Study On The Mechanism Of Pyropia Haitanensis Induced By Stressed And Lipid Metabolism Related Gene

Pyropia haitanensis has a unique heteromorphic life cycle, which contains thallus and conchocelis stages.High temperatures and desiccation are inevitable adversity for P. haitanensis during breeding proess. After hightemperature heat shock stress at35°C for30min, the fatty acids, volatile substances, superoxide dismutase,NADPH oxidase activity, H2O2content and floridoside content of P. haitanensis were measured. Meanwhile, thegene relative quantification of the heat shock protein70(hsp70) and NADPH oxidase (rboh) were analyzed.The results showed that: At normal temperature, conchocelis contains significantly higher levels of H2O2, fattyacid-derived volatiles, the copy number of Phrboh and Phhsp70genes，the activities of NADPH oxidase andfloridoside than those in thallus. After experiencing heat shock at35°C for30min, the H2O2contents, the mRNAlevels of Phrboh and Phhsp70, NADPH oxidase activity and the floridoside content in thallus were allsignificantly increased. Whereas, in conchocelis, the changes in fatty acids and their down-stream volatilespredominated, significantly increasing levels of saturated fatty acids and decreasing levels of polyunsaturated fattyacids occurred, and the8-carbon volatiles were accumulated. However, the changes in H2O2content andexpression of oxidant-related genes and enzymatic activity were not obvious. In addition, after exposure todesiccation for4h, the results showed that changes in the morphological ultrastructure of algae cells were found.Water loss in the thalli up to98%, and the emergence of partial membrane damage. The floridoside content wasincreased markedly, which involved in osmoregulation and cell wall synthesis. Meanwhile, Two genes, Phnho1,which encodes glycerokinase, and Phgpdh, which encodes glycerol-3-phosphate dehydrogenase, are involved inthe biosynthesis of a floridoside precursor, glycerol-3-phosphate. Both genes were up-regulated during desiccation.The species and content of short chain volatiles changed considerably. Phlox1and Phlox2, which facilitate theinitiation of production of downstream short-chain volatile compounds, were also up-regulated significantly.Lipoxygenases are one of the key enzymes to regulate the production of hormones and defensive metabolites.One defense-associated gene (Phlox2) was cloned from P.haitanensis gametophyte, which was encoded onelipoxygenase. Phlox2had a complete coding domain sequence (CDS) of2,700bp and could be deduced into asequence of899amino acids. The protein contained two functional domains (SRPBCC superfamily and LOXdomains). Subsequent phylogenetic analysis showed that such LOX enzymes are separated at the early stages ofevolution, establishing an independent branch. It appears that PhLOX2is a multifunctional enzyme featuring bothlipoxygenase and hydroperoxidase activities. Additionally, PhLOX2has exhibited remarkable substrate andposition flexibility, and it can catalyze an array of chemical reactions involving various polyunsaturated fatty acids,ranging from C18to C22. As a matter of fact, mono-hydroperoxy, di-hydroperoxy and hydroxyl products havebeen obtained from such transformations, and C20fatty acids seem to be the most preferred substrates. In thiswork, PhLOX2is coded as6-/9-/10-lipoxygenase against γ-linolenic acid;5-/8-/11-lipoxygenase againstarachidonic acid;5-/8-/11-/14-lipoxygenase against eicosapentaenoic acid; and4-/7-/8-/13-/16-lipoxygenaseagainst docosahexaenoic acid. It was found that triple ethylenic bonds are required for PhLOX2to function as aLOX, and the resulting hydroxyl products should be originated from the hydrogen abstraction of the carbon atombetween the second and third double bond. Presumably, the formation of di-hydroperoxides could involve a two-step pathway, in which the (n-2) positions of substrates should undergo oxygenation reaction to afford the–OOH functional group.In summary, the conchocelis and thallus of P. haitanensis have different responses to heat shock stress. Whensubjected to desiccation conditions during low tide, these physiological responses, including floridosideaccumulation, ROS production, and the generation of volatile compounds, might play a role in the extremedesiccation tolerance that P. haitanensis has. This thesis provides important experimental evidences to the studyresilience mechanisms of algal.