Preliminary Study On Molecular Mechanism Of Generating Conidia From

Arthrobotrys oligospora is a filamentous fungi, it is a model fungus to study the interaction between nematode-trapping fungi and nematode. A. oligospora is a soil fungus, it lives mainly as saprophytes, while it can live as parasites by produced adhesive three-dimentional nets to captures nematodes. In this study, the functions of two sporulation regulatory genes (fluG:AOL_s00043g361and abaA: AOL_s00080g63) in A. oligospora were analyzed during the growth and conidia production using gene knockout technology. Moreover, the expression levels between the ΔabaA mutant and wild-type were compared by digital expression profiling technology.The main results were described as follows:1. Amino acid sequences and functional domains of the two sporulation regulatory genes were analyzed. The fluG encodes a polypetide of928amino acid residues (aa), the theoretical pI and molecular weight (Mw) is5.94and104.5kDa, respectively. It contains three structural domains, including amidohydrolase2, glutamine synthetase/guanido kinase (catalytic domain) and glutamine synthetase (catalytic domain). The abaA encodes a polypetide of588amino acid residues (aa), the theoretical pI and Mw is5.50and67.5kDa, respectively. It just contains one TEA/ATTS domain.2. Screening of ΔabaA and ΔfluG mutants and phenotypic analysis. Knockout vectors of the two sporulation regulatory genes were constructed via electro-transformation of Saccharomyces cerevisiae and E. coli competent cells. CaCl2-PEG mediated transformation method was used to transform the A. oligospora protoplast, and the positive transformants of abaA and fluG were identified. Two mutants were compared with the wild type in growth rate, conidiation, trap formation and stress resistance. The results showed that the two sporulation regulatory genes are more or less involved with various biological processes. The deletion of gene fluG has no significant effect on the sporulation, but the deletion of gene abaA has a significant effect on the sporulation, the ΔfluG mutant abolished the formation of conidia.3. Expression levels of sporulation regulatory genes were analysed during the process of conidia generation. Mutants and wild-type strain were cultivated on the same medium, and the mycelia were collected at different time points. Total RNA were isolated from those samples, respectively, and the cDNA was prepared by reverse transcription. The transcriptional levels of more than30sporulation regulatory genes were determined by the Real-time PCR. The results indicated that two genes were up-regulated markedly during the sporulation in the ΔfluG mutant. Moreover, the deletion of abaA has a significant effect on the expression of12sporulation regulatory genes, it indicated that abaA gene play an important role in the formation of conidia in A. oligospora.4. RNA-seq analysis of the wild strain and ΔabaA mutant. The hyphae of ΔabaA mutant and wild-type strain were collected after the strain was cultured at5d,8d and11d, respectively. Total RNA was extracted from those samples, and the global expression profiles were compared by RNA-seq analysis. The expressional levels between the wild strain and ΔabaA mutant were compared at different time points during the formation of conidia, and seveal genes involved in the conidia production were screened.Innovations of this paper were described as follows:1. Two sporulation regulatory genes were successfully knocked out in A. oligospora. And the mutants were compared with the wild-type strain in phenotypes and physiological features. The functions of the two sporulation regulatory genes were preliminarily presumed accoding to our results.2. The expression levels between the wild strain and ΔabaA mutant were compared at different time points during the sporulation, and seveal genes involved in the conidia production were screened for further research.