Welcome to Acta Agronomica Sinica,

Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (4): 672-683.doi: 10.3724/SP.J.1006.2021.04114


Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance

TANG Rui-Min1,2(), JIA Xiao-Yun1, ZHU Wen-Jiao2, YIN Jing-Ming2, YANG Qing2,*()   

  1. 1College of Life Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2020-05-25 Accepted:2020-08-19 Online:2021-04-12 Published:2021-02-04
  • Contact: YANG Qing E-mail:ruimin_tang829@hotmail.com;qyang19@njau.edu.cn
  • Supported by:
    National Natural Science Foundation for Young Scientists of China(31900450);Science and Technology Innovation Fund of Shanxi Agricultural University(2018YJ28);Award Fund for Outstanding Doctors Working in Shanxi(SXYBKY2018034)


During potato cultivation in the field, various adverse environmental stresses would affect its growth status. Heat stress in summer always results in the decline of tuber yield and quality. Therefore, it is of great significance to reveal the response mechanism of potato to heat stress and explore heat resistant genes for potato breeding. The activity of HsfA3 (heat shock transcription factor A3) affects the expression of numerous functional genes and plays an important role in response to heat stress. In order to investigate the structure and function of potato HsfA3, the StHsfA3 gene was isolated from potato cultivar Désirée by RT-PCR. The full-length cDNA of StHsfA3 was 1506 bp encoding 501 amino acids. StHsfA3 was predicted to be a hydrophilic protein with an estimated molecular weight of 55.23 kD and a theoretical isoelectric point of 4.9. The vector of StHsfA3-pBA002 was constructed and transformed into potato plants. Totally five independent transgenic potato plants overexpressing HsfA3 were obtained. The detection of relative water content (RWC) and malondialdehyde (MDA) content showed that the RWC was significantly increased while MDA content was significantly decreased in the transgenic plants compared with the non-transgenic plants under heat stress, suggesting that StHsfA3 played a positive regulatory role in enhancing the heat resistance of potato. Furthermore, the expression levels of StHsfA3, StHsp26-CP, and StHsp70 were determined by qRT-PCR in different potato plants. The results exhibited that the expression levels of StHsp26-CP and StHsp70 in the transgenic lines overexpressing StHsfA3 were significantly higher than that in non-transgenic plants, indicating that StHsfA3 might act in synergy with StHsp26-CP and StHsp70 to increase the heat tolerance of the transgenic plants.

Key words: potato, heat stress, HsfA3, gene cloning, genetic transformation, functional characterization

Table 1

Primer sequences used in this study"

Primer ID.
Primer sequence (5′-3′)
P1-R CloneHsfA3-R GTCACGCGTCTAAAAACTATCATTCTTTGGCTG Gene cloning of StHsfA3; the restriction sites are underlined
P2-R HsfA3002-R GCTTTCTCCATTTCTACCCCAAG Transgenic verification
P6-R EF1α-R AATGTGGGAAGTGTGGCAGTCG qRT-PCR analysis; EF1α is used as the
reference gene

Fig. 1

Amplified product stripe of StHsfA3 (A) and its amino acid sequence (B) M: DNA 2000 marker; 1: amplified fragment of StHsfA3 in Fig. A; the motifs marked with red line are the four short peptide sequences containing tryptophan (W) in Fig. B."

Fig. 2

Structural analysis of StHsfA3 in potato A: secondary structure prediction; B: tertiary structure prediction; C: conservative domain prediction; D: signal peptide prediction."

Fig. 3

Hydrophobicity analysis (A) and transmembrane domain analysis (B) of StHsfA3 A: the curves above zero show that these amino acids are hydrophobic, and the higher score, the stronger the hydrophobicity; the curves below zero show that these amino acids are hydrophilic, and the lower score, the stronger the hydrophilicity. B: there is no transmembrane domain."

