A Unique orf160 Cloning and Genetic Transformation of Gossypium harknessii Cytoplasmic Male Sterile Line in Upland Cotton

Abstract

The cytoplasmic male sterile (CMS) line with the cytoplasm of Gossypium harknessii has been reported for 50 years, and hybrids have been used in commercial production, but the mechanism remains obscure. The flanking sequences of mitochondrial functional gene from “three lines” (CMS line, corresponding maintainer and restorer line) were analyzed. A specific ORF (open reading frame) of 480 bp, orf160 gene was cloned in CMS line, which located in the downstream of atp4, its N-terminal was homologous with atp6, and the C-terminal was homologous with nuclear sequence, and encoded 159 amino acids that were homologous with membrane proteins and cell cycle protein. The subcellular localization of onion epidermal cells showed the green fluorescence on cell membranes, so the mitochondria-targeting carrier can be normally expressed. The expression vectors were constructed and transformed into yeast, arabidopsis and tobacco, respectively. Compared with control, the modified yeast was abnormal and grew slowly; transformed arabidopsis and tobacco showed that the most seeds were abnormal, small and shriveled; lower seed setting rate, germinate activety and pollen grain vitality showed in individuals in T1 and T2 generations. All these showed that orf160 affects on the development of transformants, and further research should verify if the function of orf160 relates to the cytoplasmic male sterility of cotton.