Cloning And Expression Of Sublitisin-like Protease From Isaria Farinosa And Inductive Transcription Of Two Protease Genes

Entomogenous fungi as natural enemies of diverse terrestrial arthropods, has abroad application prospect due to the capacity these entomopathogens have to causenatural epizootics. They are important regulators of host populations in terrestrialecosystems and have been used to develop mycoinsecticides.Isaria farinosa parasitize a broad range of insect species representing numerousorders and are found throughout the tropics and temperate regions. Isaria farinosa issuitable for growth and infection for pests under low temperature condition, so it is moreadvanced than Beauveria bassiana and Metarhizium anisopliae to control winteringpopulations of pest. However, it also has the common shortcomings of entomogenousfungi, such as requirement for strict temperature and high humidity, poor storage, weakability to adapt to the environment, slow kill and unstable for insecticidal efficacy.Therefore, these factors blocked its wide application and commercialization ofproduction, and it is very necessary to develop more effective and more stablemycopesticides by making use of modern molecular biological techniques.According to the cDNA sequence of pr1H in the previous study，the structural geneof sublitisin-like protease, with1650bp, was cloned from Isaria farinosa, and itsupstream sequence(Named: IfPrU, GenBank accession No:GQ505371) was amplified byusing genome walking. The amplified structural gene has a51bp intron compared with itscDNA. The obtained upstream sequence was3189bp, and overlapped352bp withcDNA. Analyses indicated that the upstream sequence contained several regulatoryelements, such as GC-box、HSF and GATA, but the TATA-box and CAAT-box could notbe found.Using SMART RACE RT-PCR technology, another protease gene of the cDNAsequences was cloned from Isaria farinosa (Named: Ifpr1, GenBank accession No:JN014832). Sequence analysis showed that the full length of cDNA was1288bpencompassed an open reading frame (ORF) with960bp encoding319amino acids. Themature protein with a signal peptide consisting of20amino acids had a molecular massof33.2kDa, with a calculated pI of7.39. The upstream promoter sequence of the DNAwas obtained by gene walking technology (Named: Ifpr1-up, GenBank accession No:JN014833). The analyses indicated that the upstream sequence contained severalregulatory elements, such as NIT-2, GC-box, HSF and GATA, but TATA-box andCAAT-box were not found. Two genes from Isaria farinose, pr1H and Ifpr1, were studied at transcription levelby realtime-PCR technique. The results showed that, under inductive by cicada skin, thetwo gene expressions have greatly improved, and the Ifpr1expression is2.97×105timesthan induced before. We therefore speculate that Ifpr1plays a major role in the infectionof host and it may be a major gene of protease in Isaria farinosa RCEF0685.The expression vector pPIC9K with Ifpr1gene was transformed into Pichiapastoris GS115. After continuous methanol induction, the molecular weights of theprotease determined by SDS-PAGE were about50KDa, which doesn’t agree with theresults of the predicted values based on the amino acid sequence (33.2KDa). Enzymaticactivity assay revealed that the expression protein had bioactivity of protease. Themaximum enzymatic activity of crude enzyme liquid was26.9U/ml.