Development Of Molecular Marker Identifying The S/N Cytoplasmic Genotype And Preliminary Analysis The Function Of Orf725 In Onion(Allium Cepa L.)

Onion(Allium cepa L.) is a biennial herb which belongs to Liliaceae Allium. Onion can be eaten in fresh and processed. Because of its nutrient and medicinal value, onion has a reputation as the Queen of Vegetables in the world. Onion has a small flower organ, so it is difficult to produce hybrid by manual control. Therefore the selection of male sterile line and maintainer is particularly important for the production of onion hybrids. In this study, molecular marker was developed based on onion cytoplasmic male sterility-related gene orf725 which can distinguish S or N cytoplasm type of onion. Furthermore, the function of candidate gene orf725 causing CMS trait in onion was preliminary analysised by Agrobacterium-mediated transformation. The main results are as follows:1. A SCAR marker was developed to identify genotypes of onion cytoplasm, named OC2175, according to orf725 gene sequence-specific. Two bands were amplified in the sterile line,2175 bp and 1053bp; while only a 1053 bp band was amplified in the maintainer line. 17 groups of different genetic background CMS and its corresponding maintainer lines were used to examine the marker. And the identification results of the SCAR marker perfectly matched with the phenotypes observed in the field.2. Prokaryotic expression vector pET32a-orf725 carrying orf725 gene was constructed successfully using In-Fusion HD Cloning. The engineering strain with recombination plasmid was obtained by transformation of E.coli BL21(DE3). The expression of exogenous protein was induced by IPTG. The results indicated that the expressed protein of orf725 inhibited the growth of host cell dramatically and the growth curve was decreased significantly compared with the control. Probably because ORF725 as a competitive inhibitor of COXⅠ analogues consumed the coupling locus of COXⅠ analogues, which affected energy production. And the insufficient production capacity affected the grouth of the host cells. Another probable reason was that orf725 coded a toxic peptide, which has a lethal effect on E. coli.3. 5’-flanking mitochondrial targeting peptide was cloned according to the released rice Rf1 b sequence from GenBank. Chimeric gene Rf1b5’-orf725 was constructed by putting Rf1b5’ before orf725. Mitochondrial targeting vector for function identification of orf725 was constructed successfully by connecting Rf1b5’-orf725 into loci of BamHⅠ and SacⅠ.4. Hygromycin-resistant plantlets were obtained by floral-dip method of Agribacteriummediated transformation. Transgenic Arabidopsis mutant plants of orf725 were obtained by PCR detection, GUS staining, phenotypic examination and Alexander staining. The transgenic plants has small flower organ in which some of pollen grain has no activity and showed lower fertility. Probably because expression of ORF725 competitively inhibited the function of normal COX Ⅰ, Thus affecting the energy metabolism. This research laid a foundation for further clarifying the molecular mechanisms of orf725.