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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1697-1708.doi: 10.3724/SP.J.1006.2022.14123

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆TGA转录因子基因GmTGA26在盐胁迫中的功能分析

柯丹霞(), 霍娅娅, 刘怡, 李锦颖, 刘晓雪   

  1. 信阳师范学院生命科学学院 / 大别山农业生物资源保护与利用研究院, 河南信阳 464000
  • 收稿日期:2021-07-14 接受日期:2021-10-19 出版日期:2022-07-12 网络出版日期:2021-11-02
  • 通讯作者: 柯丹霞
  • 基金资助:
    国家自然科学基金项目(U1904102);河南省高等学校青年骨干教师培养计划;信阳师范学院“南湖学者奖励计划”青年项目资助

Functional analysis of GmTGA26 gene under salt stress in soybean

KE Dan-Xia(), HUO Ya-Ya, LIU Yi, LI Jin-Ying, LIU Xiao-Xue   

  1. College of Life Sciences, Xinyang Normal University / Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang 464000, Henan, China
  • Received:2021-07-14 Accepted:2021-10-19 Published:2022-07-12 Published online:2021-11-02
  • Contact: KE Dan-Xia
  • Supported by:
    National Natural Science Foundation of China(U1904102);Training Plan for Young Backbone Teachers in Colleges and Universities in Henan Province;Nanhu Scholars Program for Young Scholars of Xinyang Normal University

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摘要：

TGA转录因子是bZIP的一个亚家族, 在病原体和非生物胁迫反应中发挥重要作用。本研究在大豆中筛选并克隆得到1个TGA转录因子家族基因GmTGA26, 同源蛋白比对表明GmTGA26具有保守的亮氨酸拉链结构域, 与野生大豆同源性最高。基因表达特性分析表明, GmTGA26在大豆中受盐胁迫诱导表达。此外, GmTGA26编码核定位蛋白并且具有转录激活活性。通过发根农杆菌介导的大豆毛根转化, 得到过表达GmTGA26的“复合体”大豆植株, 在盐胁迫条件下, 与空载体对照相比, “复合体”大豆植株生长状态更好, 丙二醛含量和相对质膜透性明显降低(P < 0.05), 而叶绿素含量和根系活力则有显著的升高(P < 0.05)。qRT-PCR结果表明, 盐胁迫条件下在大豆毛状根中过表达GmTGA26可显著上调胁迫响应基因的表达。以上结果表明, 过表达GmTGA26显著增强了“复合体”大豆植株的耐盐能力。推测GmTGA26通过调控下游一系列胁迫响应基因从而参与调控大豆盐胁迫应激反应过程。

关键词: 大豆, TGA转录因子, 毛根转化, 耐盐性

Abstract:

TGA transcription factors are a subfamily of bZIP, which play important roles in pathogen and abiotic stress responses. A TGA transcription factor family gene GmTGA26 was screened and cloned from soybean in this study. Homologous protein comparison showed that GmTGA26 had a conserved leucine zipper domain and had the highest homology with wild soybean. The analysis of gene expression characteristics revealed that GmTGA26 gene was induced by salt stress in soybean. In addition, GmTGA26 gene encoded nuclear localization protein and had transcriptional activation activity. The “complex” soybean plants overexpressing GmTGA26 were obtained through Agrobacterium rhizogenes-mediated hairy root transformation of soybean. The growth state of “complex” soybean plants was better than the empty vector control under salt stress. Meanwhile, the MDA content and relative plasma membrane permeability decreased significantly (P < 0.05), while the chlorophyll content and root activity increased significantly (P < 0.05). The qRT-PCR results indicated that overexpression of GmTGA26 in soybean hairy roots under salt stress could significantly up-regulate the expression of stress response genes. The above results showed that overexpression of GmTGA26 significantly enhanced the salt tolerance of “complex” soybean plants. It is speculated that GmTGA26 participates in the regulation of soybean salt stress response by regulating a series of downstream stress response genes.

