Cloning and Characterization of Glycolate Oxidase and NADH-Dependent Hydropyruvate Reductase Genes in Pachysandra terminalis

Abstract

Photorespiration provides a protection mechanism in plants by diverting excessive energy accumulated from photochemical reaction, metabolizing toxic products and producing some protective molecules. The authors report cloning and characterization of a glycolate oxidase gene (GOX; NCBI accession DQ442286) and a NADH-dependent hydroxypyruvate reductase gene (HPR; NCBI DQ442287) from Pachysandra terminallis. The DQ442286 had the predicted GOX-like–Riboflavin-5′-phosphate (FMN) conserved domain and the DQ442287 had the predicted adenosine 5′-(alpha-thio)diphospho-5′-ribofuranosylnicotinamide nicotinamide adenine dinucleotide (NAD) binding domain (2-Hacid_DH_C). C-terminal peroxisome targeting signal was predicted to be -ARL for DQ442286 and –SKL for DQ442287. Both genes encoded enzyme proteins that are located in peroxisome and are involved in the photorespiration process. Real-time quantitative reverse-transcriptase polymerase chain reaction was performed to compare transcript level of the cloned genes after cold treatment. The 18s Ribosomal RNA (rRNA) was included to calibrate the data. The relative cycle threshold values (gene/18s rRNA) were 1.4, 1.5, and 1.5 for GOX and 1.2, 1.3, and 1.3 for HPR in the treatments of 4 °C 4 h, 4 °C 12 h, and control. The data revealed that gene expression was enhanced by only short-term (4-h) cold treatment. A ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco) activase gene (DQ 486905) was also cloned and analyzed following the same procedure.