The Ribosomal RNA Gene Of Nosema Antheraeae And The Proteomics Analysis On Nosema Bombycis And Nosema Antheraeae, And Comparative Proteomics Studies In Midgut Of Bombyx Mori Infected Micrsporedia

Microsporidia are obligate,intracellular single eukaryotic organisms that infect all major animal groups from invertebrates to vertebrates,including insects,fishes,and mammals.The microsporidian Nosema bombycis and Nosema antheraeae is a pathogen that infects bombyx mori and the Chinese oak silkworm,Antheraea pernyi,causing pebrine disease.Pebrine disease is a destructive disease,and has caused heavy losses in sericulture in china.Pebrine disease has increased in recent years,so it is the two grand requisites of theoretics and production to do research on microsporidia.In this paper,we used N.bombycis and N.antheraeae as experimental materials. The ribosomal RNA gene of N.antheraeae was cloned and sequenced.We presented here the molecular biological evidence of the taxonomy of N.antheraeae and the evolutional relation between N.antheraeae and N.bombycis.Proteins of N.bombycis and N.antheraeae were separated and identificated by two-dimensional electrophoresis and mass spectrometry,and analyzed proteins in the level of all cell.We characterizated and identificated the whole proteins change in the midgut of bombyx mori of fifth instar at different time after injecting N.bombycis,N.antheraeae and water by comparative proteome analysis.Immune response pattern of silkworm to microsporidia was analyzed, infection-related biomarkers and resistance-related proteins were screened in the level of proteome.1.The ribosomal RNA gene of N.antheraeaeBy PCR amplification used specific primers,cloning and sequencing,we got partial sequence of the large subunit rRNAs(LSU rRNA) gene,the complete sequence of the small subunit rRNAs(SSU rRNA) gene,the internal transcribed spacer(ITS) and 5S rRNA gene(Genbank accession number:DQ073396).The organization of the ribosomal RNA genes of N.antheraeae is LSU-ITS1-SSU-ITS2-5S,a pattern similar to those of N.bombycis.The organization of the ribosomal RNA genes differs from that of most prokaryotes as well as eukaryotes.Based on alignment and phylogenetic analysis by using the SSU rRNA gene and ITS sequences,we can conclude that N.antheraeae is a Nosema species,closely related to N.bombycis.We present here the molecular biological evidence of the taxonomy of N.antheraeae.The phylogenetic relationships of microsporidia species by combining information from SSU rRNA and ITS sequence was elucidated,which was a new attempt.2.The Proteomics Analysis of Nosema bombycis and Noseraa antheraeaeThe proteomic approach was used to investigate the proteome of N.bombycis and N.antheraeae.Purified spore were disrupted by triturating in liquid nitrogen.Total proteins were extracted in a lysis buffer.Protein samples were then analyzed by 2D electrophoresis and analyzed through MALDI-TOF-MS.16 proteins of N.bombycis were identified,among them,four were identified as enzyme,two as transcriptional regulator,two as membrane channels protein,one as cytosdeleton protein,one as signal protein,one as ribosomal protein and 5 unknown proteins.9 proteins of N.antheraeae were identified,among them,three were identified as enzyme,three as transcriptional regulator;one as envelope glycoprotein;one as cation efflux-related outer membrane exported protein;one as heat shock protein.The function of proteins was analyzed by bioinformatics and literature search.A large proportion of proteins of N.bombycis and N.antheraeae are enzyme,transcriptional regulator and membrane protein.It demonstrated enzyme,transcriptional regulator and membrane protein are high expression proteins in spore,which met the structure and physiology of spore.3.Comparative proteomics studies in midgut of bombyx mori infected N.bombycis, N.antheraeae or waterWe characterizated and identificated the whole proteins in the midgut of bombyx mori(strain:C108) of the fifth instar after infecting N.bombycis,N.antheraeae or water 24h,48h and 72h by two-dimensional electrophoresis and mass spectrometry.The whole proteins change in the midgut of bombyx mori and immune response pattern of silkworm to microsporidia at different time after injecting were analyzed by comparative proteomics.At different time after infection,expression of many enzymes involved in carbohydrate metabolism decrease,for example enolase and hydroxypyruvate isomerase.On the other hand,expression of many enzymes involved in protein and amino acid metabolism increases,for example,proteasome and fumarylacetoacetate hydrolase.The vacuolar ATP synthase(V-ATPase) is a membrane-related protein which can pump H+ outside of the cells or into the lumens of some vacuolar organelles.Expression of V-ATPase in midgut of bombyx mori infected with N.bombycis is lower than in midgut of bombyx mori infected with N.antheraeae or water.At 24h,proteins involved in immune response of silkworm to microsporidia express increase in midgut of bombyx mori infected N.bombycis and N.antheraeae than the control,while at 72h,proteins decrease in midgut of bombyx mori infected with N.bombycis than the treatments infected with N.antheraeae or the control.The result is consistent with the analysis of two-dimensional electrophoresis gel by PD-OUEST.We should pay whole attention to research expression change of actin-depolymerizing factor 4,thiol peroxiredoxin and antichymotrypsin precursor.Actin-depolymerizing factor 4 is an actin-binding protein expressed in all eukaryotic cells,which has important functions in equilibrium between monomeric and filamentous actin.In our experiment,expression change of actin-depolymerizing factor 4 is obvious.At 24h and 72h,there is little expression of actin-depolymerizing factor 4 in midgut of bombyx mori infected with N.bombycis,however there are high expression in midgut infected with N.antheraeae or water.This phenomenon explains the broken balance between monomeric and filamentous actin.Whether the balance between monomeric and filamentous actin is a precondition to microsporidian glow or a pathological process still needs to study.The actin-depolymerizing factor 4 is expected to become the target protein that identifies the silkworm infected with N.bombycis.Thiol peroxiredoxin is an antioxidant enzyme that catalyzes the reduction of peroxides,and may be involved in functions such as protection ROS released by immune effector cells and interaction between parasite and host.There are expression changes after infecting with N.bombycis,N.antheraeae or water at 24h,48h and 72h. At 24h,expression of thiol peroxiredoxin in midgut of bombyx mori infected with N. bombycis and N.antheraeae is higher than control;at 48h,expression in midgut of bombyx mori infected N.bombycis is higher than infected N.antheraeae;at 72h, expression in midgut of bombyx mori infected with N.bombycis is lower than the treatments infected with N.antheraeae or the control.The phenomenon explains thiol peroxiredoxin participate in immune response of mudgut of silkworm to microsporidia.Antichymotrypsin precursor is a precursor of antichymotrypsin,has no function, and must be processed to be active antichymotrypsin.Antichymotrypsin is a member of the serine protease inhibitor family known as serpins.Antichymotrypsin has been demonstrated a functional role in protein processing,apoptosis,restructuration of extracellular matrix and protection of cells from damage.Plasma level of antichymotrypsin has been implicated in acute-phase responses to burn injuries,surgery, and certain cancers.Expression of antichymotrypsin in midgut of bombyx mori infected with N.bombycis at 72h is far higher than the treatments infected with N.antheraeae or the control,which implicated that the expression of the Antichymotrypsin is expected to become the infection-related biomarkers of the silkworm infected with N.bombycis...