Cloning Of Gene Chit2and Pr1from Paecilomyces Fumosoroseus And Their Protein Expression In Both Prokaryotic And Eukarytic Expression System

Paecilomyces jumosoroseus is a kind of important entomopathogenic fungi. It is a pollution free insecticide which has strong contact poisoning function on insects, and with low insect resistance.Chitinase and protease could be product by fungi, including Paecilomyces fumosoroseus. They can degrade insect’s cuticle. So, chitinase and protease act as important virulence factors in insects infecting. But currently, there are just a few studies concern with the chitinase and protease in Paecilomyces fumosoroseus.With the aim of cloning the chitinase and protease gene from Paecilomyces fumosoroseus and construct the recombinant expression vector of these two genes, we completed the following work:1. The chitinase gene chit2and protease genes prl were cloned from Paecilomyces fumosoroseus successfully.2. The full length of Pfchit2gene cDNA is1744bp, has a1272bp open reading frame and3introns (56bp,46bp,50bp) with the typical sequences GT... AG.5’-UTR and3’-UTR were109bp and203bp respectively. It encodes423amino acids, the signal peptide is22amino acids long. The molecule quantity of mature protein is46.3kDa. The cloning chitinase belong to the family of Glycosyl hydrolase18including2highly-conserved regions FDGIDIDWE and SIGGW, which are the centre point of active hydrolysis of18family. The nucleotide sequence homology are87%,75%,73%,72%and71%with those of Lecanicillium attenuatum, Nomuraea rileyi, Beauveria bassiana, Trichoderma hamatum and Trichoderma harzianum respectively.3. The full-length cDNA of Pfprl gene is1632bp, with a1143bp open reading frame and three introns. The open reading frame encodes380amino acids. Its molecule quantity of mature protein is39kDa. It shows80%,76%,64%and64%nucleotide sequence homology with those of Lecanicillium psalliotae, Beauveria bassiana, Metarhizium lepidiotae and Purpureocillium lilacinum respectively.4. The pET-28a-pfchit2, pET-32a-pfchit2, pPICZB-Pfchit2and pET-28a-pf Prl, pET-32a-Pf Prl, pPICZB-Pf Prl recombinant expression vectors were constructed successfully. Both chit2and Prl can expressed in prokaryotic and eukaryotic expression system, but the expressed protein is existed as nclusion bodies.5.The optimal culture conditions of chitinase production in Paecilomyces fumosoroseus were carried out. The highest amount of chitinase production were gained when Paecilomyces fumosoroseus cultured for72hours. And2%(w/v) NaNO3can improve enzyme production.This research helps lay the groundwork for future chitinase and protease genetical modified.