Extraction Of Spore Wall Proteins And The Expression Patterns Of Main SWP Of Nosema Bombycis

Microsporidia are unicellular eukaryotes which develop as obligate intracellular parasites.They can infect a wide variety of animals ranging from invertebrates to vertebrates,including human,and they are the common pathogens for sericulture,fishery or shrimp farms.Since the first microsporidia, Nosema bombycis,had been discovered in 1857,thus far,more than 1 400 microsporidia species belonging to 150 genera have been reported.Spore wall proteins(SWPs)of microspridia contact the hosts directly,which play important role in infection.We establish a new GDGC method to extract spore wall proteins,and also analyze LC-MS/MS data of proteins acquired with different methods. For the major components of SWPs genes,we also investigate their expression pattern by RT-PCR.1.Establishment of a new GDGC method for extraction SWPs from pure spore coat after germinationCombining the biological event that the sporoplasm was ejected out of the spore after germination and combined the boiling-bath method,we establish a new method called GDGC (Germination & Density Gradient Centrifugation)to get the pure spore coats after germination by Density Gradient Centrifugation,then to extract SWPs.The SDS-PAGE electrophoresis show that SWP30,SWP25 and SWP32 are contained in this sample and SWP25,SWP32 are a little reduced, but there are more components of spore coat can be acquired with GDGC method,so it is a rigorous method for SWPs extraction.2.Comparative study on the methods of spore wall proteins extraction from N.bombycisCompared the SDS-PAGE electrophoresis of different methods including SDS extraction, boiling-bath extraction,different concentration KOH extraction,K₂CO₃ extraction and Brosssen extraction,the results show that protein sample extracted with K₂CO₃ including aboundant proteins of sporoplasm So it is unfit for extraction of spore wall proteins.But we can obtain a few SWPs that soluble in alkali from frozen spores after treated with alkaline solution.The result of SDS method is similiar to early study that only can acquire SWP30,SWP25 and SWP32.Many proteins can be collected by Brosson method which is time-consuming with many steps..3.The observation on morphology of N.borabycis followed SWP extracted.Spores treated with different methods stained by Giemsa and then observed under the microscope. Results show that fresh spores have a high germination rate(＞85%)when treated with 0.1M K₂CO₃ and 0.1 M KOH,the germination rate is about 40%when treated with 0.01 M KOH.But frozen spores can't germinate after treated with alkaline solution and the morphology of which is same as the normal spores.Both the fresh and frozen spores have complete morphology after treated with SDS and boiling-bath method and the size is same as normal spores,but the treated spores are stained easily than normal spores because of the enhanced permeability of spore wall.4.Proteomic analysis of total proteins and spore wall proteins from N.bombycis by mass spectrum.In this study,we adopted a proteomics approach LC-MS/MS to analyze protein samples acquired from fresh spores with 0.1M K₂CO₃,boiling-bath method and sample from spore coats with boiling-bath method.From LC-MS/MS analysis we obtained 316 proteins which were classified to several main parts include metabolism,protein destination,nucleic acid synthesis,protein synthesis, organization and biogenesis,cellular communication/signal transduction,cell growth,division, rescue,apoptosis and so on.We also obtain 12 N.bombycis hypothetical SWP(NbHSWP)with NbHSWP1～3 is SWP30,SWP25 and SWP32 respectively.7 NbHSWPs are single copy in N.bombycis genome.There are 3 genes share low sequence identity(identity:30%～35%)with the corresponding CDS in A.locustae and E.cuniculi respectively and the blastp result of SWPs on NCBI show a low identity(＜30%)to sequences derived from other species,which suggest that SWPs are species-specific.Promoter prediction shows that only 3 NbHSWPs have promoter motif such as TATA-BOX.EST of NbHSWP1～3 were discovered in the database.Sequence analysis of NbHSWP1～3 show that all of them have predicted N-glycosylation and phosphorylation sites,but lack O-glycosylation site.5.Analysis of SWP30,SWP25,SWP32 by RT-PCR.We feed spores to the 4th instar molted silkworm larvae with 10⁶ spores/larvae,then collected infected midgut from 12h to 10d as material to obtain RNA.The RT-PCR results ofβ-tubulin as internal reference show thatβ-tubulin transcript can be detected from 12h to 10d post infection (P.I.)and the transcript is increase obviously from 4d P.I.then increase follow the infected time. Transcripts of SWP30 and SWP25 can be detected from 36h P.I.(sporont or spore formation period) and increase obviously from 3d and then increase progressively.SWP32 shares similar pattern with the other two SWPs.The transcript of SWP32 can be detected from 12h P.I.(schizont period)and then increase obviously from 3d like SWP30 and SWP25.The increase of SWPs' transcript is obvious thanβ-tubulin from 3d,so we presume that SWPs will be geneted a lot when sporoblast stop increasing.It may be relate to formation of spore wall.In this study we acquired spore coat with purity is about 100%and establish a reliable GDGC method to extracted SWPs.Wc also acquire LC-MS/MS data by three different methods.From the analysis of LC-MS/MS data we obtained 12 NbHSWPs and the sequence analysis show only 3 of them have promoters.NbHSWP1～3 are SWP30,SWP25 and SWP32 respectively which have EST proofs in the database.Result of RT-PCR show that transcript of SWP32 can be detected at 12th after feed spores when SWP25 and SWP 30 can be detected at 36h.