Primary Study On Antioxidant System Of Pyropia Haitanensis Under High-temperature And Dehydration Stresses

Pyropia haitanensis is an important economic seaweed in southern China,has become the second cultivation alga following Laminaria in China.With the continuous development of P.haitanensis cultivation,some new cultivars with higher yield and higher tolerance were selecting.But still now,there is little research report on the difference of tolerance of the new cultivars,and the mechanism of response to heat and desiccation stresses of P.haitanensis has also not been known clearly.The aim of this study is to study the antioxidant enzyme system of P.haitanensis under heat and desiccation stresses,which is considered to be an important factor affecting the tolerance capability of P.haitanensis.The results will lay basis for understanding the response mechanisms of P.haitanensis to abiotic stresses and provide insights for selecting new tolerance strains of P.haitanensis.The main results are as follows:1.Through searching and blasting,54 Unigenes which related to antioxidant enzyme genes were found from P.haitanensis whole transcriptome data,including Mn-SOD?8 Unigenes?,Cu/Zn-SOD?4 Unigenes?,GPX?8 Unigenes?,CAT?5 Unigenes?,APX?3 Unigenes?,GR?3Unigenes?,MDAR?6 Unigenes?,TrxR?6 Unigenes?and PrxR?10 Unigenes?.2.The 10 sequences with largest query scores were selected and used as core sequences to clone the full sequences of antioxidant enzyme genes by using rapid amplification of cDNA ends?RACE?technology or direct PCR.The 10 antioxidant enzyme genes were named PhMSD,PhCSD1,PhCSD2,PhGPX,PhCAT,PhGR,PhAPX,PhMDAR,PhPrxR and PhTR,respectively.The full-length cDNAs of the 10 antioxidant enzyme genes comprised 973,1029,954,1027,1983,1800,1199,1413,838 and 1167 nucleotides,respectively.And the cDNAs encoded proteins of 224,134,216,184,542,548,224,359,162 and 388 amino acids,respectively.3.Based on the ROS-scavenging pathway in plants and microalgae cells,the cellular ROS-scavenging pathway map of P.haitanensis were draw.The main location of ROS removal network of P.haitanensis were cytoplasm and chloroplast,which was similar to plants.Moreover,there are two complete“ROS-H₂O₂-H₂O”reactions involved by PrxR and CAT in the cytoplasm of P.haitanensis,which was similar to micoralgae.4.The results of real-time quantification PCR of each antioxidant enzyme gene showed that most antioxidant enzyme genes in all three P.haitanensis strains did not actively response to heat and dehydration stresses,however,the expressions of PhCAT gene in all three strains were significantly up-regulated.So the PhCAT gene may be seemed as one of the key antioxidant enzyme genes in response to high temperature stress.The comparison of total transcription levels of all antioxidant enzyme genesshowed that Z61 was the highest,followed by WT,and Z81 was the lowest,which indicated that a positive correlation between the antioxidant enzyme gene expression and high temperature tolerance.5.By analyzing the changes of antioxidant enzyme system under high temperature stress,the possible mechanism of Z61 cellular ROS-scavenging pathway in response to high temperature was as follows:at the initial period of high temperature treatment,hydroxyl free radicals as ROS signals,activating the ROS removal pathway in cytoplasm,the ROS removal pathway in chloroplast was also activated;at 12h,the ROS removal pathway in cytoplasm and chloroplast worked togather,cellular ROS scavenging capability reached its maximum;at 24h,ROS scavenging capability in cytoplasm reduced;at 48h,ROS scavenging capability in chloroplast also decreased,cellular ROS removal pathway responding to high temperature came to an end.By the way,our study also showed that antioxidant enzyme gene was not sensitive to low rate of water loss,and the principle of ROS scavenging gene network responding to dehydration stress were redundancy and flexibility.