Key words: soybean, TGA transcription factor, hairy root transformation, saline tolerance

表1

基因表达分析和载体构建所使用的引物"

引物名称
Primer name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
GmTGA26 ATGGCTGACGCCAGTCCTA TCAGTCTCTTGGGCGGG
GmTGA26-OE CGGGATCCATGGCTGACG GGGGTACCTCAGTCTCTTGGGC
GUS GTCGCGCAAGACTGTAACCA CGGCGAAATTCCATACCTG
GmTGA26-qRT GGAGCAACAGTTAGTGGGTATCAC CTTCCATGCCCTGAGACAAAGC
ACT11-qRT GAGCTATGAATTGCCTGATGG CGTTTCATGAATTCCAGTAGC
GmDHN15-qRT TTTTGTTTTGTTGTATTGTGTAG GAAAAATCCTCCACCTGACGA
GmWD40-qRT TGCCAGTCTCGTTAGGCTTTTC CTTATTGAGTTGTTGTTTGGCAG
GmMYB48-qRT AACAACACTCTTCAGCCAGTTT GGGCAAAACAAACTTTCCTCAT
GmGST1-qRT CACAATGAGCAGCCCATAGCA CTTCAACATTCTTCTCACGCTCT
GmLEA-qRT GGTGGGTGAAACCGCACAAGA ATGGATGCCGCCACTCCGCCAG
GmNHX5-qRT GTCTGGGTTCAGTCTCGCAC ATCAGAAAGAGCAAGCCACCA
GmSOS1-qRT TTGTGCTGCATTTCTTCGAG CGTGCTTCTTCTCCTTCCAC

图1

GmTGA26与同源蛋白的序列比对及进化树分析 A: GmTGA26与其他植物中同源蛋白的保守BRLZ (碱性亮氨酸拉链区)结构域比对分析; B: GmTGA26及其同系物的系统发育树。比例尺代表遗传距离, 表示物种间的邻近关系。"

图2

不同非生物胁迫下GmTGA26基因的表达分析 *、**分别表示在0.05和0.01水平差异显著。"

图3

GmTGA26蛋白的亚细胞定位和转录活性分析 A: GmTGA26蛋白的亚细胞定位; 将重组质粒GmTGA26-GFP和NtTGA2.2-RFP共转化拟南芥原生质体, 35S::GFP和NtTGA2.2-RFP作为阳性对照; 标尺为10 µm。B: GmTGA26蛋白在酵母细胞中的转录活性分析。DDO: 二缺培养基(SD/ -Leu/-Trp); DDO/X/A: 含有125 ng mL-1 Aureobasidin A和40 μg mL-1 X-α-gal的二缺培养基; QDO: 四缺培养基(SD/-Ade/-His/ -Leu/-Trp); BD-53+AD-T: 阳性对照; BD-Lam+AD-T: 阴性对照。"

图4

转基因“复合体”大豆植株的阳性毛根鉴定 A: 阴性毛状根的GUS染色; B: 阳性毛状根的GUS染色; C: 阳性毛状根的PCR检测。标尺为200 μm; M: 1 kb DNA ladder; 1~7: GUS基因目的片段。"

图5

转基因“复合体”大豆植株在盐胁迫下的表型分析 A: 不同浓度盐处理1周后植株的生长状况, 比例尺为2 cm; B: qRT-PCR检测GmTGA26在毛状根中的相对表达水平, 以ACTIN11基因为内参; C~E: 不同盐浓度处理1周后植株的总根长、地上部以及根干重。EV: 空载体对照; OE: GmTGA26过表达植株。所有数值均以3个独立重复的平均值表示(n = 20); 误差线表示SD; *、**分别表示在0.05和0.01水平差异显著。"

图6

盐胁迫下转基因大豆的生理指标测定 EV: 空载体对照; OE: GmTGA26过表达植株。*、**分别表示在0.05和0.01水平差异显著。"

图7

qRT-PCR检测大豆毛状根中5个胁迫响应基因在正常条件以及100 mmol L-1 NaCl处理下的表达水平 EV: 空载体对照; OE: GmTGA26过表达植株。*、**分别表示在0.05和0.01水平差异显著。"

图8

qRT-PCR 检测大豆毛状根中2个盐胁迫响应基因在正常条件以及100 mmol L-1 NaCl处理下的表达水平 EV: 空载体对照; OE: GmTGA26过表达植株。*、**分别表示在0.05和0.01水平差异显著。"

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