Proliferation Of Nosema Bombycis In Insect Cells And Establishment Of Nosema Bombycis CQ1 Strain

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites.They can infect a wide variety of animals ranging from invertebrates to vertebrates,and they are the common pathogens for sericulture,fishery or shrimp farms.Thus far,more than 1400 microsporidia species belonging to 150 genera have been discovered,among which 13 species belonging to 5 genera can infect silkworms,and 14 species belonging to 8 genera can infect human.Microsporidia were classified into Microsporida,Microsporea,Microspora and Protisia.Recently,more and more evidences to support a fungal origin for microsporidia have emerged,and the latest studies suggested that microsporidia are closely to Rozella allomycis derived from Chytridiomycota.Microsporidia drew great interests of scientists because they are closely related to human health and their disputed evolutionary origin.N.bombycis is known as the pathogen of silkworm pebrine,which usually prevails in silkworm-feeding areas.Because of its vertical transmission,N.bombycis is the only one target for quarantine in silkworm-feeding countries.Most of microsporidia parasitize in their specific hosts,whereas,there are some microsporidia which can infect a wide variety of organisms.N. bombycis can infect Bombyx mori,in addition,other 40 species of insects.Microsporidia vary not only in the shape,but also in the molecular level.N.bombycis isolate CQ1 which was isolated from infected silkworms in Chongqing area,by the lab of silkworm pathology,Southwest University,was preserved in China Veterinary Culture Collection Center.On the premise of the infected systems of N.bombycis in Sf21 Cell line and BmN-SWU cell line, spores collected from a spot of silk gland heavily infected with Nosema bombycis were inoculated into Sf21 cells and cell infection were achieved,and the passages of Nosema bombycis propagated in Sf21 cell line were carried out in order to obtain a Nosema bombycis CQ1 strain whose genetic background is clear.In this study,we explored the Molecular evaluation of purity of N.bombycis CQ1 strain,by analyzing the rDNA-ITS sequences.The results are as follows:1.Establishing the infected systems of Nosema bombycis in Sf21 Cell line and BmN cell line Sf21 cells grow fast and are easy to be cultured.Under We established the infected systems of Nosema bombycis in Sf21 Cell line and observed the proliferations of Nosema bombycis in the cell line.And we also established the infected systems of Nosema bombycis in BmN cell line which constructed in our lab.These two infected systems provided additional systems for studying functional genome of Nosema bombycis.The growth of Nosema bombycis in host cells was a chronic process and Nosema bornbycis occupied all space of cytoplasm eventually.Nosema bombycis multiplied firstly in periplasm and circumferentia of cells,then extended to the centers of cells.Eventually Nosema bombycis take up all space of cytoplasm..At the same time,many infected cells broke,a lot of mature spores were released from the infected cells and the saccate vacuoles and cells-fusion can be observed.2.The proliferation and passages of N.bombycis spores from a spot of heavily infected silk glandN.bombycis from a spot of silk gland infected with Pebrine were inoculated in Sf21 cell line and BmN cell line.Proliferation of spores was observed in Sf21 cell line and passaged in Sf21 cell line and BmN cell line.In Sf21 cell line,the Nosema bombycis were passaged for eight generations.In BmN cell line,the Nosema bombycis were passaged for six generations.3.Molecular evaluation on purity of the Nosema bombycis CQ1 strainIn order to evaluate the purity of the Nosema bombycis CQ1 strain,we analyzing the sequences of rDNA-ITS and constructing phylogenetic trees based on sequences of rDNA-ITS from the Nosema bombycis CQ1 strain.The rDNA-ITS sequences of the N.bombycis CQ1 stain were amplified by PCR.The lengths of rDNA-ITS sequences are 503～516 bp.There were 12 cloned rDNA-ITS sequences,among which rDNA-ITS1 sequence had 98%identity to AY259631 reported.The other cloned rDNA-ITS sequences showed 88～96%homology with rDNA-ITS sequences derived from GenBank.In addition,sequence alignments revealed that there were transitions,transversions, insertions and deletions in rDNA-ITS,and diversity sites mainly distributed in ITS region.The results showed that heredity differentiation of ITS of N.bombycis CQ1 strain was obvious. There are sequence polymorphisms among the clones of rDNA-ITS.The reason was possible due to the multi-copy rDNA genes in N.bombycis,copy numbers of small subunit,large-subunit and 5SrDNA are 43,53,46.Sequence polymorphisms are existing in the multi-copy rDNA genes even in one spore,so we need to select a new genetic marker for evaluate the purity of N.bombycis CQ1 strain.