Cloning And Analysis Of Chitinase Gene (PoChi1) From Pleurotus Ostreatus

Chitinase is involved in the synthesis and degradation of fungal cellwall, and plays a key role in morphogenesis, growth and development of fungi. So far,there has been no research and report about chitinase gene of Pleurotus ostreatus.Chitinase gene (PoChi1) of Pleurotus ostreatus was cloned in this study, and theexpression vector was constructed and transformed into E.coli BL21(DE3). PoChi1expression pattern was analyzed by semi-quantitative RT-PCR. Overexpressionvector and antisense expression vector were constructed and transformed intoPleurotus ostreatus New831by Agrobacterium tumefaciens-mediated method withthe hygromycin resistance selection. The positive transformants were determined byPCR. The differences between transformants and original strain were analyzed. Theresults of preliminary study showed that PoChi1gene could be involved inmorphogenesis, growth and development of Pleurotus ostreatus. The mainconclusions as follows：1. Pleurotus ostreatus chitinase gene was cloned from total RNA extracted fromPleurotus ostreatus New831mycelia by PCR for the first time. Bioinformaticsanalysis indicated that the PoChi1coding sequence was1,188bp, which could encode395amino acids polypeptide including N-terminal signal peptides, three highlyconservative domains and a short C-terminal domain. The secondary structure andtertiary structure prediction of the POCHI1showed that it was similar to (beta alpha)8round barrel structure of the18family chitinase. Phylogenetic tree analysis showedthat POCHI1was a member of class Ⅲ chitinases.2. The prokaryotic expression vector was constructed and PoChi1was expressedin E.coli BL21(DE3). The SDS-PAGE analysis showed that relative molecular massof recombinant protein was approximately29kDa. Chitinase activity of crude extractswas2.172U·mL-1. The expression pattern of PoChi1was analyzed bysemi-quantitative RT-PCR in different growth stages of Pleurotus ostreatus. ThePoChi1expression level of mature stage was the highest among different growth stages of Pleurotus ostreatus. The PoChi1could play a certain role in growth anddevelopment of Pleurotus ostreatus fruiting body.3. The PoChi1eukaryotic overexpression vector pBHg-gpt and antisenseexpression vector pBHg-gfpt were constructed with the Pleurotus ostreatusendogenous gpd promoter.4. Plasmid pBHg, pBHg-gpt and pBHg-gfpt were transformed into Pleurotusostreatus New831by Agrobacterium tumefaciens-mediated method.3,4,6putativetransformants were obtained on PDA medium containing100μg·mL-1hygromycin,respectively, and3,2,1positive transformants were obtained after PCRdetermination, respectively. These transformants and original strain were cultivated onPDA medium. The growth trend of transformants and original strain were observed.The results showed that the growth trend of Pleurotus ostreatus strain pg containingthe overexpression vector was better compared to original strain and the growth trendof Pleurotus ostreatus strain pf containing the antisence expression vector was weakercompared to original strain. The PoChi1probably influence the mycelia growth anddevelopment of Pleurotus ostreatus.Above all, chitinase gene (PoChi1) of Pleurotus ostreatus was cloned firstly, andbioinformatics analysis of PoChi1indicated that it was a member of class Ⅲ chitinase.The PoChi1was expressed in E.coli BL21(DE3) and chitinase activity wasdetermined. The expression pattern of PoChi1in different growth stages of Pleurotusostreatus showed that the gene could play a certain role in growth and development ofPleurotus ostreatus fruiting body. The result of overexpression and silent expressionof PoChi1in different transformants indicated that the gene was presumed toinfluence the mycelia growth and development of Pleurotus ostreatus. The furtherstudy may reveal the relationship between chitinase gene and morphogenesis, growthand development process of Pleurotus ostreatus.