Preliminary ALSWP2 Of Nosema Gene Function

Nosema locustae is an unicellular Protozoa，and it is a special parasite of locustsin Orthoptera insects. Nosema locustae may induce epidemic of insect disease and playthe effect of persistent low population density in the locust control practice, and it hasthe advantages of non-pollution and non-toxic to human or animals as a kind of insectpathogenic microorganisms. So it is the focus on the research of the pathogenicmechanism and control application of Nosema locustae.The spore wall of microsporidia is the first part that directly contacts with the host,and the spore wall protein plays an important infection role in pathogensis process ofthe host, therefore，spore wall protein become research hot spot of microsporidian. Nowthere are13spore wall proteins to be reported, but the action mechanism and thespecific function has not been elucidated. In this study， a hypothesis apore wall protein（ALSWP2）was selected to research on the basic function of the spore wall proteinfrom five identified proteins. The sequence characteristics and the analysis oftranscription，gene cloning and prokaryotic expression，the protein function and the rolein the process of proliferation were explored to establish a theoretical basis forbiological control of locusts.The main research results are as follows:1. Sequence analysis and the analysis of transcription on ALSWP2Preliminary analysis with the biology software and online prediction methods, wefound that it’s about23KD, there are two N-glycosylation sites and one O-glycosylation sites, and there isheparin binding domain like”XBBXBX” in theN-terminal. Compared with the other microsporidian proteins, the locations of fourcysteines are conservative. Transcriptional analysis to the Nosema locustae ALSWP2show that ALSWP2can be expressed both locusts and Nosema locustae. 2. Gene cloning、prokaryotic expression and purify about ALSWP2On the basis of sequence analysis, we do the cloning to ALSWP2and purify therecombinant protein pET28a-ALSWP2with His label. We induce2、4、6and8hourswith IPTG concentration of0.1mM,0.5mM,1mM in30℃to ge the most target protein.Finally, we get the target protein through30℃、0.1mM IPTG concentration andUltrasonic centrifugal broken. And the results of Western blotting show that theantiserum specificity of it is strong, and no cross reaction with other proteins of Nosemalocustae, and it has the specificity of the species.3. Discuss the function on ALSWP2In the RNAi test of ALSWP2gene function study, the RT-PCR amplification showthat locusts without RNAi can detect the transcription in7thdays after inoculation ofNosema locustae, and locusts with RNAi can detect transcript in the third day. Itshowed that RNAi sped up the infecting ability of Nosema locustaec. And it suggestALSWP2gene has the function to resist the Nosema locustaec infection, and it isassociated with immunity of locusts.