Fig. 4

Neighbor-joining phylogenetic tree of HsfA3s from 16 plant species The Neighbor-joining phylogenetic tree of HsfA3 amino acid sequences from 16 plant species was constructed by ClustalX 2.1 and MEGA 4.0, which was bootstrapped over 1000 cycles. Abbreviations for each species and database accession numbers are as follows: MdHsfA3: Malus domestica (XP_008391748.1); ZjHsfA3: Ziziphus jujube (XP_015898712.1); CsHsfA3: Citrus sinensis (XP_006481891.1); AtHsfA3: Arabidopsis thaliana (NP_195992.2); VrHsfA3: Vigna radiata var. radiate (XP_014511066.1); GmHsfA3: Glycine max (XP_003541445.1); PeHsfA3: Populus euphratica (XP_011016541.1); NtHsfA3: Nicotiana tabacum (XP_ 016473897.1); CaHsfA3: Capsicum annuum (XP_016542894.1); SlHsfA3: Solanum lycopersicum (NP_001234854.1); OsHsfA3: Oryza sativa Japonica Group (XP_015623061.1); ZmHsfA3: Zea mays (NP_001147968.1); SiHsfA3: Setaria italica (XP_004952622.1); BdHsfA3: Brachypodium distachyon (XP_003575055.1); HvHsfA3: Hordeum vulgare (AEB26588.1). "

Fig. 5

Vector construction of 35S::StHsfA3 (A), enzyme digestion identification of 35S::StHsfA3 vector (B), regeneration of transgenic potato (C), PCR identification of StHsfA3 overexpression transgenic plant lines (D), and qRT-PCR determination of StHsfA3 in WT and StHsfA3 overexpression transgenic plant lines (E) Schematic diagram of 35S::StHsfA3 vector with site of restriction enzymes in Fig. A. M: 1 kb plus DNA marker; P: pBA002-StHsfA3 plasmid; 1, 2: enzyme digestion identification of pBA002-StHsfA3 plasmid in Fig. B. a: preincubation in darkness; b: bud formation; c: selection of transgenic lines; d: plants in pot in Fig. C. M: DNA 2000 marker; WT: non-transgenic potato plant, used as a negative control; +: pBA002-StHsfA3 plasmid, used as a positive control; L1-L5: Transgenic lines in Fig. D. The expression levels of StHsfA3 were determined in different plants in Fig. E. **: significantly different at the 0.01 probability level. "

Fig. 6

RWC (A) and MDA (B) content in the leaves of non-transgenic plants and StHsfA3 overexpression transgenic plants under different temperature treatments WT: non-transgenic plants; L: StHsfA3 overexpression transgenic plant lines; RWC: relative water content; MDA: malondialdehyde; temperature treatments: 22℃ (optimal temperature) and 35℃ (heat stress) for eight hours. Values are means ± SE of three independent experiments with three replicates in each experiment. Bars with different lowercase letters are significantly different at P < 0.05 according to Duncan’s multiple range tests."

Fig. 7

Expression levels of StHsfA3 (A), StHsp26-CP (B), and StHsp70 (C) in the leaves of non-transgenic plants (WT) and transgenic plant lines (L) under different temperature treatments WT: non-transgenic plants; L: StHsfA3 overexpression transgenic plant lines; temperature treatments: 22℃ (optimal temperature) and 35℃ (heat stress) for two hours. Values are means ± SE of two experimental replicates with three biological replicates in each experiment. Bars with different lowercase letters are significantly different at P < 0.05 according to Duncan’s multiple range tests."

Fig. S1

cDNA sequence of potato HscfA3 and corresponding amino acid sequence"

