| Hyriopsis cumingii is our country’s important freshwater mussel, is China’s pearl industry preferred shellfish and smooth pearl luster, but the actual production of pearl is mixed, resulting in the key to this problem is to Pearl deposition mechanism is not clear enough. The laboratory isengaged in the mantle and visceral mass of large nucleated pearl cultivation research, and main components of pearls and shells are Ca CO3, in order to explore the formation of calcium carbonate crystallization of calcium and carbonate ions sources in vitro and in vivo conversion, transport and synthesis. The experience on the one hand, using flow cytometry combined with calcium ion probe to explore different calcium ion concentrations in the environment under the mantle visceral mass of cells in calcium retention. In another aspect to detect different concentrations in nuclear 120 days during a Hyriopsis cumingii alpha-carbonic anhydrase gene Hc CA expression, and in the corresponding protein content and total enzyme carbonic anhydrase activity. Finally, the pearl size and the surface sediment of the Pearl River were analyzed under different concentrations of different calcium ions. The main results were as follows:1. Gene expression analysis and Blot Western protein analysis of Hc CA in the visceral mass coat after the insertion of nuclear(1)Every sampling time node. 0 m M and 0.5 m M concentration treatment group Hc CA expression are in low level, the 20 day, with the Ca2+ concentration increased Hc CA expression is also gradually increased(P< 0.05) and 50 days, 90 days, 120 days 1.25 m M, 2 m MHc CA expression was significantly higher than other concentration treatment group(P < 0.05). When the concentration of Ca2+ increased to Hc CA, the expression level of 3 m M was significantly decreased(P<0.05). From the angle of each concentration analysis showed that the 50 day the expression of Hc CA was also significantly higher than that of other groups. All Hc CA gene expression showed that was significantly lower than the corresponding period of concentration and visceral mass expression of Hc CA gene expression in mantle(P<0.05).(2)Western-blot experimental results verify the expression of Hc CA gene at different concentrations in different periods2. Detection and analysis of calcium ion retention flow cytometry in the inner cell of the mantle of the mussel shell(1) To cultivate the experiment after 6 months of Hyriopsis cumingii membrane visceral mass organizations join the trypsin 37 degrees water bath digestion and join fluo-4/AM 37 DEG C hatched sterile 30 MIN, PBS solution, trypsinization solution and incubated sterile liquid using Ca Cl2 solution of calcium ion concentration and cultivation regulation water consistent. Finally, the flow cytometry was used to stimulate the FLI-H, 488 m M fluorescence channel to collect fluorescence signals, slow to collect 104 cells. In the analysis of the loss of data before clustering according to cell gating to exclude cellular debris and bacteria to digest excessive influence. After the completion of the door, the use of FL1-H on the FL1-A mapping, once again set the door to rule out the incomplete digestion of non individual cells. At last, FL1-H was used to map the Count statistics of calcium ion content.(2) The experiment with the average fluorescence intensity, on behalf of the intracellular calcium ion content and flow cytometry data statistics found that mantle cell intracellular calcium fluorescence intensity in O MM to 2 m M concentrations were significantly increased(P < 0.05) and fluorescence intensity at 2 m M concentration reached its peak, and at 3 m M concentrations appeared decreased significantly(P < 0.05). However, the fluorescence intensity of calcium ion in the visceral mass was the same as that of the mantle cell, but the overall fluorescence intensity was larger than that of the mantle cell, and the difference was significant(P < 0.05).3. Analysis of carbonic anhydrase activity in the visceral mass of Hyriopsis cumingii mantle tissue in different environment of calcium ion concentration(1) Protein content of nitrobenzene to 348 nm after 5 min of nitrobenzene to 3 m Mol / L(buffer 15 out from the cultured for 4 months after different concentrations in mussels mantle visceral mass of tissue containing PMSF lysate proteins were extracted in subsequent spectral photometer are sequentially added 1.9 ml m Mol/LTris-H2S04 liquid p H of 7.6), 0.1 ml of enzyme solution and 1 ml of acetic acid ester, the reaction at room temperature with spectral photometer was used in the determination of the light absorption value in enzyme solution for blank control, the enzyme activity is defined as: in room temperature, each minute hydrolysis of 1 mol acetic acid vinegar enzyme amount for a enzyme activity unit. The measured samples as per unit of enzyme activity.(2) The study found that the mantle and visceral mass of carbonic anhydrase activity in calcium ion concentration in the environment is 0 m M-0.5 m M, were significantly higher than the activity 1.25 m M-3 m M environmental concentrations(P< 0.05), to 2 m M concentration treatment group was the highest. And the differences between 1.25 m M and 2 m M, the activity of carbonic anhydrase 3 m M concentration is not significant(P>0.05). In addition, the study found that in the mantle of the carbonic anhydrase activity were significantly higher than that of the visceral mass(P> 0.05), the same concentration of 0.5 m M treatment group of enzymes activity was the highest(P> 0.05), but 3 m M processing group enzyme activity is higher than that of 1.25 m M, 2 m M concentration treatment group(P> 0.05).4. Observation and Research on Pearl deposit of the pearl mussel under different environment of calcium ion concentrationThis study by scanning electron microscope analysis of pearl surface morphology, found after 180 days of mantle pearl, when the concentration of calcium ion very 0 m M, even in the freshwater environment better in the mantle tissue, there will be irregular deposition of calcium carbonate, calcium ion concentration higher than 0.5 m M, calcium carbonate in the pearl nucleus deposition became more and more regular and close concentrations up to 3 m M, the surface became rough and irregular polygons sedimentary structure began to disappear. In 180 days of visceral mass pearl pearl electron microscopy results showed both high concentration or low concentration of calcium ion environment, the deposition of calcium carbonate are very irregular, uneven surface of the Pearl, prone to cracks. |
PDF Full Text Request