Analysis of the proteins related to browning in leaf culture of Phalaenopsis

Browning often happens in Phalaenopsis leaf explant cultured in vitro. For understanding the relationship between browning and some putative related proteins in Phalaenopsis leaf explant culture, proteins from fresh and browning tissue were isolated through phenol–acetone method and separated by two dimensional polyacrylamide gel electrophoresis (2-DE). A comparison of 2-DE images revealed that there were both qualitative and quantitative differences in fresh and browning explants. An average of 433 and 395 protein spots were detected in 2-DE gels of fresh and browning explants, respectively. Among them, 19 spots differed significantly between the two samples. Three of the 19 spots were only detected in browning explants and further selected for LCQ–MS/MS analysis. Through peptide querying these proteins were identified as mitochondria ATPase, peroxiredoxin, and regulatory protein-like protein. Two cDNA genes, corresponding to the mitochondrial ATPase and peroxiredoxin, were cloned by RT-PCR with the specific primers designed against conserved regions of homologous protein codifying genes. The results indicated that these two genes were only expressed in the browning explants which were cultured in vitro for 3 days.

► This study analyzed the total proteins of browning Phalaenopsis leaf explant using proteomic method at first time.
► Proteins of fresh and browning Phalaenopsis leaf explant were isolated and separated by 2-DE.
► The protein profile of 22 spots differed significantly between the two samples.
► Three of the 22 spots were identified as mitochondria ATPase, peroxiredoxin, and regulatory protein-like protein.
► The result of RT-PCR showed that two genes only expressed in the browning explants which were cultured in vitro for 3d.