Study On Antioxidant System Of Gracilaria Lemaneiformis Under High-temperature Stress

Gracilaria lemaneiformis, as an important agarophyte, has become the thirdcultivation alga following Laminaria and Porphyra in China. With the continuousdevelopment of cultivation of G. lemaneiformis, new material07-2and cultivar981were bred by researchers, as the results the quality and the heat-resistant capability ofG. lemaneiformis were all improved. Now there is little research report on the reasonswhich caused the varieties between different strains of G. lemaneiformis and theresponse mechanism to heat stress of G. lemaneiformis has also not been knownclearly. The aim of this study is to learn the antioxidant system of G. lemaneiformisunder heat stress, which is considered to be an important factor affecting theheat-resistant capability of G. lemaneiformis. The results will lay basis for studyingthe response mechanisms of G. lemaneiformis to heat stress and provide insights forselecting new lines of G. lemaneiformis that can endure the stress of much highertemperature.Firstly, the variations of total antioxidant capacity (T-AOC), four antioxidantenzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase(GSH-PX) and peroxidase (POD), two no-enzymatic antioxidants: Vc and Ve, solubleprotein and maleic dialdehyd (MDA) of three strains of G. lemaneiformis，includingnew heat-resistant material07-2, cultivar981and wild-type under differenttemperatures (24°C,28°C,32°C) were studied. The results showed that under heatstress the T-AOC of07-2and981were stronger than that of wild type; no matter theenzymatic antioxidant activity or the non-enzymatic antioxidant activity of both07-2and981were better than that of wild type. These results mean that the ability ofscavenging ROS of07-2and981were higher than that of wild type under hightemperature. It was suggested that antioxidant system activity was responsible for theheat-resistant ability of G. lemaneiformis.Secondly, based on the sequences abtained from the suppression subtractive hybridization (SSH) cDNA libraries of G. lemaneiformis, full cDNA sequences ofmanganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-PX),glutathione reductase (GR), Vanadium bromoperoxidase (V-BPO) and peroxiredoxinⅥ (PrxⅥ), and partial sequence of catalase (CAT) of G. lemaneiformis were clonedby RACE and nested PCR. The full-length of Mn-SOD cDNA was852bp, includingan ORF of675bp encoding for224amino acid residues; the5’ UTR contained109bpand the3’ UTR was68bp. cDNA of GSH-PX was930bp，including a5’ UTR of109bp，a3’ UTR of71bp and an ORF of750bp encoding for249amino acid residues.The full-length of GR cDNA was1572bp，including an ORF of1416bp, which canencode471amino acid residues; the5’UTR was120bp and3’UTR was36bp. Thefull length of V-BPO cDNA was2372bp，it’s ORF was1830bp encoding for609amino acid residues;5’UTR was299bp and3’UTR was243bp. The complete cDNAsequence of PrxⅥ was987bp，with an ORF of672bp encoding223amino acidresidues; the5’UTR of PrxⅥ was106bp and3’UTR was209bp. After designationthe primers according to the two ends of each ORF, DNA sequences of Mn-SOD,GSH-PX, GR, V-BPO and PrxⅥ were cloned. The DNA sequences were foundconsistent with their ORF in the cDNA sequences respectively, indicating that the fivegenes did not contain introns. So mRNA of them did not require splicing duringtranscription process, which may be related to the fast response to stress ofantioxidant system. We also cloned partial cDNA sequence of CAT, which was905bpencoding for301amino acid residues.After further comparison, no differences were found between the sequences ofMn-SOD, GSH-PX, GR, V-BPO and PrxⅥ both in the W and981. Southern blottingproved that Mn-SOD and V-BPO were multicopies in both wild type and981, whileGSH-PX、GR and PrxⅥ were all single copy.Based on the cDNA sequences of the six antioxidant enzymes, the mRNAtranscription of the six antioxidant enzymes were analyzed at high temperature (32°C)of three strains of G. lemaneiformis, including new heat-resistant material07-2,cultivar981and wild-type by Real-time PCR. The results indicated that under heatstress different antioxidant enzymes played different parts in G. lemaneiformis. In981, GSH-PX and GR played important roles, whose transcriptions increased, while thetranscriptions of PrxⅥ and CAT decreased obviously. In07-2, the transcriptions ofPrxⅥ and CAT increased, while the transcriptions of GR decreased, indicating thatPrxⅥ and CAT might be the main antioxidant enzymes in07-2. In W, all of the sixenzymes were inhibited and the transcriptions of them decreased under heat stress.Compared the total transcription level of antioxidant enzymes,07-2was the highest,followed by981, and the wild type was the lowest. The result has also proven thesuperiority of heat-tolerant ability of07-2and981again. There are no differencesbetween the sequences and gene copies of antioxidant enzymes in the W and981, sothe different heat-resistant ability should have no relationship with the sequences.Maybe the regulatory sequences or some regulatory factors are the key factors,affecting the transcriptions of the antioxidant enzymes and then affecting the heatresistance of G. lemaneiformis.In this paper, genes of the antioxidant enzymes were cloned and the transcriptionpattern and activity of them under heat stress were analyzed for the first time, whichwill help us to illuminate the response mechanism to heat stress of G. lemaneiformisand also provided useful tags and sequences for screening new lines of G.lemaneiformis with stronger heat-resistant ability.