Molecular Cloning and Expression Pattern of TaAPX, Ascorbic Acid Peroxidase Gene in Wheat

doi:10.7668/hbnxb.20190762

Abstract

Abstract: The effect of ascorbic acid peroxidase (APX) in wheat under abiotic stress was studied by using the wheat of Bainong 207 as experimental material. According to the wheat TaAPX gene sequence, TaAPX was cloned by PCR, bioinformatics and tissue expression pattern analyzed. Bioinformatics and tissue expression pattern analysis were used to analyze the expression characteristics of TaAPX under abiotic stress. The results showed that TaAPX contained 876 bp open reading frame, encoded a protein composed of 291 amino acid. The molecular mass of TaAPX was 31.73 ku, the molecular formula was C₁₄₁₇H₂₂₄₆N₃₉₄O₄₂₃S₅ and the isoelectric point was 7.74, which might be a signal-free peptide, amphoteric protein and non-secretory protein. By comparing the APX protein sequences of wheat and barley and analyzing the evolutionary tree, it concluded that they had the closest genetic relationship with barley, and the maximum similarity of the full-length amino acid sequence of the coding protein was 97.25%.The tissue expression profiles detected by RT-qPCR indicated that it was differently expressed in the young roots, stems, stem nodes, young leaves, leaf sheaths, pistil and stamens. The expression level of TaAPX were highest in the young leaves, followed by that in the stem and stem node, the lowest expression level was found in the pistil and stamen. Under abiotic stress, the TaAPX expression was enhanced under drought and low temperature stress, but decreased under NaCl stress and osmotic stress.The coding sequence of TaAPX gene was obtained, and this gene responded to saline, drought and low temperature stress, suggesting that it may be closely related to anti-stress mechanism in wheat.

Key words: Wheat, APX, Gene cloning, Expression analysis, Adversity stress

CLC Number:

S512.03

WANG Runhao, YU Yongang, HU Haiyan, DING Chaojie, LI Hongmin, NIU Qingqing, LI Chengwei. Molecular Cloning and Expression Pattern of TaAPX, Ascorbic Acid Peroxidase Gene in Wheat[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(2): 48-56. doi: 10.7668/hbnxb.20190762.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20190762

