Cloning And Analysis Of Small Subunit Ribosomal RNA Sequence Of Vairimorpha Ceraces Sp.nov. (Microspora: Burenellidae) From The Insect Of Lepidoptera, Cerace Stipatana Walker

Microsporidia are a large group of unicellular, obligate intracellular parasitic protozoa without mitochondria, which can infect a wide variety of organisms including vertebrate and invertebrate. They can cause diseases to fish and insects such as silkworm, bees and locusts. As so far, there are approximately 150 described genera of microsporidia with over 1300 individual species.Microsporidia are considered to be the most primitive unicellular eukaryotes with organic evolution, so it attracts more and more attention in the molecular biology of microsporidia. Because the primary structure of their small subunit ribosomal RNA gene is conservative in all species, these sequences are used to study the eukaryotes evolution and for practical purposes to assist in species identification and to understand relationships among species. And it offers a valuable classification system of microsporidia not only including their biological characters such as morphology and life cycle, but also containing the information of molecular biology and bioinformatics.Vairimorpha ceraces sp.nov. isolated from a kind of field insect which named Cerace stipatana Walker and it can infect silkworm. After the primary study on its morphology,infection,ultrastructure and serologic reaction, it was classified as genus Nosema. Then following research found that it had disporoustic and octosporoustic form, while ocrosporoblast is more. Due to this morphological character, according to biological system for classification of the microsporidia (Sprague, 1992), the microsporidian from Cerace stipatana Walker is placed in family Burenellidae and genera Vairimorpha. A further investigation indicates it is a new species of microsporidia, named as Vairimorpha ceraces sp.nov. named after its host.At present, all the study on it from the respects of morphology,ultrastructure,pathology and biological characters, but this experiment focuses on cloning and analysis of small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov., hoping to provide evidence of molecular biology for its category and evolutional status.1 Cloning of small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov.(1)DNA was extracted by three ways, the general way,TEK and improved CTAB. All are suitable to this microsporidian. 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)Design a pair of primers with the common conserved sequence of small subunit ribosomal RNA gene , after PCR amplification, a fragment that has length of approximately 1200 nucleotides obtained.(3)Cloning of small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov. by the method of T-A cloning. In order to identify these SSUrRNA gene fragment positive bacteria clones, the recombinant plasmids were amplified by PCR and digested by enzyme EcoR I and HindIII at the same time.The small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov. is with 1228 nucleotides. It can be cut by 43 commercial enzymes at 119 sites. GC density is 34.69%, sense strand mol weight as DNA is 37.01 kilodalton. The register number of SSUrRNA of Vairimorpha ceraces sp. nov. is gi161137618|EU267796 in GenBank.2 Similarity analysis of small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov.Analyzed by BLAST, nearly all the sequences which are similar to Vairimorpha ceraces sp. are from the genera Vairimorpha and Nosema. The similarity between the type species of Vairimorpha genera Vairimorpha necatrix is 71%, while it is 99% compared with the sequences of genera Nosema that isolated from the insects of Lepidoptera.3 Homology analysis of small subunit ribosomal RNA sequence of Vairimorpha ceraces sp. nov.Phylogeny tree of microsporidia was constructed by software of ClustalX1.83 and MEGA3.1.The evolutionary distance between Vairimorpha ceraces sp.nov. and other members of Nosema which isolated from Lepidoptera is from 0.00 to 0.01. In phylogeny tree, they form a clade as is close to the type species of Vairimorpha. This phenomenon appeared often in constructing the phylogenetic tree between genera Vairimorpha and Nosema , by used of small subunit ribosomal RNA,small subunit ribosomal DNA and large subunit ribosomal RNA. Maybe there is a close relationship between genera Vairimorpha and Nosema, or its co-evolution with host because of heavy control of the host genome.It is indicated that Vairimorpha ceraces sp. nov. has highest Homology with Vairimorpha sp. German because they are in the same branch.4 Categorization and evolutional status of Vairimorpha ceraces sp. nov.The microsporidia from Cerace stipatana Walker shows their disporoustic and octosporoustic development during sporogenic development, as this experiment focusing on cloning and phylogenetic analysis its SSUr RNA gene. According to biological system for classification of the microsporidia (Sprague, 1992) and recommendation from Canning et al., Vairimorpha ceraces sp. nov. is surely the member of genera Vairimorpha, but it is more suitable to be classified into family Nosematidae.