Evolutionarily conserved untranslated regions facilitate the cloning of complete coding sequences of chondriogenes encoding NADH dehydrogenase subunits in higher plants

In plants, the mitochondrial NADH dehydrogenase(complex I) is a large protein complex transferring electrons to ubiquinone.For the nine chondriogenes encoding complex I subunits (nad1,nad2, nad3, nad4, nad4L, nad5, nad6, nad7, and nad9), an efficientstrategy for the cloning of complete coding sequences (CDSs) is important.Specific orthologous portions of untranslated regions (UTRs)were found based on multiple sequence alignments of chondriogeneorthologues encoding complex I subunits in plant species. Based onthe conservation of partial UTRs, a one-step PCR strategy was conceivedfor the cloning of CDSs of the nine chondriogene orthologues.Using this strategy, the five complete mitochondrial open readingframes (ORFs) , which encode mitochondrial NADH dehydrogenasesubunits, nad1, nad2, nad6, nad7 and nad9 respectively, were cloned inthree angiosperm species: kenaf (Hibiscus cannabinus), camphor tree(Cinnamomum camphora), and ramie (Boehmeria nivea). The fifteencloned PCR products also included 5' and 3'-UTR partial sequences.Moreover, a potential C-U RNA editing site was identified in the startcodon of kenaf nad9. In conclusion, the simple and efficient strategyavoids the use of time-consuming rapid amplification of cDNA ends(RACE) process, and facilitates the cloning mitochondrial completeORFs whose 5' and 3' flanking UTR contain an orthologous regionwith some degeneracy in higher plant species.