The Gene Cloning And Functional Analysis Of Nrcdc24 And Nrbem1 In Dimorphic Fungus Nomuraea Rileyi CQNr01

As an important entomopathogenic fungus, Nomuraea rileyi is a widely exists in Nature, has strong infectivity and pathogenicity for Noctuidae(Leptoptera) pestsand could control the insect population effectively. The regular active ingredients of N. rileyi insecticidal preparation are the conidia. However, in actual production, its sporulation requires special carbon source, long growth cycle, need of the light in the cultural processand sporulation quantity is low. All these characters limited its large-scale production and commercial use. Then our laboratory successfully induced N. rileyi Microsclerotium(MS) formation in liquid amended medium(AM). Comparing with conidia, the microsclerotium has stronger resistance to stress, durable storage and almost same virulence, and thus can be used as a potential active agent for insect control. In the earlier study, we found that the MS of N. rileyi constructed by the specialized mycelium. Also, the comparing transcriptome analysis showed that the MS differentiation is a process of oxygen stress, accompanied pigment accumulation, reactive oxygen species(ROS) producedand the expression changes of large functional genes. Study the mechanism, which gene control the process and how, could help us understand the molecular characteristics of MS formation and provide important theoretical reference basis for the MS fermentation production and formulation development later. Based on the expressed sequence tag(EST) from the data of comparative transcriptome of N. rileyi, we cloned the Nrcdc24 and Nrbem1 genes which were high expression in MS formation, and studied the genes function by the vitro RNA interference. The mainresults of research are showed as following:①The full-length c DNA sequences of the Nrcdc24 and Nrbem1 genes were obtained by SMART RACE RT-PCR. The putative coding sequences of Nrcdc24 and Nrbem1 were 3021 bp and 1689 bp, encoding 1006 and 562 amino acid residues, respectively;②Bioinformatics analysis showed that the protein of Nrcdc24 includs Rho GEF binding domain, and the protein of Nrbem1 has two Src Homology 3(SH3-1 and SH3-2) domains and a phox homology(PX) domain that binds phosphoinositides. Both Nrcdc24 and Nrbem1 have a common binding site Phox binding(phox and Bem1, PB1) domain. The phylogenetic tree analysis demonstrated that the Nrcdc24 and Nrbem1 proteins of N. rileyi are highly homologous to those of Metarhizium acridum;③We observed the process of MS formation in liquid culture with microscope synchronously and found that the MSformation began at the 84 th h when the mass of polarity hyphae partial be condensed; The transcription levels of Nrcdc24 与 Nrbem1 were analyzed by real-time quantitative PCR(q PCR) at different stages of MS development and showed that both Nrcdc24 and Nrbem1 genes transcription levels were highest at the early stage of a large number of MS formation(84 h);④To further understand the impact of Nrcdc24 and Nrbem1 on MS development, fungal growth and conidial yield, the vitro RNA interference(RNAi) method was used to silence the genes expression and the e GFP interference strains was used as a negative control. We found that the yeast-like colonies of the Nrcdc24 and Nrbem1 interference strains(named as cdc24 RNAi and bem1RNAi) growth slower than the wild type(WT) and negative control(e GFP) on the SMAY solid culture medium. The dimorphic switch(from yeast-like to hyphoid colony) was delayed by approximately 0.5 d or 1 d in the RNAi strains. Comparing with the controls, the sporulation was delayed and the conidial yield of the cdc24 RNAi and bem1 RNAi were reduced approximately 95% and 56%, respectively. While the yeast–like colonies of the double-RNAi strains were smallerthan the control strains, the sporulation was decreased 99.04%. When compared with the conidia of the controls, the spore morphology of bem1 RNAi and cdc24 RNAi were not significantly different. In AM, compared with the control, the pigmentation in fermentation broth of all the RNAi-silenced strains dropped significantly, also the fermentation broth viscosity with synchronous culture 5-7d reduced obviously. In addition, those of the both RNAi-silenced treatments mycelium become thicker and the bem1 RNAi and double-silenced strains produce many budding spores. The morphology of the hyphae in those of the cdc24 RNAi silenced strains did not exhibit observable differences. Furthermore, in the same culture conditions, the control strains began to produce MS in culture at 4th day, but the RNAi strains with no MS produced at that time. The MS formation of all the RNAi strains delayed 1-2d. Comparing with the controls, the MS dry weight of Nrbem1 and Nrcdc24 RNAi-silenced strains showed be reduced approximately 20-32% and the double disturbance strains reduced 40%. The MS yield of cdc24 RNAi, bem1 RNAi and double-RNAi strains were reduced approximately of 93.2%, 90.7% and 97.3% respectively, after 7 d formation when is the harvest time for regulated MS culture;⑤As in the case of rac ARNAi and cdc42RNAi-slienced strains, the expression levels of nox A and nox R genes in cdc24 RNAi and bem1 RNAi slienced strains were decreased obviously with the q PCR. In addition, after the Nrcdc24 gene was silenced, the transcription levels of cdc42 and rac A genes were reduced. On the contrary, the transcription levels of Nrcdc24 was increased significantly in rac ARNAi and cdc42-RNAi strains, which may indicate that the cdc42 and rac A genes are the downstream gene of Nrcdc24;⑥The virulence of the all strains were tested by inoculation of the strains onto 3rd-instar S. litura larvae. The results showed that the lethal time values for 50% mortality(LT50) was extended in the RNAi-scilenced strains. Based on the survival curve of S. litura, the average LT50 values for wild-type, e GFP, Nrbem1, Nrcdc24 and the double RNAi-silenced strains were estimated at 9.32, 9.53, 11.74, 11.06 and 13.60 days, respectively.Conclusion, we cloned Nrcdc24 and Nrbem1 from the N. rileyi and demonstrated that Nrcdc24 and Nrbem1 genes affecting the MS formation, polar growth, sporulation and virulence et al in N.rileyi. Furthermore, we found that both Nrcdc24 and Nrbem1 have some interaction relationship with NADPH complex and Nrcdc24 plays a role in the regulation of rac A and cdc42 during MS formation.