Screening The Receptors Of Serine Proteases Of Rice Blast Fungus

The rice blast fungus, Magnaporthe grisea, is an important disease fungus, is also taken as a model for the study of filamentous fingi. It's thought that serine proteases maybe one of the elicitors which will be secreted outside the cell and will interact with receptor proteins. MGG₀7965.5 gene encodes the serine proteases.In this research we found that the intron which is shown in the gene dabatase of MGG₀7965.5 gene may be an extron as the sequences of cDNA and genome show the same result. We constructed the pPICH-ser expression vector successlly, and transformed it into Pichia pastoris GS115. We expressed and purified the Serine Protease by 6×His tag which is partaked in pPICH vector.In order to study the role of serine protease in plant defense, we constructed the recombinant bait plasmid pBD-GAL4-ser. Then we transformed it into Saccharomyces cerevisae AH109 and tried to test the bait vector for automatic transcriptional activation as well as toxicity effects. The result showed that the bait vector is inactive and avirulent to AH109. AH109 strain transformed with recombinant bait plasmid mated to rice cDNA to find objected gene having interaction with bait plasmid. Thirty-two clones were selected from two series of SD/Trp^-/Leu^-/Ade^- plates and SD/Trp^-/Leu^-/Ade^-/X-α-gal plates. The vector containing objected genes were transformed into E.coli DH5α. After that, we extracted the plasmid of E.coli and retested them by yeast two-hybrid system. We got 12 contransformation clones on SD/Trp^-/Leu^-/Ade^-/X-α-gal plate. At the same time, we amplified the prey DNA fragment by PCR using consensus primers in the pAD-GAL4 vector. These clones were sequenced. At last, we obtained 12 positive clones and analysed the sequences by blast in NCBI. The results showed that 4 pAD-GAL4 vector sequences were prayed among the 12 positive clones. The residual stains were classified as follows: three of them were relative with protein metabolism, one was relative with lipid metabolism, another one was relative with ion channel, while the others were unknown. The results will facilitate further studies on interaction between the serine protease and the possible receptors and their biological functions.