Expression of a Putative Arabidopsis Laccase Gene in Pichia pastoris

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 α-factor secretion signal driven by AOX1 promoter in the pPICZαB and the recombinant vector was then introduced into Pichia by electroporation.Eighty Zeocin-resistant 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.