Cloning And Functional Analysis Of Nt19C And Homologue Genes In Nicotiana Benthamiana

Oomycetes represent a unique class of eukaryotic microorganisms that resemble fungimorphologically but are phylogenetic cousins of diatoms and brown algae within the kingdomStramenopiles. Phytophthora is one of the most important genera of oomycetes and causedepidermal and devastating plant disease. The plant disease caused by them resulted in hugeeconomic losses all over the world.During the process of its developmental stage and abiotic and biotic stressresponsiveness, plant programmed cell death often occurred. It has an irreplaceable role indevelopment, senescence and defense against pathogens. The typist reaction is HypersensitiveResponse (HR). HR is resulted from interaction between host and pathogen. The recognitionbetween them is activated by the following two aspects: interaction between plant R gene andpathogen Avr gene, combination of non-race-specific elicitor and receptor protein. HR andplant resistant signaling pathways are becoming the focus in cytology and genetics.Many plant pathogens produce lytic enzymes to breach the structural barriers of planttissues. The products generated by these enzymes may function as endogenous elicitors orDAMPs. While these families do not share a conserved sequence that makes them readilyidentifiable, they initiate conserved downstream responses. These include induced expressionof genes encoding antimicrobial/antiherbivore defense proteins, production of defense-relatedsecondary metabolites, peptides induce expression of genes encoding the biosyntheticenzymes for JA, ET and/or SA in various combinations and these phytohormones accumulatein response to DAMPs-elicitation.This study analyzed the function of Nt19C including bioinformatic analysis and geneoverexpression et al, and the achieved main results are as follows:1. By general analyses of the eight candidate genes including Nt19C, the outcome thatNt14B and Nt19D can result in Hypersensitive Response (HR) of both tobacco and potatoexplained that the two kinds of plant species may have a cross-talk in cell death signalingpathways.2. Blast search led to the identification of2homologues proteins both from Nicotianabenthamiana which were named as Nb19C-1and Nb19C-2. Nt19C, signal peptide sequencemutant gene△SP-Nt19C, Nb19C-1and Nb19C-2were cloned and constructed to the plant expression vector pMDC32.3. By bioinformatic analyses of Nt19C, the homologue from UniProtKB proteindatabase are all uncharacteristic proteins, the homologue from SGN N. benthamiana genomev0.4.4predicted protein database are NbS00035005g0001.1and NbS00010879g0006.1whoseidentity are82%and79%respectively. There are89amino acids of Nt19C whose molecularweight is9.3407kD and theoretical isoelectric point is7.95. Compared with the reportedamino acid abundance, the content of glycine from Nt19C is about doubled and content ofisoleucine and aspartic acid have increased a little. By prediction of the subcellularlocalization of Nt19C through TargetP, it localized in extracellular.4. Nb19C-1and Nb19C-2can result in yellow response in Nicotiana tobacum, whileNt19C and△SP-Nt19C are in Qin95, K346and Nicotiana benthamian. The signal peptide ofNt19C do not contribute to yellow response and the functions of Nb19C-1, Nb19C-2andNt19C are the consistent.5. Nt19C、△SP-Nt19C、 Nb19C-1and Nb19C-2can restrain the infection ofPhytophthora parasitica in Nicotiana benthamiana in different degrees.