and Expression of Calreticulin Gene of Chinese Mitten Crab Eriocheir sinensis during Molting Cycle

In this study,calcium ion contents in hepatopancreas and gill of Chinese mitten crab Eriocheir sinensis with average body weight of(5.63±2.35)g were determined during molting cycle including the molting stage(within 0.5 h after the old shell molting)and the late molting stage(0.5 h after the old shell molting until the new shell was incompletely hardened)by biochemical analysis.The structure changes in hepatopancreas and gill were histologically observed during the molting cycle by paraffin section and HE staining,and the expression of calreticulin(CRT)gene was analyzed in hepatopancreas and gill by fluorescence quantitative PCR.The results showed that there was higher calcium content in hepatopancreas in the postmolt,while lower in gill during the ecdysis and postmolt.The histological observation showed that the cell contents in hepatopancreas were changed obviously during the molting cycle.In the gill,the cavity structure of marginal vessel was not recovered at the edge of the gill lamellae.The fluorescence quantitative analysis showed that the expression levels of calreticulin gene was decreased in hepatopancreas and gill from the intermolt to premolt(minimum)and increased from the premolt to postmolt.In the ecdysis,the minimal expression level was observed in the hepatopancreas and gill,which provided a theoretical basis for the study of molting and calcium transport of Chinese mitten crab.