Mitochondrial genes atp6 and atp9 cloned and characterized from ramie (Boehmeria nivea (L.) Gaud.) and their relationship with cytoplasmic male sterility

Cytoplasmic male sterility (CMS) in plants is known to be associated with structural change and the presence of new chimeric genes in mitochondrial DNA (mtDNA). In this study, the atp6 and apt9 gene fragments were cloned from mtDNA of a CMS line and its maintainer and restorer lines of ramie (Boehmeria nivea (L.) Gaud.) using PCR and degenerate primers that were designed according to the conserved sequences within the coding region of chondriogenes atp6 and atp9 of some dicotyledons recorded in GenBank. In spite of incompleteness of the coding region, the cloned fragments showed a homology of over 94% and over 85% with atp6 and atp9 genes, respectively, from the dicotyledons in GenBank. The whole sequences of atp6 and atp9 genes including the complete open reading frames were cloned by amplifying the 3′ and 5′ end unknown sequences of these gene fragments using a DNA Walking strategy. The atp6 gene showed no difference among the CMS line, maintainer and restorer lines of ramie in DNA sequence, transcription and translation, and in the levels of protein. However, compared to the atp9 gene from the maintainer and restorer lines, within the coding region, the atp9 gene from the CMS line had a number of different nucleotides and a sequence deficiency of as many as 21 nucleotides at the 3′ end. An unusual high expression of the atp9 gene in the CMS line at the budding and full-bloom stages was revealed by RT-PCR analysis. The results indicated that the variation in DNA sequence and its encoding product, and/or the abnormal expression of the atp9 gene in the CMS line, may be closely related to male sterility in ramie. This work provides basic knowledge for understanding the function of the atp9 gene causing CMS in ramie and subsequently the molecular genetic mechanisms.