http://www.hbnxb.net/EN/Y2020/V35/I2/48

References

[1] Mittler R,Vanderauwera S, Suzuki N, Miller G, Tognetti V B, Vandepoele K, Gollery M, Shulaev V,Van Breusegem F. ROS signaling:the new wave?[J]. Trends in Plant Science,2011,16(6):300-309. doi:10.1016/j.tplants.2011.03.007.
[2] 陈露. 小麦抗氧化物质对低温的动态响应及相关基因差异表达分析[D]. 哈尔滨:东北农业大学,2017.
Chen L. Dynamic response of wheat atioxidant to low temperature and differential expression analysis of related genes[D].Harbin:Northeast Agricultural University, 2017.
[3] Zuo L, Zhou T, Pannell B K, Ziegler A C, Best T M. Biological and physiological role of reactive oxygen species-the good, the bad and the ugly[J]. Acta Physiologica,2015,214(3):329-348. doi:10.1111/apha.12515.
[4] 乔新荣,张继英. 植物谷胱甘肽过氧化物酶(GPX)研究进展[J]. 生物技术通报,2016,32(9):7-13. doi:10.13560/j.cnki.biotech.bull.1985.2016.09.002.
Qiao X R, Zhang J Y. Research progress on GPX in plants[J]. Biotechnology Bulletin,2016,32(9):7-13.
[5] Shafi A, Gill T, Zahoor I, Ahuja P S, Sreenivasulu Y, Kumar S, Singh A K. Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance[J]. Molecular Biology Reports,2019,46(2):1985-2002. doi:10.1007/s11033-019-04648-3.
[6] 李泽琴,李静晓,张根发. 植物抗坏血酸过氧化物酶的表达调控以及对非生物胁迫的耐受作用[J]. 遗传,2013,35(1):45-54. doi:10.3724/SP.J.1005.2013.00045.
Li Z Q, Li J X, Zhang G F. Expression regulation of plant ascorbate peroxidase and its tolerance to abiotic stresses[J]. Hereditas,2013,35(1):45-54.
[7] 李泽琴,李锦涛,邴杰,张根发. 拟南芥APX家族基因在植物生长发育与非生物逆境胁迫响应中的作用分析[J].遗传,2019,41(6):534-549. doi:10.16288/j.yczz.19-026.
Li Z Q, Li J T, Bing J, Zhang G F. The role analysis of APX gene family in the growth and developmental processes and in response to abiotic stresses in Arabidopsis thaliana[J]. Hereditas,2019,41(6):534-549.
[8] Liu Y J, Yuan Y, Liu Y Y, Liu Y, Fu J J, Zheng J, Wang G Y. Gene families of maize glutathione ascorbate redox cycle respond differently to abiotic stresses[J]. Journal of Plant Physiology,2011,169(2):183-192. doi:10.1016/j.jplph.2011.08.018.
[9] Maruta T, Sawa Y, Shigeoka S, Ishikawa T. Diversity and evolution of ascorbate peroxidase functions in chloroplasts:More than just a classical antioxidant enzyme?[J]. Plant Cell Physiol, 2016,57(7):1377-1386. doi:10.1093/pcp/pcv203.
[10] 王竹青,陈云,杨玉婷,苏亚春,陈珊珊,吴期滨,许莉萍. 甘蔗抗坏血酸过氧化物酶基因(ScAPX)的克隆及表达分析[J].农业生物技术学报,2015,23(2):170-180. doi:10.3969/j.issn.1674-7968.2015.02.004.
Wang Z Q, Chen Y, Yang Y T, Su Y C, Chen S S, Wu Q B, Xu L P. Cloning and expression analysis of ascorbate peroxidase gene (ScAPX) in sugarcane(Saccharum officinarum)[J]. Journal of Agricultural Biotechnology,2015,23(2):170-180.
[11] 吴文超. 棉花抗旱性评价及抗旱基因 GhAPX2 的克隆和表达模式分析[D].乌鲁木齐:新疆农业大学,2016.
Wu W C. Evaluation of cotton drought resistant, cloning of GhAPX2 and expression patterns analysis[D].Urumchi:Xinjiang Agricultural University,2016.
[12] 孙卫红,陈相燕,杜斌,宋皖,周越,王云.过量表达番茄类囊体膜抗坏血酸过氧化物酶基因(StAPX)提高了烟草种苗的抗氧化能力[J].植物生理学报,2011,47(6):613-618. doi:10.13592/j.cnki.ppj.2011.06.007.
Sun W H, Chen X Y, Du B, Song W, Zhou Y, Wang Y. Tobacco seedlings overexpressing StAPX enhanced resistance to oxidative stress[J]. Plant Physiology Journal, 2011,47(6):613-618.
