Studies On The Functional Genome Of Nosema Bombycis-characterization Of The Signal Sequence Receptor Of Nosema Bombycis

Microsporidia are a group of obligate intracellular and unicellular eukaryotic parasites,widely distribute in the nature,it can infect nearly all vertebrates and invertebrates,including human beings.Microsporidia have obtained an extremely strong parasitic adaptability in the long evolutionary process,it can depend on the host cell nutrients to complete their own metabolism and life activities.Nosema bombycis was the first microsporidia isolated and identified in 1857,which can infect silkworm and cause Pébrine.The disease is a major threat to world sericulture,ever resulted in a huge blow to the European sericulture in 19 th century,so far N.bombycis is still the only statutory quarantine object in silkworm-feeding countries.Although microsporidia belong to the eukaryote,genomes of microsporidia are highly reduced,with several characteristics of the prokaryotic genome.We identified signal sequence receptor（SSR）in the nucleotide level and protein level in N.bombycis,and analyzed alternative polyadenylation sites in the gene.The main results are as follows:1.Sequence analysis of Nbssr of N.bombycisSynteny analyses find that among several different microsporidia genomes there was a unique nucleotide fragment named NBO₃2g0035 on the No.32 scaffold in N.bombycis genome draft map,which codes an orphan protein,NBO₃2g0035.Nbssr,which is located at upstream of NBO₃2g0035,sharing the same transcription direction as NBO₃2g0035.It is interested to find that Nbssr share one base（A）with NBO₃2g0035,which means that the last base of Nbssr is the first base of NBO₃2g0035.Such case is seldom occurred in eukaryote genomes.In order to confirm the relationship between the two genes,we re-checked the reads of the genome and performed re-sequencing analysis.There are two reads passing through the zone between them,the one was the same as genome sequence assembled,that is to say these two genes share a base to coding two different proteins.The other reads indicate that there is TA deletion and thus results in Nbssr don’t have stop codon（TAA）,results in these ―two genes‖ form one open reading frame.What’s more,resequencing results showed that there was no stop codon between the two gene fragments,all resequencing reads are totally the same as the second reads with TA deletion compared to the first one.So we confirm that Nbssr and NBO₃2g0035 belong to one open reading frame with a size of 2229bp（742Aa）,there is histone promoter control 2（hpc2）domain at the N-terminal of the encoded protein.Therefore,in terms of the sequence,the Nb SSR annotated in old genome version is only part of Nb SSR,which is actually part of N-terminal named Nb SSR-N;NBO₃2g0035 is part of C-terminal named Nb SSR-C.We find that Nb SSR is relatively conserved both in size and function in microsporidia.The size of SSR is around 990bp（330Aa）for most microsporidia except Nb SSR,which size is 2229bp（742Aa）.Meanwhile they all have a conservative hpc2 domain,it was believed that SSR regulates transcription of the histone genes during the S-phase of the cell cycle by repressing transcription at other cell cycle stages.2.Expression of Nb SSR and subcellular localization analysisPolyclonal antibody against NBSSR-C prepared before was used to detect the localization of Nb SSR by indirect immunofluorescence（IFA）,results show that the protein located on nucleus membrane in the immature spores.In order to make sure the relationship between Nb SSR-N and NBSSR-C in the protein level,the polyclonal antibody against Nb SSR-N was successfully preparedand used to detect the localization of Nb SSR too,IFA results confirmed co-localization of Nb SSR-N and NBSSR-C on nucleus membrane.These results preliminarily showed that Nbssr-N and NBssr-C belong to part of Nbssr（2229bp）respectively.However Western blot shows that anti-Nb SSR-N only could recognize the protein band about 28 k Da,anti-Nb SSR-C could recognize both protein band about 28 k Da and 60 k Da,which brings a new question about the size of mature Nb SSR.3.Alternative polyadenylation of NbssrSignal sequence receptor（ssr）is a conserved gene in eukayotes.Compared to other microsporidia,the length of Nbssr is the twice as ssr in several other microsporidia.Many references showed that non-canonical splicing often occurred after ssr transcription,so it can express different size proteins to execute their biological function.In order to verify the such splicing mechanism existence in Nbssr,we designed several specific 5’ primersand found there are two polyadenylation sites in Nbssr by 3’ RACE PCR,located at the positions of 951 bp and 1166 bp.The molecular weight（MW）of these two m RNA-coding proteins are 37 KDa and 45 KDa,respectively.In summary,this study showed that the size of Nbssr was 2229 bp,but Nbssr non-canonical splicing resulting in two m RNA,which sizes were 951 bp and 1166 bp.IFA showed both Nb SSR-N and Nb SSR-C are located in nucleus membrane,which could be used as the marker of nuclear localization.