E.coli galk和gpt基因重组质粒(pIDB103)导入金鱼受精卵内的研究

Study of Eschcrichia coli Galk and Gpt Gene Introduced Into Fertilized Eggs of Goldfish

用显微注射法把含有E.coli galk和gpt基因的环状和线状重组DNApIDB103分别导入金鱼受精卵的细胞质内。这些注射过的卵子一般都能正常发育。从各不同发育时期的胚胎分离DNA与^(32)P标记的pIDB103探针杂交表明,导入的环状外源重组DNA在胚胎发育的早期,绝大部分以各种环状构型存在。从原肠胚晚期开始,它们逐渐形成串联状高分子量DNA。在尾芽期仍能检测到它们的序列。但尚未证明,它们是否与受体的染色体DNA发生整合。我们从囊胚期的胚胎中回收到了能转化大肠杆菌的环状重组DNA,它的酶切图谱和pIDB103极其相似。导入金鱼受精卵内的线状重组质粒pIDB103,除少量DNA与金鱼的染色体DNA可能发生整合外,其余绝大部分也形成高分子量DNA。

Circular and linear plasmid pIDB 103 contained E. coli galk and gpt genes were microinjected into the cytoplasm of fertilized eggs of goldfish. The exogenous DNA sequences seems to be stable in early embryonic stages of the goldfish. At the begining of embryos injected with circular plasmid, the plasmid DNA sequences were detected as circular conformation by southern blot. From gestrula stage, some plasmd DNA sequences were detected as linear conformation with high molecular weight and comigrated with cellular DNA on agarose gel electrophoresis. The circular plasmid were rescued from the injected blastula by retrasformaing E. coli HB 101. Restriction endonuclease analysis of this DNA suggested that the majority of the injected circular DNA were not modified following replication in goldfish embryos. When fertilized eggs of goldfish injected with linear plasmid pIDB 103, the most exogenous DNA sequences were detected as linear conformation with high molecular weight. Some of which seems to be integrated into the chromosome of goldfish.