三倍体皱纹盘鲍活体倍性检测

Living ploidy detection of triploid Haliotis discus hannai

以皱纹盘鲍的血细胞为材料,采用植物血球凝集素(PHA)体外浸泡法,运用正交实验设计,探讨PHA处理时间、PHA浓度、抽血时间对细胞分裂频度的影响。根据正交实验直观分析结果,得出用血细胞直接制备染色体标本进行三倍体皱纹盘鲍活体倍性检测的最优组合:PHA处理时间18h、PHA浓度0.04%,抽血时间22:00-24:00,3因素的主次顺序:PHA处理时间→PHA浓度→抽血时间。并可获得大量图象清晰、长度适中、分散良好的中期分裂相,是皱纹盘鲍活体倍性检测的最佳方法。同时,通过流式细胞仪测定皱纹盘鲍血细胞DNA相对含量进行三倍体皱纹盘鲍活体倍性检测,该方法制样简单,测试速度快。

In this paper, the orthogonal experiment of three factors and three levels L9(3~4) was adopted using blood corpuscle of Haliotis discus hannai which were in vitro immersing in PHA solution. There were three factors and three levels which included PHA treatment time: 6h, 12h, 18h; PHA concentration:0.02%, 0.04%,0.06%; sampling time:16:00-18:00, 22:00-24:00, 4:00-6:00, respectively. The tests were repeated two times. The results showed that the optimal combination of three factors and three levels in the detection of triploid Haliotis discus hannai by using chromosome specimens from blood cells of living bodies was: PHA treatment time 18h, PHA concentration 0.04% and sampling time 22:00-24:00. The order of the three factors was PHA treatment time → PHA concentration →sampling time. Under the optimal combination of the three factors and three levels many clear, proper length and well-scattered metaphase chromosomes could be obtained. It is the best method of detecting the ploidy of living Haliotis discus hannai.Determining the relatively content of blood corpuscles' DNA with flow cytometry could also make ploidy detection. But the device was expensive and the method was difficult to be spread.