The Draft Sequence And Phylogentic Analysis For The Genome Of Nosema Bombycis

Microsporidia are obligate intracellular parasitic protozoa, which can infect a wide variety of organisms including vertebrate and invertebrate (particularly insects). Some species lead to several syndromes in immunocompetent hosts and cause opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients. By now, more than 1200 microsporidia species belonging to 150 generas have been reported. However, the evolutionary origin of the microsporidia still puzzles biologists. Microsporidia were initially classified as an early branch from prokaryotes to eukaryotes, whose rRNA is of prokaryotic type without a separate 5.8S rRNA. Microsporidia have a nucleus and an intracytoplasmatic membrane system, atypical Golgi apparatus, while they lack peroxisomes and mitochondria. Recently, more and more evidence to support a fungal origin for microsporidia has emerged, and the latest studies have indicated that microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree. In particularly, it has been reported that several genes related to some mitochondrial functions, such as Fe-S cluster assembly, was found in microsporidia, suggesting that microsporidia have retained a mitochondrion-derived organelle.In the past decade years, the interest in the molecular biology of microsporidia has increased, such as many aspects of their ribosomal RNA genes have been reported, however, the molecular knowledge of microporidia is still rather limited. And there is little molecular information about the mechanism of microsporidia vertical transmission, and how the microporidia invade the host. Now, the genomic sequencing has been used widely in studing the aimed species by the method of bioinformation and comparative genomics, and through the genome, it can offer the high quantitative, large-scale and accurate information about the species. In 2001, The Encephalitozoon cuniculi genome with 2.9 Mb was published and characterised as genome compaction, and many biosynthetic pathways were lost, indicating a strong reliance on the host cell for energy and nutrients. The organization of the Antonospora locustae genome with 685 Kb size also displays the same characteristic with genome compaction and stability. So, the more genomes of microsporidia which will be sequenced completely might bring out much new knowledge and idea for us, and it can improve the research level of this phylum.As a member of the phylum microsporidia, Nosema bombycis is known as a pathogen of silkwormpebrine, which usually prevails in sericulture. Currently, N.bombycis is characterized as approximately 15.3 Mb with 18 chromosomes by pulsed-field gel electrophoreisis. Genome size of microsporidia varies from 2.3 to 19.5 Mb, and the genome of N.bombycis is 15.3 Mb, much larger than that of E.cuniculi, why cause this great divergence? So, there are many interesting questions to be disclosed about N.bombycis. In this study, we have finished the draft for the genome of Nosema bombycis, and did some analysis by means of comparative genomics, such as homologuesical gene, gene duplication, and transposable element in N.bombycis.1) Constructing library, sequencing and assemblingA plasmid library was constructed: a clone library in the plasmid vector pUC18 harboring total genomic DNA which was restricted randomly by SmaI into 1.5-2.0 kb fragments that were inserted into the unique SmaI site. And then, about 14×10~4 plasmid reads were sequencing using ABI MegaBACE 1000 which sum up to 4-fold sequencing coverage of genome. All the reads were assembling into 1096 scaffolds (>2Kb) by using RePs (including PHRED, PHRAP and CONSED) programme, which N50 of scaffolds is 32.6 Kb. The length of total scaffolds reachs 12.7 Mbp, and among them, the largest scaffold is 550Kbp length.2) Gene prediction and comparactive analysisCombining the gene predicted biosoftware and homologuesical analysis, 6356 genes were identified in the N.bombycis genome, which the average GC% of complete genes is 33%. It is found that there are abounding homologues among the N.bombycis, E.cuniculi and S.cereviase. By comparing the gene length of common homologues among three related species, it shows that the genes of N.bombycis have reduced, related to E.cuniculi or S.cereviase, suggesting gene reduction has happened in microsporidia. Analysis of the order of gene array between N.bombycis and E.cuniculi shows that most of homologues are in the same context, and even more than 72% homologues in N.bombycis fall in the syntenic blocks. The results indicate that the order of gene array in microsporidia is highly conserved and the microsporidia genomes are slowly evolving, related to other fungi, however, great genomic rearrangement has still happened after the divergence of N.bombycis and E.cuniculi. Pairwise comparison of orthologous proteins between several closely related species indicates that the highly divergence of proteins in microsporidia, and the orthologous proteins between N.bombycis and A.locustae are more divergent