[1] Zhu J K. Abiotic stress signaling and responses in plants. Cell, 2016,167:313-324.
[2] Herman D J, Knowles L O, Knowles N R. Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato ( Solanum tuberosum L.). Planta, 2017,245:563-582.
[3] Lafta A, Lorenzen J. Effect of high temperature on plant growth and carbohydrate metabolism in potato. Plant Physiol, 1995,109:637-643.
[4] Kotak S, Larkindale J, Lee U, von Koskull-Döring P, Vierling E, Scharf K D. Complexity of the heat stress response in plants. Curr Opin Plant Biol, 2007,10:310-316.
doi: 10.1016/j.pbi.2007.04.011 pmid: 17482504
[5] Scharf K D, Berberich T, Ebersberger I, Nover L. The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta, 2012,1819:104-119.
[6] Lyck R, Harmening U, Höhfeld I, Treuter E, Scharf K D, Nover L. Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors. Planta, 1997,202:117-125.
[7] Miller G, Mittler R. Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Ann Bot, 2006,98:279-288.
[8] Nover L, Bharti K, Döring P, Mishra S K, Ganguli A, Scharf K D. Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need? Cell Stress Chaperones, 2001,6:177-189.
[9] Baniwal S K, Bharti K, Chan K Y, Fauth M, Ganguli A, Kotak S, Mishra S K, Nover L, Port M, Scharf K D, Tripp J, Weber C, Zielinski D, von Koskull-Döring P. Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors. J Biosci, 2004,29:471-487.
[10] Guo J, Wu J, Ji Q, Wang C, Luo L, Yuan Y, Wang Y, Wang J. Genome-wide analysis of heat shock transcription factor families in rice and Arabidopsis. J Genet Genomics, 2008,35:105-118.
pmid: 18407058
[11] Wang F, Dong Q, Jiang H, Zhu S, Chen B, Xiang Y. Genome-wide analysis of the heat shock transcription factors in Populus trichocarpa and Medicago truncatula. Mol Biol Rep, 2012,39:1877-1886.
[12] Bharti K, Koskull-Döring P V, Bharti S, Kumar P, Tintschl-Körbitzer A, Treuter E, Nover L. Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with the plant CREB binding protein ortholog HAC1. Plant Cell, 2004,16:1521-1535.
[13] Swindell W R, Huebner M, Weber A P. Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics, 2007,8:125.
[14] Zhang J, Liu B, Li J, Zhang L, Wang Y, Zheng H, Lu M, Chen J. Hsf and Hsp gene families in Populus: genome-wide identification, organization and correlated expression during development and in stress responses. BMC Genomics, 2015,16:181.
[15] Tang R M, Zhu W J, Song X Y, Lin X Z, Cai J H, Wang M, Yang Q. Genome-wide identification and function analyses of heat shock transcription factors in potato. Front Plant Sci, 2016,7:490.
pmid: 27148315
[16] Bharti K, Schmidt E, Lyck R, Heerklotz D, Bublak D, Scharf K D. Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum. Plant J, 2000,22:355-365.
[17] Yoshida T, Sakuma Y, Todaka D, Maruyama K, Qin F, Mizoi J, Kidokoro S, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K. Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system. Biochem Biophys Res Commun, 2008,368:515-521.
[18] Link V, Sinha A K, Vashista P, Hofmann M G, Proels R K, Ehness R, Roitsch T. A heat-activated MAP kinase in tomato: a possible regulator of the heat stress response. FEBS Lett, 2002,531:179-183.
[19] Zhu M D, Zhang M, Gao D J, Zhou K, Tang S J, Zhou B, Lyu Y M. Rice OsHSFA3 gene improves drought tolerance by modulating polyamine biosynthesis depending on abscisic acid and ROS levels. Int J Mol Sci, 2020,21:E1857.
