Studies On Porphyra Haitanensis Defense Response Induced By Oligo-agar And Its Defense-associated Genes

Oligoagar, as an exogenous signal, can elicit the defense response of red alga Pyropia haitanensis.Results showed that two respiration peaks were observed at4and10minutes respectively, after additionof100μg/mL oligoagars to P. haitanensis. The increase was fourfold in the first peak and fourteen timesin the second one. After5min, the accumulation of H2O2in culture medium was detected and peaked to(102.1±6.82) nM at15min, which was ten times higher than control. The H2O2accumulation wasinhibited partially by DPI (10nM), indicating that NADPH oxidase participated in the oxidative burst.Analyzed by the fluorescence of redox-sensitive dye, found that activated oxygen species (AOS) mainlyaccumulated around the plasma membrane. Specific binding of2-AMAC labeled oligoagars on cellmembrane indicated the existence of oligoagar-recognition site. The recognition was associated with itspolymerizations degree. P. haitanensis could respond with agarotriose and agarohexaose, includingoxidative burst, H2O2accumulation, elimination of epiphytic bacteria and decline of blade rot rate, butnot with agarododecaose. In the molecular level, two defense-associated genes were cloned from itsgametophyte genome. One was respiratory burst oxidase homologue gene (designated as Phrboh) and theother was lipoxygnase gene (designated as PhLOX). Phrboh had a complete coding domain sequence(CDS) of2,853bp and could be deduced into a sequence of951amino acids which contained tentransmembrane-spanning domains (TMD). Six TMDs were at the N-terminal to harbor nonheme ironsand four amino acid residues, H111, H115, H183and H197, were conserved. Another four TMDs were atthe C-terminal between two NADPH binding sites. PhRboh had high homology with Pyropia PyRboh(91%) and lower with Chondrus CcRboh (35%). Algal Rboh clustered into a group different fromplants and animals. Up-transcription of Phrboh happened when the blade was challenged by oligoagars,showing that Phrboh contributed to the oxidative burst. Another gene, PhLOX, had complete CDS of2,697bp and encoded a98KDa protein. An extension of170amino acids harbored at its N-terminal,which was related to SRPBCC superfamily. The lipoxygense domain was at the C-terminal and fiveresidues (H583, H588, H770, N774and I898) were ligands of nonheme iron. A626determined theS-stereospecificity of hydroperoxide products. The homology between PhLOX and Pyropia PyLOX1,PyLOX2, PpLOX, Chondrus CcLOX, Gracilaria GcLOX was31%,90%,54%,34%and41%, respectively.Algal LOXs clustered well into its own group and had different sibship with other beings’ LOXs.Function analysis showed that PhLOX had wide substrate tolerance to PUFA from carbon18to20, butpreferred eicosanoid. It was up-regulated when challenged by oligoagars, suggesting that PhLOXfunctioned in P. haitanensis oxylipins metabolism. In summary, there is recognition site of oligoagars onP. haitanensis plasma membrane. It responds fit oligoagars with oxidative burst which is activatedpartially by rboh. AOS functions in both the elimination of pathogens and signaling to LOX pathway.This thesis provides important experimental evidences to the study of algal defense mechanism...