Transformation Of Cuticle-degrading Protease Gene (Cdep1) To Lecanicillium Lecanii And Enhancement Of Protease Activity

Lecanicillium lecanii is an effective bio-control fungus to pests in greenhouse. However, the application of this fungus is limited owing to slow pathogenicity and unstable effect. The extracullar enzymes, including protease, chitinase and lipase, play an important role in the process of infection. It is reported that the virulence of bio-control fungus was increased when improved the secreation of extracullar enzymes. In this article, we cloned a cuticle degrading protease gene from a Beauveriabassianastrain which has a good effect on hosts. The gene Cdep1 was then transformed into Lecanicillium lecanii via agrobacterium-mediated method and protoplast transformation method. Result showed that the protease activity was increased in the transformants. The results of this study are as follows:1. Cloning of Cdep1 gene. An effective fungus stored in the lab was identified as Beauveria bassiana. Cdep1 was cloned by the primers designed by the homologous sequences from GenBank. The sequence of Cdep1 gene was 99% homologous to Beauveria bassiana cuticle degrading protease gene(HQ840791). The amino acid sequence of Cdep1 was 99% homologous to alkalineserineprotease(KGQ12177). The 128 th amino acid was mutated to Ser from Tyr and 239 th was Val to Ala.2. Construction of expression vector. Promotor GdpA and terminator TrpC were cloned from the plasmid pAN7-1. A fusion fragment called GdpA-Cdep1-TrpC(4021 bp) was obtained through an overlap PCR accompanied by a touchdown PCR. Plasmid pBHt2 and the fusion fragment GdpA-Cdep1-TrpC were digested by restriction endonuclease KpnI and HindIII. After recovering from gel, theese two fragments were ligated by T4 legase. The recombinant plasmid was transformed to Escherichiacoli., and confirmed by restriction digestion.3. Lecanicillium lecanii was transformed by agrobacterium-mediated method or protoplast transformation method. The factors that affect protoplast transformation were investigated in this chapter. 1 mlof the conidia suspension of Lecanicillium lecanii(1.5×108 conidia/mL) was inoculated to PD media(100 mL). After culturing for 24 h, the hyphae were obtained by filtering with 240 mesh nylon net. The 30 mg/mL mixed enzymes, including lysozyme, cellulose, snailase(2:1:1) was used at the ratio of 40:1(v/w) to wet mycelia. The protoplast were generated after 3h at 28 °C 100 rpm. The amount of protoplast is 1.8×107 protoplasts/g.4. The number of gene copy was studied by realtime fluorescence quantitative PCR. The reference gene EF1α was conformed according to the previous report. Primer AM3 was designed by Beacon Designer. The relative quantification method was used for the conformation of gene copy number. After eliminating the differences(amplification efficiency, sizes of two amplified fragments, etc.) of two pairs of primer, nine single-copy transformants, one two-copy transformant and one three-copy transformant were found.5. The protease activities of transformants were tested through determining the amounts of tyrosine produced by hydrolyzingcaseinby UVspectrophotometer. The protease activities of transformants were higher than the wild-type strain, and the highest activity was 11-fold by the wild-type strain. These results indicates that the transformant with high protease activity has a sound potential use for pest biological control.