一个推测的拟南芥漆酶基因在巴斯德毕赤酵母中的表达

Expression of a Putative Arabidopsis Laccase Gene in Pichia pastoris

At5g01040基因是一个推测的拟南芥漆酶基因。根据其编码序列设计引物,利用RT-PCR方法扩增出1755bp的片段。测序结果表明,该片段存在3个突变位点,其中一个突变位点改变了所编码的氨基酸。将该片段克隆到表达载体pPICZaB上,电击转化毕赤酵母。经筛选获得80个候选重组菌株,其中18个菌株的培养液中存在漆酶活性,说明所克隆的基因在毕赤酵母中实现了分泌表达,证实了At5g01040编码的蛋白具有漆酶活性。

At5g01040 encodes a putative laccase in Arabidopsis. Primers were designed according to the coding sequence of the gene. Using of reverse transcription-polymerase chain reaction （RT-PCR） with above primers, a fragment of 1755 bp in length was amplified from total RNA of Arabidopsis. Sequencing result showed that there were three mutant sites in the cloned fragment with comparison to the sequence published in GenBank. However only one of them changes encoded amino acid, and this amino acid is not in conserved domain or motif found in functional laccase. The sequenced fragment was fused downstream of a N-terminal peptide encoding S. cerevisae a-factor secretion signal driven by AOX1 promoter in the pPICZaB and the recombinant vector was then introduced into Pichia by electroporation. Eighty Zeocinresistant colonies were identified by PCR as candidate lines carrying At5g01040. Laccase activity analysis of individual candidate lines showed that eighteen candidates had laccase activity, suggesting that cloned gene is able to express secretively in Pichia cell and At5g01040 encodes a protein with laccase activity.