[13] 郁征宇,葛晨辉,王小丽,徐晨曦,王全华,蔡晓锋. 菠菜抗坏血酸过氧化物酶基因家族的鉴定及表达分析[J].上海师范大学学报(自然科学版),2018,47(6):664-672. doi:10.3969/J.ISSN.1000-5137.2008.06.005.
Yu Z Y, Ge C H, Wang X L, Xu C X, Wang Q H, Cai X F. Identification and expression analysis of ascorbate peroxidase gene family in spinach[J]. Journal of Shanghai Normal University (Natural Sciences),2018,47(6):664-672.
[14] Livak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods,2001,25(4):402-408. doi:10.1006/meth.2001.1262.
[15] Davletova S, Rizhsky L, Liang H J, Zhong S Q, Oliver D J, Coutus J, Shulaev V, Schlauch K, Mittler R. Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis[J]. Plant Cell,2005,17(1):268-281. doi:10.1105/tpc.104.026971.
[16] Foyer C H, Shigeoka S. Understanding oxidative stress and antioxidant functions to enhance photosynthesis[J]. Plant Physiology,2011,155(1):93-100. doi:10.1104/pp.110.166181.
[17] Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, Yoshimura K. Regulation and function of ascorbate peroxidase isoenzymes[J]. Journal of Experimental Botany,2002,53(372):1305-1319. doi:10.1093/jexbot/53.372.1305.
[18] 陈国强,孟鹏,刘李黎,陈刚,王萍.高粱抗坏血酸过氧化物酶基因的电子克隆及序列分析[J].生物信息学,2011,9(2):125-130. doi:10.3969/j.issn.1672-5565.2011.02.007.
Chen G Q, Meng P, Liu L L, Chen G, Wang P. In silico cloning and characterization of sorghum APX gene[J]. China Journal of Bioinformatics,2011,9(2):125-130.
[19] 王俊斌,杨文丽,丁博,吴天文,王海凤,谢晓东.小麦 TaWRKY71a 基因克隆、生物信息学及表达分析[J].华北农学报,2018,33(3):7-13. doi:10.7668/hbnxb.2018.03.002.
Wang J B, Yang W L, Ding B, Wu T W, Wang H F, Xie X D. Cloning, bioinformatics and expression analysis of TaWRKY71a gene in wheat[J]. Acta Agriculturae Boreali-Sinica,2018,33(3):7-13.
[20] 袁珍,张鹏钰,王国瑞,曹丽茹,库丽霞,卫丽. 玉米 ZmPP2C6-01 基因的克隆及表达分析[J].华北农学报,2019,34(2):8-18. doi:10.7668/hbnxb.201751477.
Yuan Z, Zhang P Y, Wang G R, Cao L R, Ku L X, Wei L. Cloning and expression of ZmPP2C6-01 gene in maize[J]. Acta Agriculturae Boreali-Sinica, 2019,34(2):8-18.
[21] 周硕,刘永伟,董福双,杨帆,赵和,柴建芳,吕孟雨,孙果忠,王海波.小麦类钙调素 TaCML79 基因的克隆和表达分析[J].华北农学报,2016,31(6):1-6. doi:10.7668/hbnxb.2016.06.001.
Zhou S, Liu Y W, Dong F S, Yang F, Zhao H, Chai J F, Lü M Y, Sun G Z, Wang H B. Cloning and expression analysis of TaCML79 in wheat[J]. Acta Agriculturae Boreali-Sinica, 2016,31(6):1-6.
[22] 朱昀,李朝炜,刘颖,魏景芳.小麦 VP 基因的克隆、生物信息学分析及功能初探[J].河南农业科学,2019,48(2):8-16. doi:10.15933/j.cnki.1004-3268.2019.02.002.
Zhu Y, Li C W, Liu Y, Wei J F. Cloning, bioinformatics analysis and preliminary functional study of wheat VP gene[J]. Journal of Henan Agricultural Sciences,2019,48(2):8-16.
[23] 朱素琴,马红,田洋,尹文娟,许浣文,周毅,杨平.玉米 ZmGLDH 基因的克隆与表达分析[J].河南农业科学,2017,46(12):17-23. doi:10.15933/j.cnki.1004-3268.2017.12.004.
Zhu S Q, Ma H, Tian Y, Yin W J, Xu H W, Zhou Y, Yang P. Cloning and expression analysis of ZmGLDH gene in Zea mays L.[J]. Journal of Henan Agricultural Sciences, 2017,46(12):17-23.
[24] 乔枫,耿贵工,曾阳,金兰,谢惠春.枸杞抗坏血酸过氧化物酶基因的克隆与表达分析[J].中国农业大学学报,2019,24(4):64-72.
