Preliminary Study On Proteomics Response Of Porphyra Haitanensis To High Temperature Stress

Porphyra haitanensis is an economically important marine crop in southern China. As theglobal warming, over the past several years, high temperature had markedly affected thecultivation of P. haitanensis and reduced its yield remarkably. Till now, several studies have beencarried on the physiological responses and the change of gene expressed level of P. haitanensis.However, the knowledge about how the proteomics response is rather limited so far.So in this study, the response characteristics of gametophytic blades of one hightemperature tolerance line (Z-61) and a wild-type line of P. haitanensis under normal temperatureand high temperature stress for0,2,4,6,8, and10days were studied from the differentialpeotein expression. The main results are as follows:1. In order to establish a suitable protein preparation methods for P. haitanensis thalli, threesample preparation methods of plant, trichloroacetic acid/acetone precipitation (TCA/ACE)method, urea/thiourea extraction (UREA/THI) method and phenol extraction methanol/ammonium acetate precipitation method, and three protein lysis buffers were compared. Theresults showed that the optimal method of proteins preparation of P. haitanensis thalli wasextracted using TCA/ACE method and dissolved with the lysis buffer C (7mol/L urea，2mol/Lthiourea，2%CHAPS，2%TritonX-100，2%IPG Buffer pH3~10，65mmol/L DTT). Using thismethod, the yield was the highest and2-DE map have the most spots, clear backgrounds，highresolution and great repeatability.2．At the same time, from the2-DE map of gametophytic blades of P.haitanensis, we alsofound that the mainly (86.6%) protein spots distributed between pH4to pH7, so we thought thatusing the IPG strips (pH4~7) in2-DE analysis can separate the protein of P.haitanensis thallibetter than IPG strips (pH3~10). To improve the resolution of2-DE map, the IEF program of theIPG strips (pH4~7) with24cm length was optimized, the results showed that the optimal IEFprogram was10000V,90000VH.3．Compared the2-DE map of gametophytic blades of Z-61and wild-type line under normaltemperature and high temperature stress for0,2,4,6,8, and10days, a total of87protein spotswere selected for their expressed level changing (up-regulation or down-regulation) more thantwice. By analyzing with mass spectrometry and blasting with the public database andtranscriptome database of P. haitanensis,60differentially expressed proteins were identifiedsuccessfully. These differentially expressed proteins were related to metabolism, photosynthesis, respiration, antioxidant, transcription and translation, cellular signal transduction, stress proteins,cytoskeleton and other life processes.