WAT11, WAT21 yeast strains酵母菌株-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

WAT11, WAT21 yeast strains

Cat No.:NTCC504041

Introduction

The ATR1 and ATR2-1 open reading frames were isolated from pUC19/ATR1 and pUC19/ATR2-1

as BamHI-EcoRI andBamHI-BamHI fragment, respectively. TheATR1fragment was inserted at

theBamHI-EcoRI sites of pYeDP110, yielding pATR1/DP110. Similarly, the ATR2-1 fragment was inserted

at the uniqueBamHI site of pYeDP110, and a clone with the proper orientation was selected and named

pATR2-1/DP110. The NotI fragment of each plasmid encompassing URA3 and the ATR coding sequence

was isolated and used to transform WRΔ cells. Since in WRΔ the CPR1 locus is disrupted with

theTRP1 gene, integration events result in a phenotype shift from Ura− Trp+ to Ura+Trp−. This procedure

yielded two new yeast strains, WAT11 (for ATR1) and WAT21 (for ATR2-1). They express

theArabidopsis CPR instead of the yeast enzyme when grown on galactose. On glucose, WAT cells

express no CPR activity.

Analysis of the coupling between any ATR and a plant P450 requires coexpression in yeast. Genomic

integration of the ATR1 and ATR2-1 expression cassettes at the CPR1 locus was considered. For this

purpose, ATR1 and ATR2-1 coding sequences were cloned into pYeDP110 integration vector, yielding

pATR1/V110 and pATR2-1/V110. The pYeDP110 vector places the heterologous coding sequence under

the transcriptional control of GAL10-CYC1 promoter andCPR1 terminator and provides sequences from

both flanking regions of the CPR1 gene that direct the integration at this locus, resulting in the deletion of

most of the CPR1 gene. The ATR1 and ATR2-1 coding sequences were integrated at theCPR1 locus,

resulting in two new yeast strains, WAT11 and WAT21, respectively.

WAT11 genotype

MATα (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15)