Qiao F, Geng G G, Zeng Y, Jin L, Xie H C. Molecular cloning and expression patterns of LCAPX from Lycium chinense[J]. Journal of China Agricultural University,2019,24(4):64-72.
[25] 许惠滨,连玲,魏毅东,朱永生,谢华安,王宗华,张建福.水稻 OsAPX 基因的克隆及过表达载体的构建[J].福建农业学报,2013,28(2):101-106. doi:10.19303/j.issn.1008-0384.2013.02.003.
Xu H B, Lian L, Wei Y D, Zhu Y S, Xie H A, Wang Z H, Zhang J F. Cloning and construction of overexpression vector of OsAPX gene[J]. Agricultural Journal of Fujian,2013,28(2):101-106.
[26] 管清杰. 盐碱胁迫下水稻OsAPX家族基因表达特性差异研究[D].哈尔滨:东北林业大学,2007.
Guan Q J. Differential studies on the expression of rice OsAPX family genes under alkaline saline stress[D]. Harbin:Northeast Forestry University,2007.
[27] Cunha J R, Carvalho F E L, Lima-Neto M C, Jardimmesseder D, Cerqueira J V A, Martins M O, Fontenele A V, Márgis-Pinheiro M, Komatsu S, Silveira J A G. Proteomic and physiological approaches reveal new insights for uncover the role of rice thylakoidal APX in response to drought stress[J]. Journal of Proteomics, 2019,192:125-136. doi:10.1016/j.jprot.2018.08.014.
[28] Yan J Q, Wang J, Tissue D, Holaday A S, Allen R, Zhang H. Photosynthesis and seed production under water-deficit conditions in transgenic tobacco plants that overexpress an Arabidopsis ascorbate peroxidase gene[J]. Crop Sci,2003,43(4):1477-1483. doi:10.2135/cropsci2003.1477.
[29] 伍小兵,成雨洁,邓西平,郭尚洙. 叶片喷施H₂O₂以及转入Cu/Zn SOD 和 APX基因对甘薯幼苗冷后恢复的作用[J].中国农业科学,2010,43(7):1379-1388. doi:10.3864/j.issn.0578-1752.2010.07.008.
Wu X B, Cheng Y J, Deng X P, Guo S Z. Effect of foliar spraying of H₂O₂and Cu/Zn SOD and APX gene transferred in chloroplasts on the recoverability of sweet potato after chilling stress[J]. Scientia Agricultura Sinica,2010,43(7):1379-1388.
[30] Duan M, Feng H L, Wang L Y, Li D, Meng Q W.Overexpression of thylakoidal ascorbate peroxidase shows enhanced resistance to chilling stress in tomato[J]. Journal of Plant Physiology, 2012,169(9):867-877. doi:10.1016/j.jplph.2012.02.012.
[31] 段明.低温胁迫下番茄类囊体膜抗坏血酸过氧化物酶基因的表达和功能研究[D].泰安:山东农业大学,2012.
Duan M. Expression and functional analysis of the LetAPX in tomato under chilling stress[D]. Taian:Shandong Agricultural University,2012.
[32] Tiwari V, Chaturvedi A K, Mishra A, Jha B. The transcriptional regulatory mechanism of the peroxisomal ascorbate peroxidase (pAPX) gene cloned from an extreme halophyte, Salicornia brachiata[J]. Plant and Cell Physiology,2014,55(1):201-217. doi:10.1093/pcp/pct172.
[33] 王亦学,杜建中,郝曜山,张欢欢,王晓清,孙毅. 过量表达 GhAPX基因提高中林美荷杨耐盐性[J].分子植物育种,2017,15(7):2579-2583. doi:10.13271/j.mpb.015.002579.
Wang Y X, Du J Z, Hao Y S, Zhang H H, Wang X Q, Sun Y. Over expression of GhAPX gene in the improvement of salt tolerance in Populus sp.[J]. Molecular Plant Breeding,2017,15(7):2579-2583.
[34] Kornyeyev D, Logan B A, Payton P, Allen R D, Holaday A S. Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem Ⅱ in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes[J]. Physiologia Plantarum,2001,113(3):323-331. doi:10.1034/j.1399-3054.2001.1130304.
x.

Please choose a citation manager

Content to export