Analyses And Secondary Structures Of Ribosomal RNA Sequences Of Microsporidia Pathogenic To The Silkworm, Bombyx Mori

An attempt was made to classif~,?the reference nine strains of microsporidia infectious to Bombyx mori, ie, MCs, MD, ME; MG, MHa, ML, N.b, MPa and MPr. The ribosomal RNA(rRNA) genes of these microsporidia have been examined. The copies of SSUrRNA gene in genome were identified by Southern blotting. A method for extracting microsporidian RNA was described.I .Three methods for extracting microsporidian genomic DNAThe genomic DNA of adequate quality and amount for cloning and other analyses can be obtained from spores using three methods, ie, InVitro-Germination, DNA-zol and CTAB.The quality and amount of DNA related much to spores germination rate, disruption percent, spores concentration and also depended whether spores were freshly collected.2.Analyses of SSUrRNA genes of nine microsporidia Nuclotide sequences of small subunit ribosomal RNA of nine microsporidia were specifically amplified by polymerase chain reaction (PCR), employing primers complementary to the highly conserved areas in the SSUrRNA sequences.Of microsporidia examined, more than ninety percent sequences ofSSUrRNA were first obtained. Similarity of SSUrRNA sequences betweenthem was 96.4%--98.9%. Evolutionary relationships bforeen these nineland other eighteen SSUrRNA sequences representing different micro-sporidia genera in Genbank were constructed using different methods.SSUrRNA secondary structUre models derived for their sequences weredepicted. The helices showed no difference. These analyses contributed totheir somewhat limited taxonomic classification based on morphology.According to these analyses, the reference nine microsporidia weredifferent species of NOsema.3 .The complete SSUrRNA gene of Nosema. bombycisNuclotide sequence of the SSUrRNA gene 3'-end was specificallyamplified using the SSP-PCR (single specific primer -PCR) technique withan primer matching a highly conserved part of SSUrRNA genes. The entirecoding region for SSUrRNA of NOsema.bombycltr was l233bp as describedelsewhere. The sequence shared high homology with those of many micro-sporidia, especially NOsema.aPltr (l00% identity over the last l40bp of theSSUrRNA gene ).4.The so gene of MPr isolated from Pl'eris raPae L.The SSUm gene 3'-end, the illtemal transcribed spacer and LSU-AnA gene 5'-end (580R, 580 region, Escherl'chlb.coli numbering) wereobtained using PCR with primers complementary to the highly conservedareas in the am sequence.95A putative terminal sequence of the SSUasA gene of MPr wasidentified by comparison to the highly conserved terminal regions found inother NOsema and foirimorPha species. The entire coding region for SSU-rRNA of MPr was l244bp. The intemal transcribed spaces was 4lbp. TheLSUrRNA 580R was 470bp and showed high homology with the corres-ponding sequence of Nosema.aoprae.5.The copies of SSUrRNA geneWhen the radiolabelled probe synthesized by random oligonuclotide..primers using SSUrANA gene cloned as template hybridized with therestriction maP of microsporidian genomic DNA, the copies of SSUrRNAgene were identified at least l5.6:Microsporidian RNA extractionA complete procedure for extracting RNA from spores was described.This was the first time that the method has been published in details. Themethod for extracting RNA here described was Guanidine/Acid phenol/Chloroform.