[20] Wu Z, Liang J, Wang C, Zhao X, Zhong X, Cao X, Li G, He J, Yi M. Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism. J Exp Bot, 2018,69:2005-2021.
pmid: 29394377
[21] Li W, Wang B, Wang M, Chen M, Yin J M, Kaleri G M, Zhang R J, Zuo T N, You X, Yang Q. Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation. J Integr Plant Biol, 2014,56:364-372.
doi: 10.1111/jipb.12136 pmid: 24304603
[22] Jin Q, Zhu K, Cui W, Xie Y, Han B, Shen W. Hydrogen gas acts as a novel bioactive molecule in enhancing plant tolerance to paraquat-induced oxidative stress via the modulation of hemeoxygenase-1 signalling system. Plant Cell Environ, 2013,36:956-969.
[23] Rykaczewska K. The impact of high temperature during growing season on potato cultivars with different response to environmental stresses. Am J Plant Sci, 2013,4:2386-2393.
[24] Tang R M, Niu S Y, Zhang G D, Chen G S, Haroon M, Yang Q, Rajora O P, Li X Q. Physiological and growth responses of potato cultivars to heat stress. Botany, 2018,96:897-912.
[25] Ikeda M, Ohme-Takagi M. A novel group of transcriptional repressors in Arabidopsis. Plant Cell Physiol, 2009,50:970-975.
[26] Li P S, Yu T F, He G H, Chen M, Zhou Y B, Chai S C, Xu Z S, Ma Y Z. Genome-wide analysis of the Hsf family in soybean and functional identification of GmHsf-34 involvement in drought and heat stresses. BMC Genomics, 2014,15:1009.
pmid: 25416131
[27] Emani C, Garcia J M, Lopata-Finch E, Pozo M J, Uribe P, Kim D J, Sunilkumar G, Cook D R, Kenerley C M, Rathore K S. Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens. Plant Biotechnol J, 2003,1:321-336.
doi: 10.1046/j.1467-7652.2003.00029.x pmid: 17166131
[28] 贾小霞, 齐恩芳, 刘石, 文国宏, 马胜, 李建武, 黄伟. AtDREB1A基因过量表达对马铃薯生长及抗非生物胁迫基因表达的影响. 作物学报, 2019,45:1166-1175.
Jia X X, Qi E F, Liu S, Wen G H, Ma S, Li J W, Huang W. Effects of over-expression of AtDREB1A gene on potato growth and abiotic stress resistance gene expression. Acta Agron Sin, 2019,45:1166-1175. (in Chinese with English abstract).
[29] Gawel S, Wardas M, Niedworok E, Wardas P. Malondialdehyde (MDA) as a lipid peroxidation marker. Wiad Lek, 2004,57:453-455.
[30] Zhong L L, Zhou W, Wang H J, Ding S H, Lu Q T, Wen X G, Peng L W, Zhang L X, Lu C M. Chloroplast small heat shock protein HSP21 interacts with plastid nucleoid protein pTAC5 and is essential for chloroplast development in Arabidopsis under heat stress. Plant Cell, 2013,25:2925-2943.
[31] Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell, 2006,125:443-451.
pmid: 16678092
[32] Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K. Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress- responsive gene expression. Proc Natl Acad Sci USA, 2006,103:18822-18827.
[33] Li X D, Wang X L, Cai Y M, Wu J H, Mo B T, Yu E R. Arabidopsis heat stress transcription factors A2 (HSFA2) and A3 (HSFA3) function in the same heat regulation pathway. Acta Physiol Plant, 2017,39:67-75.
[1] CUI Lian-Hua, ZHAN Wei-Min, YANG Lu-Hao, WANG Shao-Ci, MA Wen-Qi, JIANG Liang-Liang, ZHANG Yan-Pei, YANG Jian-Ping, YANG Qing-Hua. Molecular cloning of two maize (Zea mays) ZmCOP1 genes and their transcription abundances in response to different light treatments [J]. Acta Agronomica Sinica, 2022, 48(6): 1312-1324.
[2] WANG Hai-Bo, YING Jing-Wen, HE Li, YE Wen-Xuan, TU Wei, CAI Xing-Kui, SONG Bo-Tao, LIU Jun. Identification of chromosome loss and rearrangement in potato and eggplant somatic hybrids by rDNA and telomere repeats [J]. Acta Agronomica Sinica, 2022, 48(5): 1273-1278.
