Effect Of Three Different Factors On The Calcium Ion Metabolism And Expression Of Alkaline Phosphatase Gene In Hyriopsis Cumingii

Hyriopsis cumingii is the major pearl mussel of china, pearl produced by it is bright and smooth, which is the first choice of freshwater mussel for producing pearls. At present, the yields of the pearls are increasing, but as the same time the quality of pearl degenerated, and the very important reason is that the knowledge of the genetic system about the pearl is still inadequate, particularly, the foundation and mechanisms of the pearls under the Ca²⁺ metabolic control have not been studied. So developing studies on the foundation and mechanisms of the pearls are the urgent thing, which not only promote scientific thesis of pearl in a depth development, but also provide the direct for creating and developing on pearl producing.The thesis includes the following two parts:1. The effect of three different factors on calcium metabolism in Hyriopsis cumingii.Extracellular and intracellular Ca²⁺ flux direction and rate of mantle from Hyriopsis cumingii were detected by non-invasive micro-test technology (NMT) and Confocal laser scanning microscope (CLSM) in the level of cell. Mantle cell were divided into three groups (3 reduplication/each group). For the first group, the different concentrations of Ca²⁺ (control, 0.5, 1.25, 3 mmol/L) were added to the culture medium; for the second group, the different concentrations of VD₃ (Control, 50, 100, 500 IU/L) were added to the culture medium; for the third group, cells were cultured under the different temperatures (20℃, 26℃, 30℃), respectively.The results showed that extracellular Ca²⁺ gradually flowed from efflux to influx, flux rate increased, and the intracellular Ca²⁺ average fluorescence intensity enhanced when adding Ca²⁺ in culture medium. The Ca²⁺ flux of 3 mmol/L Ca²⁺ adding group were highest, and intracellular Ca²⁺ average fluorescence intensity was the strongest, it had significant difference compared with the other groups (P<0.05). The flow of Ca²⁺ gradually changed from efflux to influx, and flux increased when adding VD₃ in cultured cells. Among of these, the Ca²⁺ flux of 500 IU/L VD₃ group had significant difference compared with the other groups (P<0.05). Furthermore, culture temperature significantly affected on the extracellular Ca²⁺ metabolic of the mantle cells. The flow directions of extracellular Ca²⁺changed from efflux to influx and to efflux with increasing temperature (P<0.05), and the intracellular Ca²⁺ average fluorescence intensity were increased, and then declined. The influx rate at 26℃was the biggest and intracellular Ca²⁺ average fluorescence intensity was the strongest among of them of all groups (P<0.05).2. The analysis of the expression of ALP mRNA, enzyme activity of ALP and calcium metabolic with different factors in Hyriopsis cumingiiALP has relation with the formation of nacre; it plays an important role on calcium transshipment and pearl biological mineralization. To investigate the effect of alkaline phosphatase (ALP) on calcium metabolism in Hyriopsis cumingii. The 3 ' end sequence was obtained by RACE PCR and the sequence were analysised by bioinformatics. The different expression of ALP gene mRNA in the outer mantle, gills, visceral mass, foot ax, pearl sec and the expression of ALP mRNA with Ca²⁺ and VD₃ were detected by Real-time Q-PCR testing technologyd with double the standard curve method, enzyme activity were detected by enzyme activity kit.The result showed that the target sequence about 924 bp. The test analysis of Real-time fluorescence quantitative PCR with two primer by cDNA for template showed that ALP mRNA level from mantle near by the pearl was highest (P<0.05). The result of the expression of ALP mRNA, the enzyme activity and the analysis of calcium metabolic with three different factors showed that under the temperature of 26℃, the high concentration of Ca²⁺ and VD₃ had the biggest function on the expression of ALP and the enzyme activity, it could promote more Ca²⁺ deposition within the cell.This study provides the basis of experiments and research on calcium metabolic system on mantle cell from Hyriopsis cumingii, which has a great significance on clarifing the formation mechanism of pearls and enhancing the quality of pearl, it promotes the pearl industry.