Cloning And Analysis Of Phytophthora Blight Resistance-rjelated Genes In Pepper And Cucumber

Pepper phytophthora blight caused by Phytophthora capsici, is a serious disease inpepper production. Unfortunately, the resistance gene against P. capsici of pepper has notbeen cloned. Because of the complexity of the genetics of resistance to P. capsici and the largegenome, it is difficult to isolate the resistance gene using map-based method. Fruit rot causedby P. capsici is an increasingly serious desease in some area. But there are no cultivarresistant to P. capsici. The expression change of some resistance-related genes (pathogenrecognize gene, defense-related genes, signal transduction, etc.) can dominant the plantsresistance or susceptible to pathogens. In this study, some disease resistance-related genes ofpepper cultivar “AA5” and cucumber cultivar “NW99” were isolated, and these genes can beused for molecular breeding of phytophthora blight resistance cultivars of pepper andcucumber.1. Ten new pepper NBS-RGAs were cloned using degenerate primer by PCR-basedapproach, and other53RGAs were retrived from Genbank. These RGAs can be divided intotwo classes: CNL and TNL. According70%similarity and the Polygenetic tree, the RGAs canbe divided into5groups and24subgroups. Recombination signal was found in the pepperI2-like genes. NBS domain of pepper I2-like and Bs4-like genes are under purifyingselection. We can development RGA-markers according to these RGA sequences forresistance gene mapping.2. The full length of five pepper NBS-LRR genes are isolated by TAIL-PCR and RACEapproach. These five genes designated CaRGA1~5are Rpi-blb1homologs, and share above80%similarity with the Rpi-blb1cluster members of potato. There is a LRR lack of CaRGA1and CaRGA2in contrast to the other three. There are several Rpi-blb1homolog clusters of thepotato genome. The microsynteny of this R gene cluster was interrupted in the tomato and S.phureja genome. There maybe unequal recombination occurred in this locus. The existance ofparalogs and othologs and the homologous clusters implied that replication played main rolein the evolution of this R gene family. 3. Sixty-six DE-TDFs were isolated from pepper challenged by P. capsici usingcDNA-AFLP approach. Function of Thirty-five TDFs can be predicted. They involve theprocess of signal transduction, metabolism, defense, transcription, etc. Expression change ofthese TDFs indicated the plangt strengthen its metabolism and inactive signal transduction inthe incompatible interaction between P. capsici and C. annuum.4. Three disease resistance related genes, CaCHI，CaDAD1，CaPR4，of pepper wereobtained by in silo PCR and RT-PCR. Protein encoded by CaCHI is a chitinase of ClassⅠ,which has a CBD and N-terminal signal peptide. The deduced amino acid sequence ofCaDAD1has a DAD1domain, which is conserved between animals and plants. PR4proteinencoded by CaPR4has a Barwin domain, which has the weak activity of chitinase. Theexpression changes postinoculated by P. capsici of the three genes were detected by qRT-PCR.CaCHI and CaPR4were both up-regulated by the P. capsici, however CaDAD1weredown-regulated in the early stage of P. capsici infecton.5. Two novel LOX genes designated as CaLOX2and CaLOX3were isolated andcharacterized in Capsicum annuum L. using in silico cloning and RT-PCR techniques. Thededuced amino acid sequence of the two genes contained the typical domain of lipoxygenase,and CaLOX3has putative transit peptides for chloroplast import. Phylogenetic analysisrevealed that CaLOX2clustered together with well-characterized plastidic typeⅡ13-LOXs,while CaLOX3clustered together with the typeⅠLOXs groups. Real-time quantitative PCRanalysis showed that CaLOX2transcripts were expressed in all tissues of pepper, hand thetranscripts were most abundantly expressed in the leaf but much less in flower. CaLOX3wereabundantly expressed in the germinated seeds. The expression level of the CaLOX2andCaLOX3mRNA could be up-regulated significantly after inoculation with zoospores ofPhytophthora capsici in both incompatible and compatible interaction. The comparison ofCaLOX2with the well-studied LOXs from other plant species in phylogenetic analysis andexpression patterns indicated that the most likely biochemical function of CaLOX2is in thebiosynthesis of JA through allene oxide synthase（AOS）pathway but not in the hydroperoxidelyase mediated （HPL-mediated） production of C6-aldehydes. These results indicated thatCaLOX2was probably involved in the disease resistance and defense response toPhytophthara capsici and other environment stress，such as low temperature and high salt,through SA and JA pathways.6. The comparison of the efficiency of VIGS revealed that AA5is not suit for VIGS. SoAA3was chosen for characterize the functions of CaDAD1, CaPR4and CaLOX2. Thedepletion of CaDAD1didn’t cause distinctive change in the infection of P. capsici. But thedepletion of CaLOX2caused pepper leave more susceptible to the pathgon. And knocking out of CaPR4delayed the cell death.7. Polygalacturonase-inhibiting protein (PGIP) can inhibit the activity of pathogen PGand improve the plant resistance level. Two novel PGIP genes designated as CsPGIP1andCsPGIP2were isolated and characterized in Cucumis sativus L. The deduced amino acidsequence of the two genes contained a the typical domain ofxxLxLxxNxLt/sGxIPxxLxxLxxL domain and belonged to Pgip gene family. Phylogeneticanalysis revealed the significant evolutionary homologous in the sequences of melon PGIPgene and PGIP genes in Cruciferae. Real-time quantitative PCR analysis showed thatCsPGIP1transcripts were expressed in all tissues, but the transcripts were most abundantlyexpressed in the young leaf but much less in stem. While CsPGIP2transcripts were abundantin root. The expression level of the CsPGIP1mRNA could be up-regulated significantly aftertreated with pathogen and salicylic acid (SA) compared to the normal growth environment.While CsPGIP2was not inducible by SA. UV spectrophotometry and agar diffusion analysisshowed that the protein CsPGIP1can inhibit the PG activity of P. capsici.