Isolation And Sequence Analysis Of The Mitochondrial Genome DNA Of Tilletia Indica Mitra

Tilletia indica Mitra, as an important pest around the world which causes Karnal Bunt of wheat, was very closed to T. walkeri in phylogenetic relation and morphological characteristics. Molecular biology methods applied to identification of T. indica within 10 years which has the difference of only one base with the related species-T. walkeri in the sequence of internal transcribed spacers. As a unique cytoplasmic genetic system, mitochondrial DNA (mtDNA) is a kind of complete, small, self-replicating and diverse genome,which has faster evolution of 5 to 10 times than nuclear genome, often be used to analysis the phylogenesis and genetic relationship. The study on the mitochondrial genome complete sequence of T. indica will help to analyse the genome composition, gene order, structure and evolution characters of T. indica; Moreover, phylogenetic analysis of different species in Genus Tilletia can further explain the genetic and evolutional characteristics between T. indica and other species.This paper constructs a method which isolates fungi mtDNA simple and rapid. The complete mitochondrial genome of T. indica was sequenced to analyse the evolutional characteristics and phylogenetic relationship of T. indica, including:1. Isolation of the mtDNA of T.indica. mtDNA of T. indica was obtained by the method of CsCl /Bisbenzimide density gradients ultracentrifugation, and was proved by the digestion of restriction endonuclease;2. Analysis of the complete mitochondrial genome for T. indica.(1) Mitochondrial genome and its structure, the mtDNA is 65 147 bp with 71.14 % AT (GenBank Accession No. DQ993184) by constructing the DNA library. It contains 14 kinds of protein-coding genes, two kinds of ribosomal RNAs, 24 transfer RNAs. Compared with four kinds of Basidiomycetes, its genomes composition is the same, but the gene order is greatly different.(2) Composition and second structure of transfer RNAs, It contains 24 transfer RNAs. tRNA-Ser and tRNA-Arg have two kinds each one, tRNA-Met has three kinds, and the other 17 kinds of tRNA have only one kind individually. The second structure of RNA is made by the software of tRNAscan-SE.(3) Protein-coding genes, It contains 14 kinds of hydrophobic protein altogether, such as genes coding for cytochrome b(cytb), atp synthase 6, 8, and 9 subunits(atp6, atp8, atp9), cytochrome C oxidase 1, 2, and 3(cox1, cox2, cox3), NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6(nadl, nad2, nad3, nad4, nad4L, nad5, nad6). All the initiator are ATG; 11 kinds of terminator with TAA and three with TAG 5 kinds of genes have intron(namely nad1, nad4, nad5, cox1, cytb), and 9 kinds are single gene (namely atp6, atp8, atp9, cox2, cox3, nad2, nad3, nad4L, nad6). 3. Evolution of mitochondrial genome for T.indica. Based on the similarity of genes atp6 and cytb of T. indica with related sequence of other fungi in GenBank, the phylogenetic trees of gene atp6 and cytb were made to be reference for classification and identification of related species.