简并PCR法克隆L.starkeyi苹果酸酶基因
Cloning of Malic Enzyme Gene Fragment from L.starkeyi with Degenerate Oligonucleoide Primers
Abstract
根据已报道的酵母苹果酸酶基因序列,利用COdEHOP(COnSEnSuS dEgEnErATE HybrId OlIgOnuClEOTIdE PrIMErS)软件设计两对简并引物,以斯达油脂酵母(l.STArkEyI CICC 1809)总dnA为模板做简并PCr,得到2个基因片段(ME1,ME2).对这2个目的片段进行测序,将结果翻译成氨基酸序列,blASTP结果显示其中ME2片段为油脂酵母未报道苹果酸酶基因的序列(gEnE bAnk登录号gu348991),并对巢式PCr方法检验简并PCr结果的有效性进行了评估. According to the known sequences of malic enzyme gene of yeast,two pairs of primers were designed by CodeHop(Consensus Degenerate Hybrid Oligonucleotide Primers).Two specific DNA fragments were amplified with polymerase chain reaction(PCR) assay from genome DNA of L.starkeyi CICC 1809.Those DNA fragments(ME1,ME2) were sequenced and translated into corresponding amino acid sequences.The ME2 sequence of the two fragment was determined as the unknown malic enzyme gene fragment by alignment to the reported malic enzyme sequences(Genebank number is GU348991).This study also verified the accuracy and effectiveness of the results with the nested-polymerase chain reaction(PCR).