摘要
The genomic library of a cold-resistant fresh water teleost, Leuciscus waleckii,was constructed by using phage EMBL3 as vector. Three positive liybridized recombinant phage plaques were screened out by AFP gene probe of a marine teleost, North American winter flounder(Pseudopleur-onectes americanus).Three positive hybridized bands of 2.7 Kb/Sac I, 8 Kb/BamHI and 20 Kb/Hind III DNA segments obtained from these positive hybridization recombinant phage DNA were identified when a AFP gene and a 0.4 Kb cDNA of AFP gene of a marine teleost,the winter flounder, were used respectively as the probes for Southern analysis.These preliminary results show that a homologous sequence of AFP gene of winter flounder seems to be present in the genome of Leuciscus waleckii.
极地海域中的鱼类非常耐寒。它们的血液里常含有两种能降低其体内冰点的蛋白质：抗冻糖蛋白（ＡＦＧＰ）和不含糖的抗冻蛋白或抗冻多肽（ＡＦＰ）。这些蛋白的编码基因已经被克隆，并将黄盖鲽ＡＦＰ导入虹鳟鱼和大西洋鲑鱼，而改变了它们的抗冻性能。为开展中国的抗冻转基因鱼研究，本项目对中国北方的耐寒淡水鱼的抗冻基因进行了分离研究。所用实验材料鱼为中国内蒙古北部生活的瓦氏雅罗鱼（Ｌｅｕｃｉｓｃｕｓｗａｌｅｃｋｉｉ），属鲤形目（Ｌｙｐｃｉｎｉｆｏｒｍｅｓ），鲤科（Ｌｙｐｒｉｎｉｄａｅ），雅罗鱼亚科（Ｌｅｕｃｉｓｉｎａｅ）。基本按标准方法构建瓦氏雅罗鱼的基因文库，只在包装反应至３０分钟时，再加一份ＢＨＢ２６９０制备物，并延长３０分钟反应时间。所得文库的成斑率大于１×１０５ｐｆｕ。已知ＡＦＰ基因常常多至４０个拷贝，故已够用。采用通用的原位分子杂交和Ｓｏｕｔｈｅｒｎ分析进行瓦氏雅罗鱼ＡＦＰ基因同源序列的分离及其亚克隆分析。以北美黄盖鲽ＡＦＰ基因片段为探针，从瓦氏雅罗鱼ＤＮＡ文库中筛选了三个阳性克隆。图一所示为其中一个的双份膜及其复筛结果。图二为这个阳性克隆ＤＮＡ经过三种限制酶切后，同时与北美黄盖鲽ＡＦＰ基因片段和该基因０．４ＫｂｃＤＮ?
