Cloning And Functional Analysis Of Carnitine Acyl Transferase Genes From Phytophthora Capsici

Phytophthora capsici is an important oomycete plant pathogen that causes blight and fruit rot of peppers and other important commercial crops. Now, the prevention of Phytophthora blight of capsicum is still based on traditional chemical-based prevention causing excessive heavy, metals, pesticide residues problem, coupled with slow progress in breeding for disease resistance. The sequencing problems brought a great threat to human health and environment security, seriously affect the pepper production. Therefore, the use of advanced molecular biology techniques to explore important pathogenic factor and pathogenesis of Phytophthora capsici become the hotspot and keystone of the current researches.According to the relevant literature, Magnaporthe grisea invades plant tissue due to the action of appressoria, which are used to breach the leaf cuticle and allow development of intracellular, infectious hyphae. Targeted deletion of PTH2 encode peroxisomal Carnitine Acyl Transferase genes, generated mutants that were completely non-pathogenic, demonstrate that peroxisomal carnitine acetyl transferase (CAT) activity is necessary for appressorium function. Now there is less study on the appressorium-related genes of oomycete, therefore, the research on this type gene can be of great significance.In this study, the highly pathogenic strain of Phytophthora capsici strain SD33 saved in our lab was used as experimental material, and combinating with previous researches, we carried on research about the CAT homology gene in P. capsici. The details are as follows:1. Multiple sequence alignment, gene cloning, bioinformatics analysis and phylogenetic analysis of carnitine acetyl transferase genes derived from P. capsici strain SD33. According to the CAT gene reported in the literature, we filter the CAT gene from Phytophthora capsici genome by biological software, and successfully cloned the sequences of two CAT genes, we named it as PcCATl and PcCAT2. Using a variety of bioinformatiics software to predict and analyze the two gene sequences. We selected the CAT gene sequences of different species to construct a phylogenetic tree, that shows different species of CAT gene gathered in the corresponding different branches, demonstrate that the CAT gene has a certain conservative in oomycete.2. To analyze expression of PcCATl and PcCAT2 gene in different growth and infection stages. We analyzed expression pattern during the early interaction of the PcCAT1 and PcCAT2 genes by the qRT-PCR technology, the results show that the two genes were efficiently expressed at 4 h after inoculation, at this time the expression of the two genes were significantly up-regulated.3. Stable silencing and over-expressing transformations for PcCATl and PcCAT2 genes in P. capsici strain SD33. In order to further study the function of PcCATl and PcCAT2 genes in Phytophthora capsici strain SD33, with pHam34 vector to build PcCAT1 and PcCAT2 full-length gene silencing and over-expressing vectors. By PEG-mediated protoplast cotransformation techniques, we got stable silencing and over-expressing transformats of PcCATl and PcCAT2 genes, and then determined the expression levels by RT-PCR and real-time quantitative PCR.4. Biological analysis and pathogenicity identification of the silencing and over-expressing transformats of PcCATl and PcCAT2 genes. We observed and comparatively analyzed the different developmental morphology of all kinds of transformation and P. capsici strain SD33, such as mycelium, sporangia, appressorium, induced zoospore of all kinds of transformation and P. capsici strain SD33, inoculated the leaf of pepper to detect the pathogenicity. Observed and analyzed the infect structure and process of all kinds of transformation and P. capsici strain SD33 by Aniline blue staining.Through this study, we can see the function of CAT in the pathogenesis of P. capsici, and we proved that PEG-mediated protoplast co-transformation technology can be implemented in P. capsici and it was determined that stable transformation successfully supply a molecular tool to exploit the pathogenic mechanism of Phytophthora blight of capsicum.