Cloning And Functional Analysis Of Na⁺-Ca²⁺ Exchanger And Calcium Channel Inhibitor In Nomuraea Rileyi

Nomuraea rileyi as an insect pathogenic fungi, can cause insects epidemic in the fields thus received extensive attention of people. But as the main effective components of insect fungus- conidia, the sporulation of N. rileyi required culture conditions are harsh, which limits its industrial production and large-scale application. The research group of Zhongkang Wang in Chongqing University used the induction method of plant pathogenic fungi MS successfully induced microsclerotium(MS) of N. rileyi. MS as a special infect structure, with the advantages of industrial production, high temperature resistant, resistant storage, strong resistance and others can replace traditional conidia. Then they used the method of comparative transcriptome to explore the formation mechanism of MS. At the same time in the optimization of induction medium found that adding different concentrations of Ca2+ MS yielded remarkably different. So this paper selected genes related to Na+-Ca2+ Exchanger(NCX) and Calcium Channel Inhibitor(CCI) from the comparative transcriptome of N. rileyi to study the relationship between calcium and MS of N. rileyi. The main results are as follows:① By adding Ca2+ in the induction medium found that in AM-Ca2+ free medium the size of mycelium pellet was heterogeneity and decreased by 34.4%. And the hyphae twined loosely, there also are a lot of hyphae, puce pigment accumulation decreased obviously. The biomass statistics found that adding Ca2+ or not biomass was not significantly change, so it didnt affect the growth of N. rileyi;② According to the NCX and CCI EST sequence of the comparative transcriptome, obtained the NCX and CCI full-length c DNA and genomic DNA sequences by narmal PCR. open reading frame(ORF) of NCX and CCI are 3006 bp and 405 bp, encoding 1001 and 134 amino acids respectively, Using the bioinformatics software and web site analysis the NCX and CCI found: the protein molecular weight of NCX was predicted to 109.563 k Da, mainly composed of alpha helix and Random coil, the theory of isoelectric point is 5.98, it has no signal peptide, but transmembrane structure; the protein molecular weight of CCI wsa predicted to 13.63 k Da, mainly composed of alpha helix, beta sheet and random coil. its theory of isoelectric point is 7.5. It has no transmembrane domain but signal peptide; Using the Clustal X and MEGA4.0 homologous comparison and phylogenetic analysis revealed that the closest phylogenetic relationship of NCX and CCI protein in N. rileyi are metarhizium anisopliae;③ Transcription levels analysis of NCX and CCI by q PCR: NCX and CCI expressed in each period of MS formation, NCX reached the maximum at 72 h, then in microsclerotia maturation(72-120h) the transcription level was gradually decreased. The expression level of CCI in MS development period(48-120h) increased gradually, reached the peak at 120 h. This shows that NCX and CCI involved in the formation of MS. But the regulation they involved was different, NCX mainly plays an important role in the early stage of MS formation to activated development process, while CCI expressed highly in the late stage of MS development, may be involved in the maturation process of MS;④ RNA interference(RNAi) researched the functions of NCX and CCI: By screening the most suitable interference concentration of NCX and CCI was 400 n M, the interference efficiency were 55.5%, 93.6% respectively. In NCX interference strain, the expression level of CCI increased by 2.1 times; in CCI interference strain expression level increased by 5.3 times. Comparing to the wild type strain(WT) and negative control(EGFP), the colony morphology of NCX and CCI interference strain became smaller, sporulation time delayed, and sporulation were reduced to 60.6% and 88.2% respectively. Statistical MS production found that interference strains formed a sharp reduction in the number of MS, decreased respectively 81.8% and 90.4%. And at the same time statistical biomass found that biomass of NCX and CCI interference strain respectively decreased 25.8% and 30.5%.⑤ The MS of all the interference strain was inoculated onto the cuticle of third-instar Prodenia litura to detect the influence on virulence by NCX and CCI, found that the lethal rate of RNAi strains decreased by 4.45% and 6.1% respectively, LT50 extended over 2d comparing to wild type strain(WT) and negative control(EGFP).Conclusion: NCX and CCI gene of N. rileyi were involved in the formation of MS, NCX was involved in the initiation and development process of MS, CCI mainly participated in the process of MS maturity. They not only affected MS formation, But also obviously influenced on the spores production and virulence of N. rileyi, but its mechanism needs further study.