Cloning and Expression Analysis of TaGPX8 Gene in Wheat (Triticum aestivum)
ZHANG Hua-Dong1,2, LI Ya-Qian1,2, DONG Fei-Yan1,2, DING Fang-Cao3, LIU Meng-Wei2, XU Jia-Sheng1, GAO Chun-Bao1,2, WANG Xiao-Ling1, LIU Yi-Ke2,*, FANG Zheng-Wu1,*
1 College of Agronomy/MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River (Co-construction by Ministry and Province), Yangtze University, Jingzhou 434025, China; 2 Food Crops Institute/Hubei Provincial Key Laboratory of Germplasm Innovation and Genetic Improvement of Food Crops/Wheat Disease Biology Research Station on Central China, Ministry of Agriculture and Rural Affairs, Hubei Academy of Agricultural Sciences, Wuhan 430064, China; 3 Wuhan University of Bioengineering, Wuhan 430403, China
Abstract：Glutathione peroxidase (GPX), as one of the most important antioxidant enzymes in plants, plays an important role in plant stress response. In the analysis of the transcriptome data of the wheat (Triticum aestivum) GPX gene family, it was found that the TaGPX8 (IWGSC accession No. TraesCS4D02G162000.1) had the characteristics of being induced by stress. In this study, TaGPX8 was cloned, its protein was analyzed by bioinformatics and subcellular localized, and the expression of TaGPX8 in roots and leaves under salt and drought stress were analyzed.The results of gene structure analysis showed that TaGPX8 had a full length of 14 754 bp, including 6 exons and 5 introns, and the full length of CDS was 564 bp, encoding 187 amino acid residues. The relative molecular mass of the encoded protein was 21.330 kD, its theoretical isoelectric point was 6.62, and its average hydrophilicity was -0.664. Promoter sequence analysis found that the promoter sequence of TaGPX8 contained some cis-acting elements involved in hormone response, light response, and stress response. qPCR results revealed that TaGPX8 gene responded to drought and salt stress. Under drought stress, the expression level of TaGPX8 in leaves at 12 h had no significant difference with the control, but was significantly induced at 24 h, its expression level was up regulated, which was 12 times that of the control, and the expression level returned to no significant difference with the control in 48 h. In addition, the expression in roots also increased at 48 h of drought stress treatment. Under salt stress, the expression of TaGPX8 in leaves was inhibited to a certain extent at 12 h of treatment, but the expression level increased at 24 h of treatment, and then recovered to no significant difference with the control at 48 h. However, the expression in the root was suppressed. The yeast transcription activation experiments verified that TaGPX8 did not have transcriptional activation activity. Subcellular localization showed that the TaGPX8 protein was localized in the nucleus and cell membrane. This study provides a reference basis for further research on the function of TaGPX8 gene.
[1] 蔡琼, 丁贵杰, 文晓鹏, 等. 2016. 马尾松谷胱甘肽过氧化物酶PmGPX6基因cDNA克隆及转化拟南芥耐旱性初步研究[J]. 林业科学研究, 29(6): 839-846. (Cai Q, Ding G J, Wen X P, et al.2016. Cloning of glutathione peroxidase PmGPX6 gene from Pinus massoniana and the study on drought tolerance of transgenic Arabidopsis thaliana[J]. Forest Research, 29(6): 839-846.) [2] 刘萌萌, 韩立军, 刘宝玲, 等. 2022. 陆地棉GhSDP1及其启动子的克隆与表达分析[J]. 生物技术通报, 38(2): 34-43. (Liu M M, Han L J, Liu B L, et al.2022. Cloning and expression analysis of GhSDP1 and its promoter in Gossypium hirsutum[J]. Biotechnology Bulletin, 38(2): 34-43.) [3] 马超, 张均, 王学平, 等. 2018. 外源MeJA对花后干旱胁迫下小麦光合特性的影响[J]. 麦类作物学报, 38(5): 563-571. (Ma C, Zhang J, Wang X P, et al.2018. Effect of exogenous methyl jasmonate on photosynthetic characteristics in wheat under drought stress after anthesis[J]. Journal of Triticeae Crops, 38(5): 563-571.) [4] 马亭亭, 周宜君, 高飞, 等. 2012. 盐芥谷胱甘肽过氧化物酶基因(ThGPX6)的克隆及表达分析[J]. 植物遗传资源学报, 13(2): 252-258. (Ma T T, Zhou Y J, Gao F, et al.2012. Cloning and expression analysis of glutathione peroxidase 6 (ThGPX6) in Thellungiella halophile[J]. Journal of Plant Genetic Resources, 13(2): 252-258.) [5] 肖蓉, 罗慧珍, 张小娟, 等. 2015. 干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因(ZjGPX)的差异表达及功能分析[J]. 