Expression Of Activator Protein PeaT1 From Alternaria Tenuissima In Pichia Pastoris And Studying Of Its Interacting Protein

A activator protein PeaT1 from Alternaria tenuissima JH505 can activate plant resistance against tobacco mosaic virus and can also enhanced a wide range of crop growth. The protein-coding gene was cloned using RT-PCR and RACE techologly. In this study, PeaT1 was expressed successfully in Pichia pastoris. To explain the molecaular mechanism of PeaT1, the PeaT1 interacting protein was studyed by phage display techologly and yeast two-hybrid system. The results are as follows:1. PeaT1 gene was subcloned into the P. pastoris expression vector pPIC9K, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pPIC9K-peaT1. The recombinant plasmid was linearized by Sal I or Bgl II and transformed into P. pastoris GS115 by electroporation method, respectively. The gene was in frame integrated into the Pichia genome through homologous recombination. Recombinant strain was screened by MD and further confirmed by PCR. Regulated by theα-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant protein was expressed and secreted into the culture supernatant. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 35 kD. The supernatant was collected and then purified by anion exchange chromatography. This puried protein can promote wheat seedling growth obviously.2. A 15-mer phage display peptide library was screened for specific binders against PeaT1. After 3 rounds of panning, positive phage clones were enriched. 10 out of 90 selected phage clones could bind specifically with PeaT1 and their affinity was identified by ELISAs. The 10 sequenced clones represent 9 kinds of amino acid sequence. Comparison of the short-peptides sequence with Arabidopsis thaliana's protein database, six proteines containing one of the 15-peptide sequence were obtained.3. High-quality ds cDNA were synthesized from total RNA of Arabidopsis thaliana using SMART technology. Then ds cDNA were transformed into yeast with the Sma I-linearized plasmid pGADT7-Rec, in which repair enzymes will restore the linearized plasmid to its circular form through homologous recombination. 5 Ade+/His+ colonies were found through cotransformation. They can also activate the expression of a third reporter gene-MEL1. Sequencing analysis indicated that four colonies have similar sequence. Finally, we obtained two interacting protein named as PIP-1 and PIP-2.4. PIP-1 is ATLFNR1, tranfering electrons within the cyclic electron transport. PIP-2 is a nascent polypeptide-associated complex domain-contained protein. PIP-2 is up to 80.6% similar to Nicotiana benthamian NbBTF3, aβ-NAC, which affected development and/or physiology of chloroplasts and mitochondria in plants.