Isolation and Characterization of Mitochondrial DNA from Arehicityars and Metacitrus

Archicitrus와 Metacitrus로부터 Mitochondrial DNA의 분리 및 특성

The purity of mtDNAs isolated from Archicitrus and Metacitrus leaves was higher in percoll density gradient centrifugation than differential and sucrose density gradient centrifugation. The most clear mtDNAs were obtained from mitochondria included in the Interface band of between 21% and 45% under isomotic, low viscosity conditions in the three step discontinuous percoll density gradient centrifugation. DNase treatment to the crude mitochondrlal suspension still more increased purity of mtDNA by the effective removal of the nuclear and chloroplast DNA and mtDNAs were appeared as a single band at middle position of tube by EtBr /cscl density gradient centrifugation. Agarose gel electrophoresis of mtDNAs resolved a single, broad band containing high molecular weight DNAs In all preparation. Yield of mtDNAs was about 110 and 2 ug Per 2009 in mature and immature leaves respectively. The mtDNA fragment patterns showed by EcoR I treatment were indistinguishable with respect to nom bet and position of bands in Archicitrus and Metacitrus. In the pattern of Hind E restriction, the Metacitrus displayed the unique band between 5.0 and 4.0kb, in addition to four fragments about 5.0, 2.4, 2.15, and 2.0kb, respectively, different from Archicitrus. Also the pattern of total mtDNAs fragment by the treatment of Pst I showed that the distinguishable fragment pat tern was not appeared in Archicitrus(C. iyo Tanaka), but about 6.0, 5.5, 5.0 and 2.Bkb fragments were appeared only in Metacitrus(C. junos Sieb). Therefore it was indicated that two species in intra-subgenus were identical each other, whereas considerable difference was revealed for inter-subgenus.