半滑舌鳎血淋巴细胞体外培养及其染色体制备在性别鉴定中的应用

摘要

研究建立了半滑舌鳎(Cynoglossus semilaevis Giinther)外周血淋巴细胞体外培养及染色体制备方法,确定了最佳条件为:在24℃的环境中半滑舌鳎血淋巴细胞在添加20%胎牛血清的MEM培养基中,用终浓度为0.3 mg/mL的LPS为刺激源培养72h,在结束培养前3h加入终浓度0.08μg/mL秋水仙素,可获得较多、较好的染色体分裂相.利用这种方法对半滑舌鳎遗传性别进行了鉴定,同时与生理解剖观察、性腺切片、性腺细胞培养、雌性特异标记等方法进行了比较,确定了适宜各阶段不同类型鱼的性别鉴定方法,丰富了半滑舌鳎活体遗传性别鉴定的方法.%The method of the peripheral blood lym-phocytic cell culture and chromosome preparation of the tonguefish (Cynoglossus semilaevis Günther) was established in this research. The conditions of the peripheral blood lymphocytic cell culture and chromosome preparation were well searched from culturing temperature and time, effect of PHA, ConA,Lipopolysaccharide as immune enhancers, treating time and concentration of colchicines and so on. The result showed that 20％ FBS with MEM and LPS (final concentration 0.3 mg/mL) was the best blood lymphocytic cell medium. Cultivating for 72h, treating the cells with colchicines (final concentration 0.08 μg/mL) for 3h before culture ending could get better chromosome samples. We also presented a comparison of several methods to identify the sex of the tonguefish,including the preparation of chromosome from cultured peripheral blood lym-phocytic cell, female-specific DNA marker, Physiological anatomy, gonadal biopsy and gonad cell culture. The first two methods could identify genetic sex of tonguefish exactly, while the other three methods could identify physical sex of tonguefish. However, there were big limitations of anatomical observation, Gonadal biopsy and gonadal cell culture. Preparation of blood lymphocyte chromosome and female-specific marker methods could guarantee in vivo identification applicable, and the accuracy was also remarkable. The method of chromosome preparation could also be used to identify super female individual fish,while the method of female specific marker could not achieve. In a word, this experiment enriched the methods of genetic sex identification of Günther in vivo.