【作者】 陈琳玲；

【导师】 李凯； 廖端芳；

【作者基本信息】 南华大学 ， 药理学， 2006， 硕士

【摘要】 目的：1)开发出新的基因克隆方法，2)对几种鱼类、鸟类的FBN1基因进行克隆，3)对这些基因进行遗传进化学研究，分析R1170H为马凡综合症致病突变的可能性。 方法：通过对现有物种原纤维蛋白-1（Fibrillin-1，FBN-1）蛋白氨基酸序列分析，设计可用于多物种FBN1基因克隆的摇摆引物，运用反向巢式PCR对鸡、火鸡、鲤鱼、虹鳟鱼、黑牛头鱼的cDNA进行扩增，测序分析PCR产物。运用GENERUNNER软件对新克隆的FBN1cDNA序列进行分析，并由各cDNA序列推测其氨基酸序列。采用invitrogen公司的Alignx软件对NCBI中人类、牛、猪、大鼠、小鼠、鸡以及新克隆的鸡、火鸡、鲤鱼、虹鳟鱼、黑牛头鱼FBN1蛋白序列中1107位氨基酸在遗传进化史上的保守性进行分析。 结果：运用我们新开发的反向巢式PCR成功克隆了鸡、火鸡、鲤鱼、虹鳟鱼、黑牛头鱼的FBN1基因片段，鸡的FBN1序列与GENBANK中gi|50752970|ref|XM₄13815.1|Gallus gallus fibrillin 1基因的部分序列完全相同，其余四个物种的FBN1基因片段为新克隆基因序列，已登录GENBANK，其登录编号分别为：DQ314744、DQ314745、DQ314743、DQ314746。 运用GENERUNNER软件对我们克隆的鸡、火鸡、鲤鱼、虹鳟鱼、黑牛头鱼的序列进行分析，结果显示：鸡的FBN1蛋白中编码1170位氨基酸的碱基为GTA，其对应的氨基酸为更多还原

【Abstract】 Aims: To develop a DNA-recombination independent cloning method; 2) To clone FBN1 genetic fragments of several types of birds and fishes; and 3). To analyze the evolutionary conservation of the amino acid 1170 in order to determine the possibility of the causal relationship of a rare Rl 170H identified in an atypic Marfan syndrome.Method: Degenerated primers targeting the conserved amino acids flanking the R1170H relevant point mutation were used to clone the DNA fragments of FBN1 of chicken, turkey, carp, rainbow trout, and blackbull head. The amino acid sequences of the cloned DNA fragments of the newly cloned FBN1 were deduced and aligned using the program of Alignx.Results: An inverse nested PCR was developed and successfully applied in the cloning of 4 pieces of new FBN1 genes of turkey, carp, rainbow trout, and blackbull head. These four newly cloned sequences were submitted to NCBI with the accession numbers of DQ314744, DQ314745, DQ314746, and DQ314747 issued. The inverse nested PCR was also used to confirm the FBN1 sequence of chicken. The amino acids at the location of 1170 were histamine (H) for chicken and turkey, glutamine (Q) for the rest three species. The 1170 amino acid is not a highly conserved during evolution.Conclusion: 1). Developed and confirmed the application of using inverse nested PCR for cloning of genes with similar or conserved amino acid sequences within or between gene families during evolution; 2). Cloned 4 new FBN1 fragments from 4 species and submitted to genbank with access numbers issued; 3). The rare mutation of R1170H is not highly conserved during evolution and may not be the cause responsible for the atypic Marfan syndrome recently identified.更多还原