Cloning and expression analysis of the chitinase gene Chit1 from entomopathogenic fungus Nomuraea rileyi CQ strain

Abstract:

Abstract: [Objective] In order to deeply understand the role of the entomopathogenic fungal chitinase in invasion and pathogenic process, we cloned and expressed a chitinase gene from Nomuraea rileyi CQ strain, and detected chitinase activity in recombinant Pichia pastori fermentation supernatant. [Methods] Specific primers were used to amplify the complete sequence of chitinase gene and open reading frame (ORF) from genomic DNA of N. rileyi Cq Strain extracted by CTAB. The fragment of ORF was linked with vector plasmid pPIC9K to construct the pPIC9K-chit1 expressing vector and transferred into Pichia pastori. The recombinant yeast P. pastori was screened by 1.5mg/L G418 and PCR check. Activity of chitinase in recombinant P. pastori fermentation liquid was verified by transparent zone test and OD540 determination, and the molecular weight of chitinase was analyzed by SDS-PAGE. [Results] The complete sequence length of chitinase gene of N. rileyi CQ strain is 2756bp (GenBank accession number EU795711) that contains a 1827bp of ORF chit1, a 76bp uncoding region at the 5’end, a 240bp uncoding region at the 3’end, and 3 introns. The ORF chit1 encoding 424 amino acids of chitinase precursor, and the theoretical restriction site of single peptide is between Gly(20) and Leu(21). Activity of chitinase expressed by recombinant P.pastoris increased with fermentation time, and reached the peak of 482.5U/100μL at 72h. Hydrolysis test of chitin showed clear transparent zone at the 1% colloidal chitin plate. SDS-PAGE analysis suggested that the molecular weight of chitinase was 41.0 kDa. [Conclusion] This research cloned the chitinase gene Chit1 from N. rileyi CQ strain and expressed in recombinant P. pastori successfully. It has great significance for in-depth study on the infection and lethal mechanism of pathogenic fungi to insects.