Screening For The Interacting Proteins Of Activator Protein PemG1 From Magnaporthe Griesea And Their Functional Analysis

Activator protein PemG1 is a type of protein elicitor isolated from Magnaporthe griesea. It can strongly induce the resistance to disease and promote the plant growth. At present, its receptor proteins and signal transduction pathway in plant become hot study. In this paper, the interacting proteins or peptides of the PemG1 were screened by yeast two-hybrid and phage display technology, and analysis whether they have the characters of the receptor proteins. The key experimental results are as follows:(1) Yeast two-hybrid LexA system screening: Firstly, we constructed the DNA-BD fusion vector pLexA- pemG1 by cloning activator protein gene pemG1 from Magnaporthe griesea into pLexA vector; and tested for expression of bait protein. The result shows that neither PemG1 has the autonomous reporter gene activation, nor yeast cell toxicity by transforming the fusion vector pLexA- pemG1 into yeast strain EGY48[p8op-lacZ]. By screening for interacting protein of PemG1 from tomato cDNA library cloned in pB42AD vector using sequence transformation and repeatly restreaking on SD inducible medium (SD / Raf / Gal /-His /-Trp /-Leu /-Ura / X-Gal + BU salts) to eliminate the false positive, finally, 10 groups of the interacting protein were obtained. After blasting the sequences by NCBI BLAST,an interacting protein MGIP-18 with twice transmembrane structure is deduced to be the receptor of the activator protein PemG1.(2) Yeast two-hybrid GAL4 system screening: We also constructed the DNA-BD fusion vector pGBKT7- pemG1 by cloning pemG1 into pGBKT7 vector and transformed into yeast strain AH109 for testing no autonomous reporter gene activation and cell toxicity. A cDNA library was generated with SMART III and CDS III sequence from rice leaf by SMART? cDNA Synthesis technology including the processes such as total RNA extraction, RT-PCR and LD-PCR. By cotransforming with the fusion vector pGBKT7- pemG1, ds cDNA generated by SMART? cDNA Synthesis technology and Sma I-linearized pGADT7-Rec, screened on the SD medium (SD/–Ade/–His/–Leu/–Trp/X ?α-Gal), the interacting protein MGIP 4-1 (or MGIP 4-6) is obtained, which is highly homologous with the heat shock proteins DnaJ. DnaJ as a molecular chaperone protein is involved in post-translational modification, protein folding process, and regulate the combination between receptor and ligand.(3) Phage display technology biopanning: Using purified protein PemG1 as an antigen, screened with phage 15-mer peptides library by four circles of"adsorption- renounce - amplified". Randomly selected 90 clones for ELISA, measured the absorption value of OD450, and then sequenced the positive clones. Finally, two subunits peptides sequence B2,C2(or D2)of NADH dehydrogenase were obtained, which is a key enzyme in the electron transport chain on the mitochondrial inner membrane. Another peptide D3(or E2)was also obtained, which is the protein-binding domain of DnaJ family of heat shock protein.(4) Functional analysis: As a receptor protein, it usually combines with the ligand molecular, has the structure of transmembrane, and is involved in the ligand-mediated signal transduction process. By blasting their sequences on NCBI and analyzing the structure of the interacting proteins, MGIP-18 was considered to have the character of a receptor. Moreover, interacting protein MGIP 4-1 (or MGIP 4-6) was deduced to be involved in regulation of the receptor and ligand combination.