than that between N.bombycis and E.cuniculi. In addition, the orthologous proteins between N.bombycis and E.cuniculi are slightly less divergent than that between S.cereviase and S.pomb ,which both belong to ascomycota, suggesting that divergent times between N.bombycis and E.cuniculi might be shorter than that between S.cereviase and S.pomb, whose divergent time is reckoned about 330-420 millionary years.3) The characteristics of gene family and gene duplication in Nosema bombycisThe clustering of all the genes show that a mass of gene families exists in Nosema bombycis, and the families which contain only double genes sum up to 71.2% of total gene families.In the same families, many genes are found to show high similar to another gene, and more than 80% of all closest gene pairs is higher than 80% identities at the level of amino acids. This indicates that gene duplication has happened in N.bombycis genome, which is one of factors resulting in gene family. Through calculating the Ks(the number of synonymous substitutions per synonymous site) value of pairs of gene duplication, it shows that most Ks value of pairs of gene duplication are distributed at 0-0.25, furthermore, in the Ks value of 0.125, there appear an obvious peak. This suggests that most of gene duplication have happened simultaneously in close evolutionary time, and genomic duplication might have occurred in the N.bombycis. Of common genes (the gene in N.bombycis that homologuesical with that of E.cuniculi), it was found that the number of families which contain more than two genes is much fewer than that of specific genes (the gene in N.bombycis that non-homologuesical with that of E.cuniculi), indicating that specific genes are easier to engender into gene families, for adapting to their specific host. The Ka/Ks value of most pairs of gene duplication is small than 1, suggesting that most genes are suffered from the pressure of purifying selection after duplication. However, the pressure of purifying selection of specific genes after duplication is less than that of common genes, indicating that after duplication, specific genes are more inclined to mutate to change their gene function. All these results suggest that the form of gene family and gene duplication is closely correlative to the parasitical liftstyle of Nosema bombycis, and these charateristics should be the result of evolutionary selection that N.bombycis is obligately parasitized in its host, Bombyx mori.4) Characterization and genomic evolution of transposable element in Nosema bombycisTwelve Tel-like (Tc1Nb) and one piggyBac-like (NbPB1) DNA transposons are characterized in N.bombycis genome. The structures of all the Tc1Nb transposons are very conservative, such as their ITR sequence, and D,D34E catalyse motif in ORFs of transposase. Analysis of sequence divergence for all copies of each element shows that Tc1Nb6 have the lowest average nucleotide diversity. And RT-PCR shows that Tc1Nb6 element can be transtribed in vivo. All these suggest that Tc1Nb6 should be still active in N.bombycis, which can have valuable implications for the design of new, high-efficiency vectors to be used in transfection studies in microsporidia. NbPB1 transposon is identified firstly in microsporidia which is piggyBac-like element, and its structure is very similar to that of BmPBLE12 element which characterized as piggyBac-like element in Bombyx.mori. Southern blotting suggests that NbPb1 has at least three copies in N.bombycis. Phylogentic tree of transposase shows that NbPB1 is closest to BmPBLE 12, and other piggyBac-like transposase segments in N.bombycis are also closest to that in B.mori, indicating that NbPb1 should be horizonal transferred from B.mori genome.The eight complete N.bombycis long-terminal repeat retrotransposons (Nbr) had been identified in the genome. All of these Nbr elements are Ty3/gypsy members and have close relationships to Saccharomycetes LTR retrotransposons identified previously, providing further evidence of their relationship to fungi. To explore the effect of retrotransposons in microsporidian genome evolution, their distribution was characterised by comparisons between two N.bombycis contigs containing the Nbr elements with the completed genome of the human parasite Encephalitozoon cuniculi, which is closely related to N.bombycis. The Nbr elements locate between or beside syntenic blocks, which are often clustered with other transposable-like sequences, indicating that they are associated with genome size variation and syntenic discontinuities. The ratios of Ka/Ks of the open reading frames among members of each of the eight Nbr families were estimated, which reveal the purifying selection acted on the N.bombycis LTR retrotransposons. These results strongly suggest that LTR retrotransposons play a major role in reorganization of the microsporidian genome. The present study presents an initial characterization of some transposable elements in the N.bombycis genome and provides some insight into the evolutionary mechanism of microsporidian genomes.