Cloning Of 10 Pectate Lyase Genes From Phytophthora Capsici And Functional Research Of Pcpel1

Phytophthora blight of peppers 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 role in the relation of pathogenesis action by pepper blight in pepper, including cutinase, pectinase, cellulose, hemicellulase, protease and so on. And pectinase can be classified as polygalacturonase (PG), Pectinmelythesterase (PME), and pectate lyase(PL) . PL is a key roll in infection process of P. capsici. , through degrading plant cell wall , and is ubiquitously secreted in fungi, bacteria and Oomycets. The research is primary on pel gene cloning and expression.The paper expatiated selecting pel genes through a DNA genetic library constructed basing on a strong infection strain of P. capsici, and eukaryotic expression ,Real-time fluorescent quantitative PCR , western blot of pcpel1 .Ten pel genes were isolated by screening genomic DNA library with PCR method. Then pcpel1 recombinant protein was obtained through eukaryotic expression, and purified through Ni-NTA system. Western blot with anti-His antibody showed that pcpel1 can express efficiently in Pichia pastiris. The process and results are as follows: 1. Selecting DNA libraty whith Pool-PCR, 10 pcpel obtainedA serious of primers were designed according to data including nucleotide sequences of fungi, Oomycets, bacteria, and plants from Genbank , and the resultant comprised 4 pairs. Repeating PCR to screen the DNA library with different primer pair respectively, we got three full length genes and seven fragments. Blast result of the 10 gene on NCBI, showed they were all pectate lyase genes.Analysis of sequences indicated that there were highly conserved sequence in the ten pel genes and various N-glycosylation sites. The pel genes of P. capsici showed high homogeneity compared with pel 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 1800bp in length and encoded a deduced amino acid sequence of 600 residues, and the molecular weight was about 50kd. The signal peptide cleavage site was predicted by SignalP 3.0 server(http://www.cbs.dfu.dk/service/signalP), showing their signal peptide was around 21-22 residues .The N-glycosylation sites were predicted by NetNGlyc 1.0 server (http://www.cbs.dfu.dk/service/ N-glycosylation), which also dipted difference among the ten gene, they had 1-6 N-glycosylation sites. And the deduced isoelectric point was between 6-10, through analyse online programe (http://us.expasy.org).2. Amplification of the 3′or 5′end sequence of pel genes There are two ways for amplificating the 3'or 5'end of pel genes.(1)RACE: collect 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. There are two genes obtain the full length.(2)TAIL-PCR: collect mycelium of the phytophthora capsici strain after cultivation in liquid shake medium in conical flask for five days. Then grind the mycelium in liquid nitrogen and extract total DNA using TCAB. Then process the Thermal asymmetric interlaced PCR (TAIL-PCR) whose model was product of the first-round PCR ,and primers are GSP1 and AD. 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. We obtained the full length of other 4 fragments.3. Eukaryotic expression and purified of pcpel1 Gene-specific primers were designed and synthesized to amplify the pcpel1 mature peptide sequence according to known sequence. The amplified fragment was inserted into pPIC9K. Recombinant expression plasmid pPIC9K/pcpel1 was constructed. The plasmid was transferred into E. coli DH5αto obtain positive clones and to amplify culture. The plasmid pPIC9K/ pcpel1 was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme Sal 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 65KDa by SDS-PAGE .4. RT-PCR and Western BlotThe leaves of peppers with 4-6 fully expanded were inoculated with SDPH-33 and then the total RNA of infected leaves was isolated and cDNA was synthesized. Specific primers were designed based on the sequence of pcpel1 and the expression of pcpel1 in pepper leaves was detected by Flurogenic Quantitative Polymerase Chain Reaction (FQ-PCR). The result indicated that pcpel1 can express in infected peppers respectively and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on the 7th day. However, these was no expression of pcpel in the healthy peppers. Based on the result of FQ-PCR.PCPEL1 was used to prepare antibodies in Taishan white rabbits, then the antibody was used to tested the pcpel1 of expression. Western blot with anti-His antibody showed that expressed protein could specially react with this antibody, which can express in infected pepper and the expressed level became stronger with the time prolong after inoculation. The pcpel1 was determined to be the most important pathogenetic gene.