Cloning Of Four Pectin Methylesterase Genes And Eukaryotic Expression Of Pcpme6 From Phtophthora Capsici

Pepper blight is a worldwide disease, which impacted on agriculture almost in every country, and caused enormous loss on economy. The disease was caused by phytophthora capsici , a soilborn Oomycete, which can survive for several months even longer time in the soil as oospores or chlamydospore. In the optimum condition, the pathogen can not only impact pepper, and pumpkin, cucumber, tomato, and other plant can be its host as well.At present, the pepper blight was controlled mainly by chemical methods and breeding resisitant cultivars. However, strong variability in pathogenicity of P. capsici caused the instability of resistant breeds. It is very important to research infection mechanism, resistance biochemistry mechanism, resistance genetics, and quantitative trait loci and gene clone, as well, for resistance identification and application in breeding for disease resistance. Cell wall degrading-enzyme plays very important roll in the relation of pathogenesis action by peper blight in peper, including cutinase, pectinase, cellulose, hemicellulase, protease and so on. And pectinase can be classified as polygalacturonase (PG), Pectinmelythesterase (PME), and pectinlyase(PL) . PME is a key roll in infection process of P. capsici. , through degrading plant cell wall , and is ubiquitously secrated in fungi, bactera and Oomycets. The research is primary on pme gene cloning and expression.The paper expatiated selecting pme genes through a DNA genetic library constructed basing on a strong infection strain of P. capsici, and eukaryotic expression and western blot of pcpme6 .Four pme genes were isolated by screening genomic DNA library with PCR method. Then pcpme6 recombinant protein was obtained through eukaryotic expression, and identified by western blot with multiclone antibody. The process and results are as follows:1 Selecting DNA libraty whith PCR, 4 pme obtainedA serious of primers were designed according to data including nucleotide sequences of fungi,Oomycets, bacteria, and plants from Genbank ,and the resultant comprised 8 pairs, P230+ AP650,P230+ P920,P230+ P795,P445+ P920,P445+AP650, P620 + P920 ,P650+P920,P445+ P795. Repeating PCR to screen the DNA library with different primer pair respectively, we got three full length genes and one fragment. We got pcpme6 with P230+AP650,and pcpme7and pcpme8 with P650+P920, with P445+P920 and P445+P795 having the same gene pcpme9. Blast result of the 4 gene on NCBI, showed they were all pectin melythesterase genes.Analysis of sequences indicated that there were highly conserved sequence in the four pme genes and various N-glycosylation sites. The pme genes of P. capsici showed high homogeneity compared with pme genes of other Oomycetes and consequently defined the station of Oomycete in the nature.They had the same basic structure through analysis using DNAman soft ware, the biggest open reading frame was around 1100bp in length and encoded a deduced amino acid sequence of 345-348 residues, and the molecular weight was about 37-38kd. The signal peptide cleavage site was predicted by SignalP programe (http://www.expasy.org), showing their signal peptide was around 16-20 residues .The N-glycosylation sites were predicted by scan prosite (http://www.cbs.dfu.dk/service/signalP) , which also dipcted difference among the four gene, they had 4-7 N-glycosylation sites. And the deduced isoelectric point was between 5-9, through analyse online programe (http://us.expasy.org).2 Amplification of the 3′end sequence of pcpme9 by 3′raceCollect mycelium of the phytophthora capsici strain after cultivation in liquid shake medium in conical flask for seven days. Then grind the mycelium in liquid nitrogen and extract total RNA using Trizol. Use reverse transcriptase to synthesis cDNA as model for the first-round PCR in which the primer were USP and GSP. Then process the second-round PCR whose model was product of the first-round PCR ,and primers NUSP and NGSP. The target DNA fragment recovered from agarose gel and cloned into E.coli DH-5α,and then sequenced in biology company. Recombine the full length gene, and analyze using DNAman soft ware.The result showed that its full length was 1041bp, and the biggest opening reading frame contained 1038bp, encoding 346 residues. The deduced molecular weight was about 38kd. The signal peptide 16 residues and N-glycosylation sites number was seven, according to the online programe (http://us.expasy.org).3 Eukaryotic expression and western blot of pcpme6Gene-specific primers were designed and synthesized to amplify the pcpme6 mature peptide sequence according to known sequence. The amplified fragment was inserted into pPIC9K. Recombinant expression plasmid pPIC9K/pcpme6 was constructed. The plasmid was transferred into E. coli JM109 to obtain positive clones and to ampify culture. The plasmid pPIC9K/ pcpme6 was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme stu I. After screening on plates with density gradient of G418, transformants'DNA was extrcted and PCR was performed for identification . The expressed protein was detected about 50KDa by SDS-PAGE .The protein secreated from recombinant strains was purified through SDS-PAGE to prepare polyclonal antibody by immunizing rabbits. Western blot showed that expressed protein could specially react with this antibody, which dipcited that the recombinant successfully expressed, accompanied with prepared antigen's relative immunocompetence, and the sensitivity and specificity of polyclonal antibody were high, as well.