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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)

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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"

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