| Microsporidia is an obligated intracellular parasitic organisms that can infect almost all animals from protozoa to mammals,including insects,fish and humans.The Nosema antheraea is a pathogen of pebrine disease to Antheraea pernyi,which can bring great hazard to the production of Antheraea pernyi.The current research of Nosema antheraeae at the cellular and molecular level is still seldom involved in.In this thesis,we firstly analyze the nuclear apparatus and chromosomal DNA of Nosema antheraeae.Secondly,we sequenced the genome of Nosema antheraeae and presented the comparative genomics results between Nosema antheraeae and other closely related species.The spores of Nosema antheraeae were collected from the silkglands of Antheraeae pernyi,and then were purified by the percoll gradient centrifugation.The purified spores that stained by the dye of DAPI or SYBR Green I were observed under the fluorescent microscopy.The observation show that spores stained DAPI and SYBR Green I can be distinguished clearly under the microscope.It can be found that the 3-5?m spores have the thick spore wall and contained double nuclears.And then,the ulstrustrure of Nosema antheraeae were observed under the electron microscope.The results show that the spores contain common structures,however,some important organelles such as mitochondria,peroxisomes,Golgi apparatus and microtubule structure cannot be seen under the current technique in this study.To preliminarily analyze the genomic size of N.antheraeae,the MFI measured by flow cytometry of the presumed diplokaryotic microsporidian N.antheraeae was compared with that of N.bombycis,a diplokaryotic microsporidian whose karyotype and total genomic DNA content is well characterized.Using the MFI ratios calculated from flow cytometric readings,the absolute DNA content of N.antheraeae spores was calculated to be lower than that of N.bombycis.Because the haploid genome size of N.bombycis is known to be 15.3 Mbp,the haploid genome of N.antheraeae is calculated to be lower than 15.3Mbp.About 109 spores of Nosema antheraeae were collected after percoll gradient purification,and the plugs were made after the spores were embedded in the low melting point agarose.After the plugs were digested in the solution of protease enzyme,PFGE were conformed to estimate the numbers and size of each chromosome in Nosema antheraeae.Pulsed-field gel electrophoresis showed that N.antheraeae exhibited a pattern consisting of several bright and faint bands of variable staining intensity,and that the chromosomes were between 375-and 960 kb.Because co-migration of similarly sized molecules can occur,it was difficult to visualize all chromosomal DNAs in a single separation step.Therefore,two alternative PFGE conditions were used to improve the resolution of particular zones of the molecular karyotype.Electrophoregrams permitted the resolution of 13 bright chromosomal bands and two faint ones(i.e.?,?).Therefore,the haploid karyotype of N.antheraeae was determined to be 15 chromosomes.A total of 657 Mb of Illumina reads were obtained after filtering ambiguous reads.Assembly resulted in 6,215 scaffolds for a total of 6.6 Mb of unique sequence,from which 3,413 coding were identified.For the average gene length in a genome-wide scale,we used N.antheraeae as reference and compared five different microsporidian species with N.antheraeae.Considering that the genome size of N.antheraeae is smaller than that of N.bombycis,compactions of gene length and expansion of intergenic region compaction contradictorily exist in the microsporidia Nosema.Gene ontology classification of three Nosema indicated that no distinct diversity of gene functional categories,except for the genes involved in energy metabolism.Further analysis of gene variation involved in energy metabolism of Nosema antheraeae,it was found that the Nosema antheraeae lack of genes that synthesize the ATP/NADPH,and lost several key genes involved in glycolysis,the citric acid cycle,trehalose metabolism and oxidative phosphorylation. |
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