Creation Of Tracer Nosema Bombycis

Microsporidia is a class of obligate intracellular single-cell eukaryotic microorganisms existing in nature widely.Microsporidia has a wide range of hosts,affecting the growth and development of the host,and can cause death of the host in serious cases.Nosema bombycis is the earliest known as a kind of microsporidia,and is one of the important members of the genus Nosema.It can infect the silkworm,an important silk economic insect,and cause Pébrine disease,which cause huge economic losses to the sericulture industry.Therefore,the study of the life cycle of Nosema bombycis and the labeling and tracing of its infection path and proliferation process will lay the foundation for the basic research and effective prevention and control of Nosema bombycis.Nosema bombycis has a strict parasitism and can only proliferate in the host cell to complete the life cycle.At present,most of the tracers of microsporidia use the method of marking mature microsporidia,however,immature microsporidia in intracellular stage are difficult to detect.Bsides,microsporidia can not be labeled and observed in real time,which limits the study of its life cycle and the mechanism of infection and proliferation.Visualized tracer technology is an effective means of studying pathogen invasion,proliferation,and interaction with the host.The methods of genetic manipulation to achieve exogenous fluorescent protein in vivo tracing have been widely used in other pathogens.However,due to the lack of microsporidia genetic manipulation system,tracking of microsporidia in vivo has not been obtained at present.In view of this,this thesis firstly analyzed the characteristics of BmE-SWU1 cells infected by Nosema bombycis and then combining with the structural characteristics of different developmental stages,we explored the construction of tracer Nosema bombycis by attempting different transgenic methods.The main results are as follows:1.The characteristics of BmE-SWU1 cells infected with Nosema bombycisTransmission electron microscopy showed that the sporeplasm could be observed in the BmE-SWU1 at 3 h after infection.At 9 h after infection,the volume of the sporeplasm increased.At 48,72 and 84 h aftert infection,schizont,sporoblast with thick plasma membrane and neonatal Nosema bombycis with complete structure were observed in turn.The transcription of Nb?-Tublin,NbSWP5,NbCs-1,NbPTP2 and NbMCM6 genes were analyzed by RT-PCR at different time after infection with Nosema bombycis.It is found that the expression level of the related genes is corresponded with to the characteristics of the development process of Nosema bombycis.Using NbPTP2 antibody,DAPI and fluorescent brightener 28,the Nosema bombycis in BmE-SWU1 cells were labeled and observed.The Nosema bombycis could be distinguished between extracellular infective phase,schizont phase,sporoblast phase and mature spore phase.The above results indicate that after infecting host BmE-SWU1cells,Nosema bombycis completed a life cycle at 84 h,and the infective phase is at 0³ h after infection.The proliferative phase is at 3⁴8 h after infection,and it has entered the sporognic phase at 72 h after infection.2.Creation of tracer Nosema bombycisFour fluorescent protein expression vectors of pIZ-OpIE2-DsRed,pig-A3-EGFP,pSL1180-NbCDCP23-DsRed and pSL1180-NbPTP2-DsRed which contain different promoters ware constructed.Firstly,the plasmid was transfected into the infected cells by multiple liposome transfection.The fluorescent tracer Nosema bombycis were observed only in the experimental groups transfected with pIZ-OpIE2-DsRed and pig-A3-EGFP.No tracer Nosema bombycis were obtained through introducing mature Nosema bombycis by electroporation.Electroporation was carried out by electroporation of cells infected with Nosema bombycis.Tracer Nosema bombycis were found in the experimental groups at 9 and 48 h after infection,but not in the experimental group at 60 h after infection.We introduced pig-A3-EGFP expression vector into silkworm body infected with Nosema bombycis by intravitreous injection transfection.The results showed that a part of Nosema bombycis showed green fluorescence.The above results indicate that the OpIE2^prm and A3^prm can initiate the expression of fluorescent proteins in the Nosema bombycis.The proliferative period is beneficial to the transformation of exogenous genes.It is feasible to transfer exogenous genes into Nosema bombycis by transfecting infected cells and silkworm body,besides,electroporation of infected cells is also feasible.3.The collection and identification of tracer Nosema bombycisThe genome of tracer Nosema bombycis which obtained from the silkworm of transfection experime was extracted and detected by PCR.The specific band of EGFP was amplified.The collected tracer Nosema bombycis were further inoculated into the cells,and it was found that the tracer Nosema bombycis can normally infect the host cells and proliferate in the host cells to produce new spores.The tracer Nosema bombycis could be observed within 8 days after infection.The above results indicate that the A3^prm-EGFP expression element entere the genome of Nosema bombycis,and activated the expression of fluorescent protein.However,the fluorescence signal intensity is weak,which may be related with the low efficiency of introduction or the low activity of promoters.In summary,this study clarify the characteristics of the silkworm embryonic cell line BmE-SWU1 infected with Nosema bombycis and explore various methods to create tracer Nosema bombycis.As a result,we obtain the tracer Nosema bombycis with fluorescence signal.At the same time,we screen the available fluorescent protein expression vector,and determine the optimal time for vector introduction and detection.We also initially establish the method of creating tracer Nosema bombycis.