Studies On Expression, Preparation, Characterization And Function Of Tobacco PR-1a

A group of plant-coded proteins induced by different stress stimuliincluding viruses, fungi, bacteria, physiological, chemical or physicalfactor, named "pathogenesis-related proteins"(PRs) is assigned animportant role in plant defense against pathogenic constraints and ingeneral adaptation to stressful environment. Tobacco PR-1a was the firstpurified and characterized pathogenesis-related protein and was regardedas the type member of PR-1 family.In order to establish an effective method of procaryotic expressionand purification for PR-1a protein, coding sequence of tobacco maturePR-1a was sub-cloned into Escherichia coli expressing vectorpGEX-5X-3 for Glutathione S-transferase-fusing expression. Using thedesigned forward and reverse primers containing BamH1 and XhoI site,respectively, cDNA(0.43kb) for coding tobacco mature PR-1a proteinwas amplified successfully by PCR, with pBluescript SK vectorcontaining tobacco PR-1a gene as the template. Then PCR productionswere directly inserted into T/A cloning vector and the recombinants weredigested with restriction enzymes BamH1 and XhoI. The PR-1a fragmentwas ligated with pGEX-5X-3 which was digested with the same enzymes,and then transformed E.coli JM109. The construction of pGEX-PR1a expressing vector was validated by double enzyme digestion andbidirectional DNA sequencing, and tobacco PR-1a proteins were expressedin heterologous systems for the first time.The clone with the highest expression was chose for subsequentexperiments. The optimized condition for induction was at IPTG finalconcentration 1.0 mmol/L, 29℃for 4 h. A majority of fusion protein,named GST-PR1a was soluble perhaps owing to Glutathione S-transferasetag.Induced cells were homogenized by sonic disruption and then thesupernatant was collected by centrifugation. Because fusion proteincontaining GST-tag, GST-PR1a(42 kDa) was isolated and purified byGlutathione Sepharose 4 Fast Flow affinity column. The yield was about6mg of recombinant proteins/1L of bacterial culture. The N-terminalGST moiety was removed from the recombinant protein by Factor Xa,then Factor Xa was captured by agarose Xarrest^TM. At last, purifiedPR-1a protein(15 kDa)was acquired.Although PR-1a was the first discovered PR protein and the type ofPR-1 family, its three dimensional structure and active site had not beenreported. The 3D-structure of the protein determines its function to ahigh degree. The 3D-structure of tobacco PR-1a was modeled by usinghomology and molecular dynamics methods. P14a protein was searchedout by probing with tobacco PR-1a coding sequence and percentagesimilarity to tobacco PR-1a was 58.7ï¼…. Initial model was constructed byauto-modeling program named Modeler, then optimum conformation oftobacco PR-1a was acquired after energy-minimum and moleculardynamics simulation. The conformation at 500ps was chose to be thefinal conformation. Compatibility of amino acid residues was reviewedby Profile—3D program, and geometry characteristic of the model wasexamined by Prostat program. Results showed that the 3D-structure weconstructed was reasonable and creditable.On the base of the modeling and a highly conserved sequence GHYTQVVW in PR-1 families, two possible active sites were predictedby Binding-Site module. Analyses from sequences and geometric figureand angle showed that Site 1 which comprised Leu44, His46, His48,Trp72, Asn84, Cys86, His94, Thr96, Gln97, Trp100, Asn129, Tyr130,Pro135, Tyr136 was more likely to be the active site than Site 2.Purified GST-PR1a and PR-1a proteins were incubated at 20℃～100℃for 30min, fluorescence spectra indicate that the fluorescenceintensity of GST-PR1a at 20℃～90℃and PR-1a at 20℃～60℃hadsmall changes. Results showed that the conformations of GST-PR1a andPR-1a were similar to natural state at above temperature range. PR-1aprotein was stable under 60℃and GST-tag improved the stability of thefusion protein.The purified GST-PR1a was used to immunize New Zealand rabbitsand the polyclonal antibody was obtained, whose titer against GST andGST-PR1a were 1:64 and 1:32 respectively. Double-immunodiffusionand western blotting both confirmed the preparation of anti-rabbitGST-PR1a polyclonal antibody of high specificity. The anti-rabbitGST-PR1a polyclonal antibody we obtained can immuno-react not onlywith recombinant GST-PR1a protein, but also with native tobacco PR-1a,-1b and-1c. We can conclude that the recombinant GsT-PR1a protein isof identical immunochemical properties with native tobacco acid PR-1.Studies on antifungal activities of recombinant GST-PR1a proteinsbefore and after removement of GST-tag were carried out. The results invitro showed that neither GST-PR1a nor cleaved PR-1a could inhibitmycelium growth of Phytophthora nicotianae. The inhibitory tests ofPhytophthora infestans zoospore germination in vitro revealed thatrecombinant proteins had consistent antifungal activities with naturetobacco PR-1. The germination of the zoospores and germ-tube lengthwere reduced in the presence of GST-PR1a at a concentration of 600μg/ml, and the germination of the zoospores was almost stoppedcompletely in the presence of cleaved PR-1a at a concentration of 250μ g/ml. Dose-response curve of inhibition of Phytophthora infestanszoospore germination by cleaved PR-1a proteins showed that IC₅₀ andIC₉₀ of cleaved PR-1a were 100μg/ml and 250μg/ml respectively. It wasnoted that GST-PR1a protein had much more lower inhibition againstPhytophthora infestans than cleaved PR-1a protein. When theconcentration of GST-PR1a protein reached up to 600μg/ml, it causedno more than 50ï¼…growth inhibition. These results suggested that theGST-tag in recombinant protein probably interfere in antifungal activity.However, the effectiveness of PR-1a With N-terminal GST-tag ininhibiting P. infestans suggests that the free N-terminus of PR-1a is notnecessary in the antifungal mechanism.The fusion GST-PR1a protein and the corresponding antiserum weobtained could be used for GST pull-down assay to detect interaction ofPR-1a with its targetsin further studies, in order to provide some cluesfor finding out the relationships of PR-1a and other proteins. Moreover,the recombinant proteins will be used as ligand for binding studies andaffinity chromatography to identify and isolate the PR-1 receptors.Finally, the three dimensional structure and the possible active sites oftobacco PR-1a offer very significant and important information forprobing into the antifungal mechanism further.