双壳贝类染色体标本制备技术的优化

Optimization of Methods for Preparation of Metaphase Chromosome in Bivalve

本研究以牡蛎、咬齿牡蛎鳃细胞为材料,采用PHA处理法、低温同步化法及活体去壳法进行预处理,制作成染色体标本;以马氏珠母贝、企鹅珍珠贝担轮幼虫为材料,采用PHA促繁殖获取胚胎法,制作胚胎染色体标本。对不同时间不同处理方式获得牡蛎染色体分裂相比例、PHA促繁殖获取胚胎法获得马氏珠母贝染色体分裂相比例进行统计。结果发现,PHA处理法在24h时能获取最多分裂相(3.50‰),低温同步化法在72h时能获得最多分裂相(2.20‰),活体去壳法始终保持较高分裂相比例(3.60‰),PHA促繁殖获取胚胎法获得最大分裂相比例(10.00‰)。4种方法都能获得理想的中期分裂相数目。其中,低温同步化法缺点是温度难控制、预处理时间长;活体去壳法易导致贝类死亡;PHA促受精获取胚胎法缺点是胚胎的获取受繁殖季节限制。PHA处理法预处理时间短、获取分裂相数目多,无疑是贝类成体染色体制备的最好的方法。同时,PHA也为贝类人工繁殖提供了一种更为有效的手段。

Three methods, PHA, low temperature synchronizing and ripping a shell, were used to treat the gill of Crassostrea sp. and Saccostrea mordax. PHA was used to treat the gill cells ofPinctada martensi and Pteria penguin, early embryos were used for chromosome preparation. Then the metaphases obtained in four different methods were counted respectively. The proportion of metaphase increased after the different tissues were treated by all the four methods. Then it is the good method of artificial breeding. It was found that PHA led to the most metaphases after 24 hours （3.50‰）; low temperature method produced the most metaphases after 72 hours （2.20‰）; shell-ripping method always resulted in high percentage of metaphase （3.60‰） and larvae from inducting spawning with PHA produced the highest percentage （10.00‰）. All the four methods produced quite satisfactory results, among which the low temperature method is time-consuming in preparation and hard to control the temperature; shell-ripping method often leads to death; larvae from inducting spawning with PHA is restrained by reproduction season. In comparison, PHA method needs short time in preparation but produces relatively large number of metaphase, thus it is the best way for preparation ofmetaphase chromosome. What＇s more, PHA is also an effective way for inducting bivalve spawning.