| Entomogenous fungi will not cause environmental pollution, residual and resistance in control of insect pests, which is a kind of environmental-friendly fungal pesticides. As peole of environmental protection awareness enhancement, the research of environment-friendly pesticides is widely strengthened. Nomuraea rileyi can natueally infect a variety of insect pests and domain epidemic in the field, so it is an important entomopathogenic fungi. As an important pesticide, the conidia are the active ingredient. However, its sporulation requires fastidious growth conditions, which limit its mass commercialization and application. Therefore, it needs to find easy-to-scale production of effective propagules to instead of conidiospore. Liquid spores because its cell wall is thin, low toxicity and storage can not be used in production. Microsclerotium(MS) is a pseudoparenchyma formed by pigmented, compact aggregation of hyphae which is highly resistant to desiccation, strong resistance, in a suitable environment to hypha and spore. Microsclerotium(MS) of N. rileyi had been successfully accomplished by liquid amended medium(AM). The resistance and stability of microsclerltia can act as propagules withstand adverse environment and exhibited highly insecticidal activity against Spodoptera litura, and thus can be used as an alterative active instead of conidia in pest biocontrol. Our preliminary experiments had proven that the formation of N. rileyi MS is closely related to oxidative stress and a large amount of reduced enzyme or synthetic reduced substance genes are up-regulated during the MS differentiation. In the present study, we cloned the Nraoxgene and characterized their involvement in MS development by RNA interference(RNAi) technology. The results of main research are as follow:①Gene cloning and sequence analysis of the Nraox geneUsing the available transcriptome library of CQNr01, we cloned and obtained the full-length c DNA sequence of Nraox(Gen Bank No. KM978957). Sequence analysis showed that the full-length c DNA of Nraoxcontained a 1068-bp ORF encoding a protein of 355 amino acids with a predicted molecular mass of 40.517 KDa and isoelectric point 9.53. There is no a signal peptide AOX. In addition, the Target P analysis showed that AOX was located in the mitochondria. The genomic sequence of Nraox was approximately 1512-bp long and consists of three exons interrupted by two introns, corresponding to the c DNA sequence. To examine the phylogenetic relationship of the AOX of CQNr01 with other species, a phylogenetic tree was established using MEGA 4.0 program. AOX proteins were highly homologous to the sequence of the entomopathogenic fungus Metarhizium anisopliae, belonging to the Clavicipitaceae. And, the homology comparison by Blast P showed that Nraox had up to 88.17% homology with M. anisopliae AOX.②Nraox expressionThe expression levels of Nraox gene during MS development were analyzed by RT-q PCR. The results showed that the Nraox expressed in all developmental stages, while the highest expression in the MS initiation stages(84 h), when compared with that in the germinating spore stag. Expression profile of Nraox during oxidative stress was analyzed by q PCR. Compared to the control, the Nraox expression were up-regulated in varying degrees when induced by both H2O2 and menadione. The Nraox expression was increased by 7-fold with H2O2 treated, whereas the transcript level up-regulated by almost 20- fold with menadione treated.③The function of alternative oxidase was tested using enzyme inhibitionAt the same time, the intracellular H2O2 concentration measuring after treatment with the inhibitor, showed that the content of the intracellular H2O2 was higher after the treatment of SHAM berore 10 min, and after 15 min, have no significant difference. Overall, the clean rate was a little slower than that the control.The hyphal morphology and the MS formation of CQNr01 were examined after treatment with SHAM. Compared with the controls, the hyphal morphology of treatment with inhibitor was abnormal, curve and more bud-like structure. Meanwhile MS formation was delayed and the sizes bigger and more fluffy than that of the control. Besides, the MS yields were reduced to approximately 95%.④The function of alternative oxidase was verified by RNA interferenceAfter interference efficiency with 800 n M si RNA, the m RNA level in the RNAi-silenced strains(treated by 800 n M si RNA) was declined by 76.1%. A variety of morphological features were observed in the Nraox RM. When grown on SMAY media containing different stress(0.3 m M H2O2 and 0.3m M menadione), there were no significant differences between wild type and egfp RM strains. While, the growth of the Nraox RM were slower, mycelium formation was delayed, the conidial yields were reduced, and the colonies of Nraox RM were smaller compared to the wild type. In AM medium, MS was formed normally in the controls while the Nraox RM could not produce normal MS successfully after 3-3.5 day. In addition, the sizes of MS formed were bigger than those of the control. Meanwhile, the abnormal hyphae in the Nraox RM were observed under the microscope and both MS yields and biomass were measured. The data revealed that the biomass and MS yields of Nraox RM were significantly reduced, which suggested that Nraox was required for normal hyphal growth and MS differentiation of CQNr01.⑤Pathogenic identification of target pestsThe virulence of the all strains were tested by inoculation of the strains onto the cuticle of third-instar Spodoptera litura larvae and found mortality was reduced, the lethal time values for 50% mortality(LT50) was extended and reduced to approximately 26.2% in the RNAi-scilenced strains.Conclusion: At the present study, the Nraox gene was cloned from N. rileyi. The epression profile analysis and Nraox silencing demonstrated that the Nraox plays an important role in MS differentiation by regulating the intracellular redox balancing and hypha growth. Furthermore, the Nraox was found to essential for virulence in CQNr01. Studies are currently underway to provide a gist insight into both the molecular mechanism of MS differentiation and the control of insects by MS in N. rileyi. |
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