Abstract: 【Objective】Cytoplasmic male sterility (CMS) is controlled by the cytoplasmic genome, mainly the mitochondrial genome, which plays an important role in breeding and production of hybrids. Sterile lines and hybrids are the main types of tobacco cultivation in China, accounting for more than 80% of the total tobacco cultivation area. The sterile type of tobacco in China is sua-CMS, which is also the only sterile type of tobacco production, but the control gene and sterility mechanism of sua-CMS are not clear. COXⅡ is the second subunit of cytochrome oxidase (COX), encoded by the mitochondrial genome. Nucleotide base variants occur at the terminal of the COXⅡ in sua-CMS lines, which resulted in the replacement of the C-terminal of COXⅡ Thr²⁵⁷ by Ser. The function analysis of COXⅡ in sua-CMS tobacco provides a foundation for the elucidation of the molecular mechanism of sterility in tobacco.【Method】In this study, four sets of sua-CMS lines and fertile controls were used as materials, and RNA blot hybridization and qRT-PCR methods were used to analyze the transcript and gene expression of COXⅡ between sua-CMS lines and fertile controls, and western hybridization and COX enzyme activity experiments analyzed COX protein expression and COX enzyme activity, and ATP content were measured. The COXⅡ of fertile tobacco was overexpressed in sua-CMS line, and the effects of COXⅡ overexpression on COXⅡ expression, COX enzyme activity, ATP content and stamens development of tobacco were studied.【Result】COXⅡ multi-sequence alignment showed that Thr²⁵⁷ at C-terminal was highly conserved between species. Expression analysis showed that the COXⅡ transcript in sua-CMS line was significantly lower than that of the fertile control, and quantitative expression of four sets of sua-CMS lines and fertile controls showed that the expression amount of COXⅡ in sua-CMS lines was only 0.3%-0.4% of the fertile controls, the COX protein abundances and enzyme activity were significantly lower than that of the fertile control, and the ATP content of sua-CMS line was 52% of that of the fertile control. When COXⅡ was overexpressed in sua-CMS line, the expression levels of COXⅡ in transgenic plants increased by up to 171 times, the COX enzyme activity increased by 70%, the ATP content increased by 90%. However, all indicators including COXⅡ expression levels, COX enzyme activity and ATP content in transgenic plants were lower than those of fertile tobacco. We also observed significant changes in anther morphology in transgenic plants, and the differentiation of the anther wall appeared, but there was no pollen grains in the anthers.【Conclusion】This study shows that the mutant COXⅡ of sua-CMS line affects COX function, which is a main reason of anther energy deficiency, and it is also an important factor affecting the development of anthers.

Key words: tobacco, cytoplasmic male sterility, COXⅡ, ATP level