Fosmid Library Construction And Assembly Analysis Of The Mitochondrial Gene Of Rhizoctonia Solani AG1 IA

Rhizoctonia solani is a worldwidely important disease of rice and one of three important diseases of rice in China, but studies on its genome is underdeveloped, which restricts many research work's development. In this study, the first Rhizoctonia solani AG1 IA fosmid library was constructed and the initial analysis has been presented. We sequenced Rhizoctonia solani AG1 IA's mtDNA through Illumina GAⅡtechnology and spliced the sequencing results, finally we got a complete mitochondrial genome sequence.Then assembled and predictied the mtDNA, and analysed the evolutional characteristics of different species in Rhizoctonia solani and the phylogeny between Rhizoctonia solani and other species. Main results are as follows:1. Construction and characterization of Rhizoctonia solani AG1 IA fosmid libraryThe Rhizoctonia solani AG1 IA genome has been sequenced before the library's construction, and it was 36.4Mb. The genome DNA was extracted from Rhizoctonia solani AG1 IA,and electrophoresis. Selected and recovered the fragments which were lager than 36kb from low melting point gel using agarose enzyme. After puritied, the prepared DNA was end-repaired. The collected fragments were ligated into the fosmid vectors and then packaged to construct the library. The final fosmid library was consisted of 5000 clones, and the frequency of recombinant clones was 100%.16 clones was selected randomly from the library, digested with NotI enzyme and detected through pulse electrophoresis. The results suggested that the inserted fragment was almost 35-45Kb,with an average size of 35kb, the coverage was 4.81 times of Rhizoctonia solani AG1 IA genome.8 clones were picked out from the 16 clones to end-sequencing and blast in ncbi. As a result, one was not successfully sequenced and all the other sequences were related with the fungi genes. The fosmid library will serve as a useful resource for genetic mapping, physical mapping, and positional cloning of Rhizoctonia solani AG1 IA。It will be important to better understanding of Rhizoctonia solani genome.2. Isolation of the mtDNA of Rhizoctonia solani AG1 IAl)mtDNA of Rhizoctonia solani AG1 IA was obtained by the method of SDS and DNAse. Speeding centrifugal separation was used to get Rhizoctonia solani AG1 IA mtDNA;2) The mitochondrial genome was identified by blasting the sequences in NT database.An154kb mitochondrial genome sequence with 28 gaps was gained by SOAPdenovo. These gaps were filled up by PCR,and the primers were designed according to the upstream and downstream sequences of each gap. Finally, we got an 146kb mtDNA.3. Analysis of the complete mitochondrial genome of Rhizoctonia solani AG1 IA.We got 21 ORFs which are high similar to mitochondrion genes of fungi. Some conserved mitochondrial genes such as COX1, COX2, ATP6 were found. In the prediction process, BLASTX was used to identify candidate homologous genes from Swiss-Prot database, and some useful ORFs were got by using exonerate aligning sequence from Swiss-Prot proteins. From the known DNA sequence,1 tRNA genes,1 ribosomal RNA gene and 4 ribozyme gene were identified by Rfam v9.1. And other 25 tRNAs were detected by using tRNAscan-SE.1) Mitochondrial genome and its structure:the mtDNA was 146118bp and had 33.8% GC. It contained 26 tRNA genes,1 ribosomal RNA gene and 4 ribozyme genes,21 kinds of protein-coding genes.2) composition and second structure of transfer RNAs:mtDNA contained 26 transfer RNAs, and had 2 tRNA-Leu and tRNA-Arg,3 tRNA-Met, and 2 tRNA-Undet whose function were unknown. The other 17 kinds of amino acid had only one tRNA. The second structure of tRNA was made by the tRNAscan-SE. 3)Protein-coding genes:we predicted that there were 21 kinds of protein peptide, corresponding to 21 genes:cytochrome b(ctyb cyb), atp6 and 8 subunits (atp6,atp8), NADH dehydrogenase (ndl,nd3,nd4,nd5,nd6,nd6)cytochrome,C oxidasel,2 and 3(coxl, cox2, cox3), BI(BI1, BI1, BI2), and five unknow genes(IAmt2, IAmt6, IAmt7, IAmt8, IAmt18).3.Evolution of mitochondrial genome in Rhizoctonia solani AG1 IA:Based on the similarity of atp6 and cytb of Rhizoctonia solani AG1 IA with related sequence of other fungi in GenBank, the phylogenetic trees of gene atp6 and cytb were made,which could be referenced for classification and identification of related species.