青岛文昌鱼（Branchiostomabelcheritsingtauense）染色体荧光原位杂交条件研究

摘要

The 18S rDNA sequence labeled as probe was applied for fluoresce in situ hybridization. According to the signals quantity and specificity on blastula cells, the key factors were discussed on chromosomal fluoresce in situ hybridization of amphioxus Branchiostoma belcheri tsingtauense. The results showed that optimum conditions were as follows: the pretreatment was 5ng/μl pepsin, 3-5 minutes incubation and 10ng/μl RNase, 1 hour incubation; the concentration of ratio salmon sperm DNA/probe DNA was 100-300; the wash stringency was 50% formamide/2×SSC, 1×SSC, 2×SSC at 42℃; 3% BSA blocking; 12-18h hybridizing at 37℃; counterstain with 20μl 1.5μg/ml PI per slide.%采用青岛文昌鱼18SrDNA序列为探针进行荧光原位杂交，根据囊胚晚期细胞上的核杂交信号多少及特异性确立青岛文昌鱼染色体荧光原位杂交的最佳反应条件。结果表明适合于青岛文昌鱼染色体荧光原位杂交的条件为：预处理采用5ng／μl的胃蛋白酶处理3—5min和10ng／μlRNA酶处理1h；100—300倍探针浓度的鲑鱼精DNA进行封闭；50％甲酰胺／2×SSC，1×SSC，2×SSC，42℃的洗脱强度；3％BSA进行抗体封闭；杂交时间可以控制在12—18h；杂交温度保持37℃；PI（1．5μg／ml）的量每片20μl以内。