Expression of Alternaria tenuissima peaT2 gene in Pichia pastoris and its function

Abstract:

A positive clone was screened from Alternaria tenuissima expression library . The result of sequencing and gene analysis indicated that the cloned DNA fragment has a complete ORF，which was named peaT2 (Protein Elicitor from Alternaria tenuissima 2) with the GenBank accession number EF212880. The gene was amplified by PCR and subcloned into the pPIC9K of Pichia pastoris expression system. The resulting recombinant plasmid pPIC9K/peaT2 was verified by sequencing and digested by SacⅠ. The linearized DNA was transformed into P. pastoris GS115 by electroporation. By means of MD and G418-YPD plates and PCR, the recombinant P. pastoris strains (his^- mut⁺) were obtained. A recombinant clone cultivated on YPD plate with high concentration of G418 was randomly selected as expression strain. The protein expression was induced by methanol and analyzed by 12% SDS-PAGE. The results of SDS-PAGE and Western blot indicated that this gene was expressed successfully in P. pastoris with the induction of methanol. In BMMY culture medium, the expressed protein reached its maximum amount at 72h, whereas no corresponding protein was detected in the negative control. Bioassay was performed with the expressed protein. After soaking with the expressed protein for 8h, wheat seeds were cultured, the height of seedlings was measured after 24h, 36h, 48h, 7d, respectively, and the root length was measured after 7 days. The results showed that the expressed protein can promote seeding growth and root length obviously in appropriate concentration. It is revealed that PeaT2 can act as protein elicitor.