Pathogenicity And Genetic Variation, Cloning And Expression Of Polygalacturonase Genes From Phytophthora Capsici

Pepper blight is a worldwide disease, which impacted on agriculture of China and other countries badly, and caused enormous loss on economy. The disease was caused by phytophthora capsici. P. capsici is a soilborn Oomycete, which can survive for several months even longer time in the soil as oospores or chlamydospore. At the optimum condition, the pathogen can cause stem rot, root rot and wilt of pepper. The host range of the pathogen is wide including the pepper, pumpkin, cucumber, tomato, and so on.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. Important pathogenic factors of P. capsici are investigated. Polygalacturonase (PG) plays an important role in infection process of P. capsici. The paper took the P. capsici as resources, analysing their genetic diversity and relationship between pathogenicity and their origins in China. High level PG activity isolate of P. capsici was selected. Seven pg genes were obtained by screening genomic DNA library and Pool PCR method. Then pcpg2 recombinant proteins was acquired according to eukaryotic and heterologous inducing expression. At last, the expression level of pcpg2 in pepper tissues was investigated by RT-PCR and Northern Blot. The purified fusion protein was inoculated into leaves of pepper to observe the diseased symptom. Main researchs were as follows:Pepper blight plant and soil were collected from Shandong, Shanxi, Hubei, Zhejiang, Guangdong, Sichuan and Yunnan provinces of China from 2004 to 2005. Genomic fingerprints of 56 strains of P. capsici from different pepper cultivated areas were analysed by using 12 RAPD primers. The result of the RAPD fingerprints showed that the polymorphism existed among different strains. And all strains were divided into eight genotypes. According to the occurrence situation of peper blight in China, this study confirmed that most of the genotypes of being tested isolates were not obviously related to localities, climate and planting style, except for small number of isolates were connected with all those factors.The pathogenicity of 31 isolates of P. capsici was identified by soaking root in spore suspension. The result showed that the 31 isolates were divided into nonpathotype, weak pathotype and strong pathotype . And the analysis of RAPD of P. capsici indicated that the polymorphism existed among isolates with different pathotype. And division of isolates was not related to their different pathotype. In general, it was concluded that there existed obvious differentiation of pathotype and genetic diversity within the 31 isolates of P. capsici, but that there was not opposite relationship between pathotype and genotype.The population genetic diversity of pathogenic and nonpathogenic isolates of P. capsici was assessed by RAPD. The result of RAPD showed that the isolates were divided into two groups. The pathogenic isolates were in a group, where there were two nonpathogenic isolates. Sequence analysis of the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene indicated that pathogenic and nonpathogenic isolates were also separated into two clusters. But the population of pathogenic isolates was less diverse than that of the nonpathogenic isolates, suggesting that the pathogenic isolates was monophyletic origin. For both pathogenic and nonpathogenic P. capcisi, no relationship was observed between their genetic profiles and pathogenicities. Finally, there are high genetic diversity and obvious pathogenecity variation in P. capsici from different areas in China.The activities of PG were determined with spectrophotometer. The genome library of P. capsici with highest activity was constructed in Huada Jiyin Institute. Isolating and identifying pg genes with Pool PCR method, and structural features were analysized. The results of activities showed that level of PG was higher before rejuvenation than that of after rejuvenation. And the levels of PG of pathogenic isolates were higher than those of nonpathogenic isolates. The genome DNA library of P. capsici isolate was constructed. Seven pg genes were cloned from the genome DNA library, and their GenBank accession numbers were DQ365796, DQ415987, DQ415988, EF558848, EF558847, EF558846 and EF558845. Analysis of their sequences indicated that there were high similarities of the seven pg genes and existed various N-glycosylation sites. The predicted proteins had a typical PG consensus fragment and protein active site (NNYCSGGHGISIGS), and had a similar tertiary structure. Finally, the result showed that pg genes of P. capsici gathered with pg genes of other Oomycetes and defined the station of Oomycete in the nature.Gene-specific primers were designed and synthesized to amplify the polygalacturonase mature peptide sequence according to known sequence. The amplified fragment was inserted into pPIC9K. Recombinant expression plasmid pPIC9K/pcpg2 was constructed. The plasmid was transferred into E. coli JM109 to obtain positive clones. The plasmid pPIC9K/pcpg2 was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme Sal I. After screening on plates with G418, PCR was performed to amplify and identify the genome DNA of these transformants. Several recombinant strains that could express pcpg2 were found. The expressed protein was detected by SDS-PAGE. Specific antibody was prepared by immunising rabbit. Western-blotting demonstrated that the recombinant protein could specially react with this antibody at 37KDa size. Finally, pcpg2 was expressed in P. pastoris, and specific antibody was prepared.Gene-specific primers were designed and synthesized to amplify the polygalacturonase mature peptide sequence. The amplified fragment was inserted to pET-22b(+). Then recombinant plasmid pET/pcpg2 was constructed. The plasmid was transferred into E. coli BL21(DE3) and induced by IPTG. The result of SDS-PAGE and western-blotting showed that the recomnant has expressed a 37KDa specific protein. And western-blotting further conformed the specificity of pcpg2-antibody.Leaves of four-six leaves pepper were inocubated with P. capsici spore suspension. Expression of pcpg2 in leaves was detected by RT-PCR with designing the gene-specific primers. The result showed that level of expressin increased with extending of time, and the level is highest at the seventh day. DNA fragment amplified by PCR was synthesized to probe. The result of northern-blotting indicated that expressions of pcpg2 were detected in inocubated leaves, although there was no expression in uninocubated leaves.The fusion protein PG2 was purified. The the cell wall was extracted from pepper leaves. The analysis of DNS indicated that purified PG2 could act with the cell wall. The result showed that leaves inoculated by PG2 were diseased, however, the leaf inoculated by sterile water was no symptom.