[3] SHI Yan-Yan, MA Zhi-Hua, WU Chun-Hua, ZHOU Yong-Jin, LI Rong. Effects of ridge tillage with film mulching in furrow on photosynthetic characteristics of potato and yield formation in dryland farming [J]. Acta Agronomica Sinica, 2022, 48(5): 1288-1297.
[4] ZHOU Hui-Wen, QIU Li-Hang, HUANG Xing, LI Qiang, CHEN Rong-Fa, FAN Ye-Geng, LUO Han-Min, YAN Hai-Feng, WENG Meng-Ling, ZHOU Zhong-Feng, WU Jian-Ming. Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane [J]. Acta Agronomica Sinica, 2022, 48(4): 1017-1026.
[5] FENG Ya, ZHU Xi, LUO Hong-Yu, LI Shi-Gui, ZHANG Ning, SI Huai-Jun. Functional analysis of StMAPK4 in response to low temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(4): 896-907.
[6] ZHANG Xia, YU Zhuo, JIN Xing-Hong, YU Xiao-Xia, LI Jing-Wei, LI Jia-Qi. Development and characterization analysis of potato SSR primers and the amplification research in colored potato materials [J]. Acta Agronomica Sinica, 2022, 48(4): 920-929.
[7] JIN Rong, JIANG Wei, LIU Ming, ZHAO Peng, ZHANG Qiang-Qiang, LI Tie-Xin, WANG Dan-Feng, FAN Wen-Jing, ZHANG Ai-Jun, TANG Zhong-Hou. Genome-wide characterization and expression analysis of Dof family genes in sweetpotato [J]. Acta Agronomica Sinica, 2022, 48(3): 608-623.
[8] TAN Xue-Lian, GUO Tian-Wen, HU Xin-Yuan, ZHANG Ping-Liang, ZENG Jun, LIU Xiao-Wei. Characteristics of microbial community in the rhizosphere soil of continuous potato cropping in arid regions of the Loess Plateau [J]. Acta Agronomica Sinica, 2022, 48(3): 682-694.
[9] ZHANG Hai-Yan, XIE Bei-Tao, JIANG Chang-Song, FENG Xiang-Yang, ZHANG Qiao, DONG Shun-Xu, WANG Bao-Qing, ZHANG Li-Ming, QIN Zhen, DUAN Wen-Xue. Screening of leaf physiological characteristics and drought-tolerant indexes of sweetpotato cultivars with drought resistance [J]. Acta Agronomica Sinica, 2022, 48(2): 518-528.
[10] XIE Qin-Qin, ZUO Tong-Hong, HU Deng-Ke, LIU Qian-Ying, ZHANG Yi-Zhong, ZHANG He-Cui, ZENG Wen-Yi, YUAN Chong-Mo, ZHU Li-Quan. Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea [J]. Acta Agronomica Sinica, 2022, 48(1): 108-120.
[11] JIAN Hong-Ju, SHANG Li-Na, JIN Zhong-Hui, DING Yi, LI Yan, WANG Ji-Chun, HU Bai-Geng, Vadim Khassanov, LYU Dian-Qiu. Genome-wide identification and characterization of PIF genes and their response to high temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 86-98.
[12] XU De-Rong, SUN Chao, BI Zhen-Zhen, QIN Tian-Yuan, WANG Yi-Hao, LI Cheng-Ju, FAN You-Fang, LIU Yin-Du, ZHANG Jun-Lian, BAI Jiang-Ping. Identification of StDRO1 gene polymorphism and association analysis with root traits in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 76-85.
[13] ZHAO Wen-Qing, XU Wen-Zheng, YANG Liu-Yan, LIU Yu, ZHOU Zhi-Guo, WANG You-Hua. Different response of cotton leaves to heat stress is closely related to the night starch degradation [J]. Acta Agronomica Sinica, 2021, 47(9): 1680-1689.
[14] ZHANG Si-Meng, NI Wen-Rong, LYU Zun-Fu, LIN Yan, LIN Li-Zhuo, ZHONG Zi-Yu, CUI Peng, LU Guo-Quan. Identification and index screening of soft rot resistance at harvest stage in sweetpotato [J]. Acta Agronomica Sinica, 2021, 47(8): 1450-1459.
[15] SONG Tian-Xiao, LIU Yi, RAO Li-Ping, Soviguidi Deka Reine Judesse, ZHU Guo-Peng, YANG Xin-Sun. Identification and expression analysis of cell wall invertase IbCWIN gene family members in sweet potato [J]. Acta Agronomica Sinica, 2021, 47(7): 1297-1308.
Full text



No Suggested Reading articles found!