欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (4): 672-683.doi: 10.3724/SP.J.1006.2021.04114

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析

唐锐敏1,2(), 贾小云1, 朱文娇2, 印敬明2, 杨清2,*()   

  1. 1山西农业大学生命科学学院, 山西太谷 030801
    2南京农业大学生命科学学院, 江苏南京 210095
  • 收稿日期:2020-05-25 接受日期:2020-08-19 出版日期:2021-04-12 网络出版日期:2021-02-04
  • 通讯作者: 杨清
  • 作者简介:E-mail: ruimin_tang829@hotmail.com
  • 基金资助:
    国家青年科学基金项目(31900450);山西农业大学科技创新基金项目(2018YJ28);山西省优秀博士来晋工作奖励资金科研项目(SXYBKY2018034)

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 Published:2021-04-12 Published online:2021-02-04
  • Contact: YANG Qing
  • 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)

RichHTML

32

PDF

350

摘要：

马铃薯在田间生长时常会受到各种不利环境的影响。夏季高温常导致马铃薯块茎产量和质量的下降。因此, 阐明马铃薯对热胁迫的响应机制, 发掘耐热相关基因, 对马铃薯耐热性的提高意义重大。热激转录因子(heat shock transcription factor A3, HsfA3)在植物机体内的活动影响到大量功能基因的表达, 在植物响应热胁迫的过程中发挥重要的作用。为了研究马铃薯中HsfA3的结构和功能, 本研究通过RT-PCR从马铃薯品种Désirée中克隆到长度为1506 bp的StHsfA3基因, 编码501个氨基酸。StHsfA3的相对分子量为55.23 kD, 理论等电点为4.9, 属于亲水性蛋白。构建StHsfA3-pBA002过表达载体, 并转化马铃薯植株, 共鉴定得到5个独立的StHsfA3过表达转基因马铃薯株系。通过对转基因植株和非转基因植株叶片中的相对含水量(relative water content, RWC)以及丙二醛(malondialdehyde, MDA)含量的测定发现, 在高温胁迫下, 转基因植株叶片中的RWC显著高于非转基因植株, MDA含量显著低于非转基因植株, 说明StHsfA3在耐热过程中起到正调控作用。对StHsfA3、StHsp26-CP和StHsp70在不同马铃薯植株中的表达分析显示, StHsfA3过量表达诱导了StHsp26-CP和StHsp70的表达, 预示着StHsfA3可能协同StHsp26-CP和StHsp70来增强过表达转基因株系的耐热性。

关键词: 马铃薯, 热胁迫, HsfA3, 基因克隆, 遗传转化, 功能分析

Abstract:

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

表1

本研究所使用的引物"

引物编号
Primer ID.		 名称
Name		 引物序列
Primer sequence (5′-3′)		 用途
Function
P1-F CloneHsfA3-F GCGCTCGAGATGAACCCATTTGATAAAAATCAAG StHsfA3的克隆, 酶切位点用下画线标出
P1-R CloneHsfA3-R GTCACGCGTCTAAAAACTATCATTCTTTGGCTG Gene cloning of StHsfA3; the restriction sites are underlined
P2-F HsfA3002-F GGGTAATATCCGGAAACCTCCT 转基因验证
P2-R HsfA3002-R GCTTTCTCCATTTCTACCCCAAG Transgenic verification
P3-F DLHsfA3-F CGGAAGTTCTTCTGGATCATCTG 荧光定量分析
P3-R DLHsfA3-R CCATCTGCTTCTGTCTTTGTTCT qRT-PCR analysis
P4-F DLsHsp3219-F CGTCCAATGTCAGTGTTGGTGG 荧光定量分析
P4-R DLsHsp3219-R CAGTGCCTTGGTTGTTTCCTCC qRT-PCR analysis
P5-F DLHsp70-F GTGGCAGTAATGGAAGGTGGGA 荧光定量分析
P5-R DLHsp70-R TTCTCCGGATTCACCACCGATT qRT-PCR analysis
P6-F EF1α-F CTGTTAAGGATCTGAAGCGTGGT 荧光定量分析, EF1α为内参基因
P6-R EF1α-R AATGTGGGAAGTGTGGCAGTCG qRT-PCR analysis; EF1α is used as the
reference gene

图1

StHsfA3的扩增产物条带(A)及其氨基酸序列(B) A图中: M: DNA 2000分子标记; 1: StHsfA3的扩增片段。B图中: 红线部分为4个中心是色氨酸(W)的短肽基序。"

图2

马铃薯StHsfA3的结构分析 A: 二级结构预测; B: 三级结构预测; C: 保守结构域预测; D: 信号肽预测。"

图3

StHsfA3疏水性分析(A)和跨膜域分析(B) A: 图中曲线得分在0以上说明这部分氨基酸为疏水性, 分值越高疏水性越强; 得分在0以下说明氨基酸为亲水性氨基酸, 分值越低亲水性越强。B: 没有跨膜结构域。"

图4

16个物种HsfA3的系统发育树 利用ClustalX 2.1和MEGA 4.0软件构建来自16个物种的Hsf A3氨基酸序列的系统发育树, 重复计算次数设为1000。各物种名称缩写及NCBI数据库中的序列号如下: MdHsfA3: 苹果(XP_008391748.1); ZjHsfA3: 枣(XP_015898712.1); CsHsfA3: 柑橘(XP_006481891.1); AtHsfA3: 拟南芥(NP_195992.2); VrHsfA3: 豇豆(XP_014511066.1); GmHsfA3: 大豆(XP_003541445.1); PeHsfA3: 杨树(XP_011016541.1); NtHsfA3: 烟草(XP_016473897.1); CaHsfA3: 辣椒(XP_016542894.1); SlHsfA3: 番茄(NP_001234854.1); OsHsfA3: 水稻(XP_015623061.1); ZmHsfA3: 玉米(NP_001147968.1); SiHsfA3: 狗尾草(XP_004952622.1); BdHsfA3: 二穗短柄草(XP_003575055.1); HvHsfA3: 大麦(AEB26588.1)。"

