【摘要】 目的:克隆猴胆碱乙酰转移酶(ChAT)cDNA,并将猴ChAT cDNA测序结果与猪、大鼠、小鼠、原鸡以及人的ChAT cDNA序列相比较。方法:以改良的酸苯酚方法提取成年日本猴脑纹状体总RNA。以RNA为模板经逆转录获得cDNA第一链,设计基因特异性引物扩增cDNA链上不同片段。经序列分析仪获取ChATcDNA完整序列。结果:在猪、大鼠、小鼠、原鸡中作为起始密码的ATG密码子的位置在猴ChAT cDNA中由ACA所取代,而猪、大鼠、小鼠、原鸡及人类cDNA中终止密码子TGA的位置在猴ChATcDNA中则由TAA取代。猴ChAT序列与人类、猪、小鼠、大鼠以及原鸡ChAT序列的相似程度分别为96.36%,87.96%,85.47%,84.88%和71.84%。氨基酸序列显示,ChAT中乙酰辅酶A结合区域和两个可能的钙/钙调蛋白激酶的磷酸化位点在猴ChAT中未发生改变。结论:猴ChAT cDNA与人ChAT序列最相似,与重要功能基团作用相关的氨基酸序列在猴ChAT中具有保守性。更多还原

【Abstract】 Objective: To clone the cDNA of monkey choline acetyltransferas(ChAT) and compare the sequence of monkey ChAT cDNA with that of pig,rat,mouse,gallus and human. Methods: Total RNA from Striatum of adult Japanese macaque was abstracted with the modified acid phenol method. RNA was reversely transcribed using superscript RNase H-reverse transcriptase to obtain the first-strand of cDNA,which was amplified with gene-specific primer pairs for different part of ChAT. All of the final polymerase chain reaction (PCR) products of different sizes were sequenced using the ABI 310 sequencer (Perkin Elmer,CA,USA) according to the manufacturer’s instructions. Results: In monkey ChAT cDNA,the ATG codon assigned to the putative initiation codon for pig,rat,mouse and gallus was replaced by ACA and the TGA codon assigned to the termination codon for pig,rat,mouse,gallus and human was replaced by TAA. The percentage of similarity in the ChAT sequence between monkey and human,pig,mouse,rat,and gallus is 96.36%,87.96%,85.47%,84.88% and 71.84%,respectively. The amino acid sequence showed the acetyl CoA binding domain in ChAT and two potential phosphorylation sites (Arg-Xaa-Xaa-Ser) of calcium/calmodulin protein kinase were preserved in monkey ChAT cDNA. Conclusion: The monkey ChAT cDNA has the highest similarity to the human ChAT. The amino acid sequences related to the important functional groups were conserved in the cDNA of mokey ChAT.更多还原