Gene Cloning Of Protein Elicitor PeaT2 From Alternaria Tenuissima And Studying Of Its Interacting Protein

Protein elicitor is a specific signal protein which activates pathogen defense and plant growth pathways.The protein elicitor from Alternaria tenuissima. has showed good properties of growth promotion and disease resistance. It is important to do some research concerning its gene function. This paper was mainly focused on protein elicitor of Alternaria tenuissima. The entry library of Alternaria tenuissima and antiserum of anti- Alternaria tenuissima protein were acted as experimental material. After constructing Alternaria tenuissima cDNA expression library, immnoscreening was performed and nine positive clones were obtained. One of the positive clones was chosen and sequenced which had a complete ORF. The gene was subcloned in Pichia pastoris expression system, and expressed successfully with the induction of methanol. Bioassay results showed that the expressed protein can act as protein elicitor. The protein encoded by the gene was acted as baiting protein in the yeast two-hybrid system. After screening tomato cDNA library, its interacting proteins were obtained. Major results were as follows:1. Construction Alternaria tenuissima cDNA expression library. After transfering entry library into Gateway vector to creat expression library by LR Clonase enzyme, the recombinational library was transformed into E. coli BL21 (DE3) competent cells and the library genes were expressed by IPTG induction.2. Immnoscreening the expression library strain by antiserum of anti- Alternaria tenuissima protein, nine positive clones were obtained and sequenced. The sequence of positive clones was compared by Blast in GenBank. The results indicated that five positive clones had similarity with genes in the database; one had 89% amino acid sequence identity to NAD-dependent formate dehydrogenase AciA/Fdh protein; one had 73% amino acid sequence identity to D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase; there were three other positive clones which had same sequences and 76% amino acid sequence identity to Prohibitin. The result of 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 of EF212880.3. peaT2 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 Sac I. 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.4. To search interacting protein of Alternaria tenuissima PeaT2 protein, a bait vector plexA-peaT2 was constructed in yeast two-hybrid system 1, and assay whether PeaT2 activated the reporter genes was carried in the yeast two-hybrid system. The peaT2 gene was amplified by PCR from pDEST17- peaT2, and cloned correctly info yeast vector plexA for constructing bait vector plexA-peaT2. The resulting recombinant plasmid was transferred into yeast cell EGY48 [p8op-lacZ] by PEG/LiAC, and tested whether PeaT2 could activate the reporter genes in the yeast two-hybrid system 1. The bait vector plexA-peaT2 was successfully constructed, and PeaT2 did not activate the reporter genes. The bait vector plexA-peaT2 can be used for yeast two-hybrid system 1. The recombinant plasmid could be used as "bait plasmid" to screen tomato cDNA library and search PeaT2-interacting protein. Choose blue clones, diversed plasmid AD, and the plasmid was transferred into yeast cell EGY48 [p8op-lacZ] by PEG/LiAC repeatly, finally, five positive clones were obtained.5. The positive clones were cloned from tomato cDNA AD library and sequenced. The result of sequencing and gene analysis is as follows. One was ferredoxin-I with function of transfering electrons in a wide variety of metabolic reactions; one had 99% amino acid sequence identity to AVA-P2, ATPase of Arabidopsis thaliana. with its function of hydrogen-transporting ATPase activity and rotational mechanism; one was a gene with unknown function; one had 99% amino acid sequence identity to 40S ribosomal protein of Solarium tuberosum; one had 92% amino acid sequence identity to RNA-binding protein of Nicotiana glutinosa. The results indicated that there were receptor proteins in leaf of plant.6. To search more function of peaT2 gene, the gene was ligated into pET28a (+) to construct a recombined vector pET28a-peat2, which was subsequently transformed into E. coli BL21 (DE3) competent cells. Peat2 was expressed by IPTG induction. The protein was purified with AKTA system and further confirmed with ELISA assay. The purified protein will be used in peptide display cloning system.These results above provide some fundamental information for the subsequent research on genetic manipulation and molecular biology of peaT2 gene.