图5

StHsfA3过表达载体的结构示意图(A)、过表达载体的酶切验证(B)、转基因马铃薯植株的再生(C)、StHsfA3过表达转基因植株的PCR检测(D)、不同植株中StHsfA3表达量的qRT-PCR检测(E) A: 带有酶切位点的35S::StHsfA3载体示意图。B: M: 1 kb plus DNA marker; P: 含有pBA002-StHsfA3载体的农杆菌质粒; 1, 2: 含有pBA002-StHsfA3载体的农杆菌质粒酶切结果。C: a. 预培养; b. 芽分化; c. 生根筛选; d. 盆栽苗。D: M: DNA 2000 marker; WT: 非转基因植株, 作为阴性对照; +: pBA002-StHsfA3农杆菌质粒, 作为阳性对照; L1~L5: 转基因株系。E: 不同植株中StHsfA3的表达量测定。**表示在0.01水平差异显著。"

图6

不同温度处理下非转基因植株与StHsfA3过表达转基因植株叶片中的相对含水量(A)和丙二醛含量(B) WT: 非转基因植株; L: StHsfA3过表达转基因株系(L1、L3、L5三个株系); RWC: 相对含水量; MDA content: 丙二醛含量; 正常生长温度: 22℃; 高温胁迫: 35℃; 处理时间: 8 h; 图中的值是3个独立试验的结果, 每个试验3个重复, 以平均值±标准误表示。根据Duncan’s多重测试, 柱形图上不同小写字母表示P < 0.05的显著性差异水平。"

图7

不同温度处理下非转基因植株(WT)和转基因植株(L)叶片中StHsfA3 (A)、StHsp26-CP (B)和StHsp70 (C)的表达水平 WT: 非转基因植株; L: StHsfA3过表达转基因株系(L1、L3、L5三个株系); 正常生长温度: 22℃; 高温胁迫: 35℃; 处理时间: 2 h; 图中的值是2次试验重复的结果, 每个试验3个生物学重复, 以平均值±标准误表示。根据邓肯多重测试, 柱形图上不同小写字母表示P < 0.05的显著性差异水平。"

附图1

马铃薯HsfA3基因的cDNA序列和对应的氨基酸序列"

[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] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[2] 王海波, 应静文, 何礼, 叶文宣, 涂卫, 蔡兴奎, 宋波涛, 柳俊. rDNA和端粒重复序列鉴定马铃薯和茄子体细胞杂种染色体丢失和融合[J]. 作物学报, 2022, 48(5): 1273-1278.
[3] 石艳艳, 马志花, 吴春花, 周永瑾, 李荣. 垄作沟覆地膜对旱地马铃薯光合特性及产量形成的影响[J]. 作物学报, 2022, 48(5): 1288-1297.
[4] 周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[5] 冯亚, 朱熙, 罗红玉, 李世贵, 张宁, 司怀军. 马铃薯StMAPK4响应低温胁迫的功能解析[J]. 作物学报, 2022, 48(4): 896-907.
[6] 张霞, 于卓, 金兴红, 于肖夏, 李景伟, 李佳奇. 马铃薯SSR引物的开发、特征分析及在彩色马铃薯材料中的扩增研究[J]. 作物学报, 2022, 48(4): 920-929.
[7] 谭雪莲, 郭天文, 胡新元, 张平良, 曾骏, 刘晓伟. 黄土高原旱作区马铃薯连作根际土壤微生物群落变化特征[J]. 作物学报, 2022, 48(3): 682-694.
[8] 余慧芳, 张卫娜, 康益晨, 范艳玲, 杨昕宇, 石铭福, 张茹艳, 张俊莲, 秦舒浩. 马铃薯CrRLK1Ls基因家族的鉴定及响应晚疫病菌信号的表达分析[J]. 作物学报, 2022, 48(1): 249-258.
[9] 荐红举, 尚丽娜, 金中辉, 丁艺, 李燕, 王季春, 胡柏耿, Vadim Khassanov, 吕典秋. 马铃薯PIF家族成员鉴定及其对高温胁迫的响应分析[J]. 作物学报, 2022, 48(1): 86-98.
[10] 许德蓉, 孙超, 毕真真, 秦天元, 王一好, 李成举, 范又方, 刘寅笃, 张俊莲, 白江平. 马铃薯StDRO1基因的多态性鉴定及其与根系性状的关联分析[J]. 作物学报, 2022, 48(1): 76-85.
[11] 谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.
[12] 苏亚春, 李聪娜, 苏炜华, 尤垂淮, 岑光莉, 张畅, 任永娟, 阙友雄. 甘蔗割手密种类甜蛋白家族鉴定及栽培种同源基因功能分析[J]. 作物学报, 2021, 47(7): 1275-1296.
[13] 李鹏程, 毕真真, 孙超, 秦天元, 梁文君, 王一好, 许德蓉, 刘玉汇, 张俊莲, 白江平. DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘[J]. 作物学报, 2021, 47(4): 599-612.
[14] 秦天元, 刘玉汇, 孙超, 毕真真, 李安一, 许德蓉, 王一好, 张俊莲, 白江平. 马铃薯StIgt基因家族的鉴定及其对干旱胁迫的响应分析[J]. 作物学报, 2021, 47(4): 780-786.
[15] 杨阳, 李淮琳, 胡利民, 范楚川, 周永明. 白菜型油菜srb多室性状的遗传分析与分子鉴定[J]. 作物学报, 2021, 47(3): 385-393.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2