Studies On Nosema Bombycis Genomics

Microsporidia are unicellular eukaryotes which contain 70S ribosomes and develop as obligate intracellular parasites. They can infect a wide variety of animals such as insect, fishery, shrimp, rabbits, rodents and primates, ranging from invertebrates to vertebrates, including human. Nosema bombycis, which is the first microsporidian, has been identified in the middle of the nineteenth century. From then on, more than 1,300 microsporidia species which belong to 160 genera have been reported. Among them, there are 14 species belonging to 8 genera can infect humans, which lead to serious syndromes in immunocompetent hosts and can cause opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients. Since scientists find microsporidian not only can infect insects and anminals. but also human beings, scientists pay more attention to microsporidian. For a long time, researchers characterized microsporidia as "primitive" eukaryotes without mitochondria, but now they are widely recognized as being fungi which have undergone reductive evolution as so to appear to be primitive organisms. Nowadays, a highly reduced mitochondria organelle has been identified, which is so-called "mitosome". N. bombycis is known as the pathogen of silkworm pebrine, which usually prevails in sericulture and cause large loss in economy. The prevention and treatment of pebrine still is a difficult problem in sericulture. In 1968, using sucrose density gradient centrifuge, Ishihara and Hayashi found the ribosomal sedimentation coefficient of N. bombycis is 70S, which includes 50S and 30S subunits. As a eukaryote. microsporidian has prokaryotic 70S ribosome. We still don't know what cause this phenomenon. For the study on ribosome of microsporidian, there are many reports mainly on the ribosomal RNA and ribosomal protein aspects, especial on the ribosomal RNA.In this research, we perform a genome-wide survey for ribosomal protein genes and rDNA sequence of N. bombycis. At first, we identified all ribosomal protein genes in N. bombycis genome, and investigated the distribution of these genes in genome, then analyzed the gene order among microsporidian. Also, we explored the distribution of rDNA in genome and the chromosomal location of rDNA units. We analyzed the polymorphism of rDNA sequence of N. bombycis isolates from different regions of China. Additionally, we performed some experiments on activity of rDNA which harbors the MITEs sequence. At last, using the genes'length and structural analysis, we give an initial explore on the cause of the 70S ribosome formation.1. Analyses and identification of the ribosomal protein genes in N. bombycisAs the result we identified 130 CDSs corresponding to 73 ribosomal protein genes, and N. bombycis has one more ribosomal protein CDS than Encephalitozoon cuniculi. Among them, three ribosomal protein genes (RPL19, RPS4 and RPS18) with short introns (23 or 24bps) were verified by N. bombycis ESTs, and they have the same structure as E. cuniculi, Nosema ceranae and Octosporea bayeri. The core promoter and regulatory elements of partial ribosomal protein genes were predicted on the 500 bps upstream sequences. The arrangements of'AAATTT-like signal-CCC-like (or GGG-like) motif-transcription start site'are present in the upstream sequences. Several regulatory elements, such as SP1, GATA-1 motif, were detected, which may-have synergy with introns of ribosomal protein genes for its high transcriptional frequency. There are 56 (about 76.7%) ribosomal protein genes of N. bombycis located to syntenic blocks, which suggested their position were conserved among microsporidian species. And phylogenic trees of ribosomal protein genes do not support the fungi origin of Microsporidia, which suggest this group genes may be not suitable for phylogeny analyses.2. Polymorphism of rDNA sequence and chromosomal localization of rDNA units in N. bombycis.Using the RepeatMasker program, we perform a genome-wide survey in N. bombycis genome for rDNA units. Totally, we detected 42 LSU rDNA,40 SSU rDNA and 37 5S rDNA units. At the same time, we perform Southern blotting which use three different rDNA probes to hybrid with separated chromosomes of N. bombycis. We got the same result that rDNA units distributed on all chromosomes of N. bombycis. The rDNA polymorphism analyses for N. bombycis isolates from different regions Chongqing, Guangdong, Guangxi and Yunnan in China identified the differentiation in species is obvious existed in the N. bombycis. This phenomenon may be result from the genetic variation in passage or the tissue specificity of its host. At the same time, all sequences of Sichuan isolate form a sole branch. This indicates that the difference of geographical population does exist. The sequences from other regions were mixed in phylogeny tree, which may be caused by crossing transmission. The sequence polymorphism indicates the diversity and complexity of the genetic background in N. bombycis. This may be adaptability for living environment in the evolution of N. bombycis. In general, we think this result from three aspects:genetic mutation in passages, host tissue specificity and different geographical environment.3. The activity of rDNA inserted with MITEs sequence in N. bombycis.Using the genome data of N. bombycis, four families of miniature inverted-repeat transposable elements (MITEs) in ribosomal DNA (rDNA) were characterized in the N. bombycis and were named LSUME1, ITSME1, SSUME1 and SSUME2. The genome-wide investigation shows that these MITEs families distribute randomly in N. bombycis genome. All insertion sequences have conserved characters of MITEs, such as high AT content, the direct repeat (DR) sequence and terminal inverted repeat (TIR) sequence at both ends of each MITEs sequence. Additionally, secondary structures of rRNA contained MITEs sequence were predicted and find that these MITEs sequences locate in variable region or expansion segment (SSUME1 locates in V2 region. SSUME2 locates in V4 region and LSUME1 locates in D9 region) of rDNA. At the same time, northern blotting and dot blotting have been performed to detect the transcriptional and functional activity of the rDNA containing MITEs insertion. Fortunately, we found the rDNA which harbors the MITE not only can be transcripted but also can be assembled into a ribosome. However the biological and structural significance of this observation is not readily apparent.4. The formation of 70S ribosome in N. bombycis.Through analyses of rDNA length among different species (including prokaryote and eukaryote), we find the rDNA length of N. bombycis is the shortest. Further analyses of rRNA secondary structure indicate that some stems or loops are reduced in N. bombycis. At the same time, using the same method to analyze the ribosomal proteins, we find that the length of the majority of ribosomal proteins in N. bombycis is between that of the prokaryote and the eukaryote. Secondary structure comparison indicates that the number ofα-helix andβ-strand is slight smaller than that of other eukaryote. According to this information, we can conclude that the ribosomal proteins and rRNA of N. bombycis both have some degree of reduction or decrement, and maybe this reduction result in this special 70S ribosome present in eukaryote N. bombycis.