中国农业科学, 48(14): 2806-2817. (Xiao R, Luo H Z, Zhang X J, et al.2015. Differential expression and functional analysis of glutathione peroxidase gene from jujube (ZjGPX) under drought and salt stresses[J]. Scientia Agricultura Sinica, 48(14): 2806-2817.) [6] 张晗晗, 徐燕, 纪德华, 等. 2016. 坛紫菜谷胱甘肽过氧化物酶基因的克隆及表达特征[J]. 中国水产科学, 23(4): 791-799. (Zhang H H, Xu Y, Ji D H, et al.2016. Cloning and expression analysis of the glutathione peroxidase gene from Pyropia haitanensis[J]. Journal of Fishery Sciences of China, 23(4): 791-799.) [7] 张贺. 2020. 三个ROS应答基因在流胶病菌侵染桃树过程中的功能分析[D]. 博士学位论文, 华中农业大学, 导师: 李国怀, pp. 77-99. (Zhang H.2020. Functional analysis of three responsive genes of Lasiodiplodia theobromae during infection[D]. Thesis for Ph.D., HuaZhong Agricultural University, Suppervisor: Li G H, pp. 77-99) [8] 张丽丽, 徐碧玉, 刘菊花, 等. 2012. 香蕉谷胱甘肽过氧化物酶基因MaGPX的克隆和表达分析[J]. 园艺学报, 39(8): 1471-1481. (Zhang L L, Xu B Y, Liu J H, et al.2012. Isolation and expression analysis of a cDNA encoding glutathione peroxidase from banana[J]. Acta Horticulturae Sinica, 39(8): 1471-1481.) [9] 朱佳莉, 诸葛斌, 方慧英, 等. 2015. 新型渗透压调控的工业酵母启动子[J]. 微生物学报, 55(11): 1385-1391. (Zhu J L, Zhu G B, Fang H Y, et al.2015. New osmo-regulational promoters in the industrial yeast[J]. Acta Microbiologica Sinica, 55(11): 1385-1391.) [10] Avsian-Kretchmer O, Eshdat Y, Gueta-Dahan Y, et al.1999. Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in Citrus[J]. Planta, 209(4): 469-477. [11] Chen M Y, Li K, Li H P,et al.2017. The glutathione peroxidase gene family in Gossypium hirsutum: Genome-wide identification, classification, gene expression and functional analysis[J]. Scientific Reports, 7: 44743. [12] Criqui M C, Jamet E, Parmentier Y, et al.1992. Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases[J]. Plant Molecular Biology Reporter, 18(3): 623-627. [13] Depège N, Drevet J, Boyer N, et al.1998. Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-like proteins[J]. European Journal of Biochemistry, 253(2): 445-451. [14] Feng J L, Wang L Z, Wu Y N, et al.2019. TaSnRK2.9, a sucrose non-fermenting 1-related protein kinase gene, positively regulates plant response to drought and salt stress in transgenic tobacco[J]. Frontiers in Plant Science, 9: 2003. [15] Hossain M A, Bhattacharjee S, Armin S M, et al.2015. Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: Insights from ROS detoxification and scavenging[J]. Frontiers in Plant Science, 6: 420. [16] Islam T, Manna M, Kaul T, et al.2015. Genome-wide dissection of Arabidopsis and rice for the identification and expression analysis of glutathione peroxidases reveals their stress-specific and overlapping response patterns[J]. Plant Molecular Biology Reporter, 33: 1413-1427. [17] Kumar R, Masthigowda M H, Kaur A, et al.2020. Identification and characterization of multiple abiotic stress tolerance genes in wheat[J]. Molecular Biology Reports, 47(11): 8629-8643. [18] Li W J, Feng H, Fan J H, et al.2000. Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa[J]. Biochimica et Biophysica Acta, 1493(1-2): 225-230. [19] Margis R, Dunand C, Teixeira FK, et al.2008. Glutathione peroxidase family-an evolutionary overview[J]. The FEBS Journal, 275(15): 3959-3970. [20] Miao Y, Lv D, Wang P, et al.2006. An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses[J]. Plant Cell, 18(10): 2749-2766. [21] Mittler R.2002. Oxidative stress, antioxidants and stress tolerance[J]. Trends in Plant Science, 7(9): 405-410. [22] Paiva A L S, Passaia G, Jardim-Messeder D, et al.2020. The mitochondrial isoform glutathione peroxidase 3 (OsGPX3) is involved in ABA responses in rice plants[J]. Journal of Proteomics, 232: 104029. [23] Sugimoto M, Furui S, Suzuki Y, et al.1997. Molecular cloning and characterization of a cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase from spinach[J]. Bioscience Biotechnology and Biochemistry, 61(8): 1379-1381. [24] Sugimoto M, Sakamoto W.1997. Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress[J]. Genes Genetic Systems, 72(5): 311-316. [25] Ullah A, Manghwar H, Shaban M, et al.2018. Phytohormones enhanced drought tolerance in plants: A coping strategy[J]. Environmental Science and Pollution Research, 25(33): 33103-33118. [26] Zhu D, Luo F, Zou R, et al.2021. Integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses[J]. Journal of Proteomics, 234: 104097.
