Expression and Characterization of a Mitochondrial Gene orf216 from the CMS-Honglian Rice Expressed by Pichia pastoris Yeast

Plant mitochondrion is featured with a great many of repeats and redundant sequences. A lot of unknown orfs involved in CMS are often characterized. Correspondingly, multiple CMS mitotypes carrying unique mitochondrial CMS genes have been identified in some of plant species. Although the characterization of abnormal mitochondrial open reading frames (ORF) associated with cytoplasmic male sterility (CMS) has been well documented, the functional domain of this kind of ORFs’ encoded proteins remains unclear. We studied the ORF216 protein, related to the CMS trait in CMS-HL rice, which is constructed to three different fragments and were introduced into the yeast Pichia pastoris to investigate the possible functional domain. The result showed that the full length and contained PD020792 domain fragment expressions of ORF216 protein inhibited the growth of yeast cells. And in the transformants that expressed the full lengths or contained PD020792 domain fragments of ORF216 protein, the content of reactive oxygen species (ROS) was increased and ATP level was decreased compared with the control. These results showed orf216 gene could have common function as others CMS-associated genes and PD020792 domain could be the functional domain of ORF216 protein. Furthermore, expression of ORF216 and Red fluorescent protein (RFP) as a fused protein in yeast cell showed that ORF216 was able to anchor to mitochondria. These observations provide important evidence for the functional domain and mechanisms of orf216 gene and other CMS-related genes.