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作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2171-2182.doi: 10.3724/SP.J.1006.2023.24193

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

烟草转录因子NtNAC080在非生物胁迫下的表达分析及功能鉴定

文利超1,2(), 熊涛3, 邓智超1,2, 刘涛1,2, 郭存4, 李伟1,*(), 郭永峰1,*()   

  1. 1 中国农业科学院烟草研究所 / 烟草行业基因资源利用重点实验室, 山东青岛 266101
    2 中国农业科学院研究生院, 北京 100081
    3 湖北省烟草公司恩施州公司利川市分公司, 湖北恩施 445000
    4 云南省烟草公司昆明市公司石林分公司, 云南昆明 650000
  • 收稿日期:2022-08-23 接受日期:2023-02-10 出版日期:2023-08-12 网络出版日期:2023-02-28
  • 通讯作者: 李伟,郭永峰
  • 作者简介:E-mail: 82101201624@caas.cn
  • 基金资助:
    国家自然科学基金项目(31400267);国家自然科学基金项目(31970204);中国农业科学院科技创新工程项目(ASTIP-TRIC02)

Expression and functional characterization of NtNAC080 transcription factor gene from Nicotiana tabacumin under abiotic stress

WEN Li-Chao1,2(), XIONG Tao3, DENG Zhi-Chao1,2, LIU Tao1,2, GUO Cun4, LI Wei1,*(), GUO Yong-Feng1,*()   

  1. 1 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong, China
    2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 Enshi Tobacco Company, Enshi 445000, Hubei, China
    4 Kunming Tobacco Company, Kunming 650000, Yunnan, China
  • Received:2022-08-23 Accepted:2023-02-10 Published:2023-08-12 Published online:2023-02-28
  • Contact: LI Wei,GUO Yong-Feng
  • Supported by:
    National Natural Science Foundation of China(31400267);National Natural Science Foundation of China(31970204);Agricultural Science and Technology Innovation Program(ASTIP-TRIC02)

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摘要：

NAC作为植物特有的转录因子, 广泛参与植物生长发育、衰老及胁迫响应等生物学过程。为探究烟草NtNAC080在非生物胁迫响应中的功能, 利用qRT-PCR技术分析了不同胁迫处理下NtNAC080的表达模式, 结果表明, NtNAC080的表达受干旱、高盐胁迫以及ABA、MeJA和SA激素的诱导; 以NtNAC080基因的敲除突变体及野生型(K326)烟株为材料, 分析敲除株系在高盐和干旱胁迫下抗逆表型。试验表明, 与野生型相比, 2个敲除株系的耐盐抗旱能力均明显增强; 干旱和盐胁迫下敲除株系的抗氧化酶(SOD、POD、CAT)活性以及可溶性蛋白、脯氨酸含量显著高于野生型, 而丙二醛含量显著低于野生型。相反地, 异源表达NtNAC080的转基因拟南芥与野生型(Col-0)相比对盐和干旱的耐受性明显减弱。qRT-PCR分析发现在干旱和盐处理后胁迫相关基因(NtDREB1A、NtKAT2、NtNHX1等)在NtNAC080基因敲除株系中表达水平显著高于野生型。以上结果表明, NtNAC080在烟草的非生物胁迫响应中起负调控作用, 这可能是通过调控抗氧化酶活性及胁迫相关基因的表达来实现的。

关键词: 烟草, NtNAC080, ROS, 表达分析, 非生物胁迫

Abstract:

NAC proteins, which constitute one of the largest plant-specific transcription factor families, are widely involved in the regulation of plant development, senescence and stress responses. To explore the function of NtNAC080 in abiotic stress response, qRT-PCR was used to analyze the relative expression pattern of NtNAC080 genes under different abiotic stress treatments. Results showed that the relative expression level of NtNAC080 genes was induced by drought, salt, ABA, MeJA, and SA treatments. The NtNAC080 mutant and WT (K326) plants were used to analyze the phenotypes under salt and drought stresses. Results showed that NtNAC080 knockout tobacco lines had increased tolerance to salt and drought stresses. Under salt and drought stresses treatments, the antioxidant enzymes (SOD, POD, and CAT) activities, soluble protein, and proline contents of these two knockout lines plants were significantly higher than those of WT, while MDA content was significantly lower than that of WT. In contrast, NtNAC080-overexpressing Arabidopsis plants had more sensitive to salt and drought stresses. Furthermore, NtNAC080 mutation in tobacco resulted in the up-regulated expression of abiotic stress-related genes (NtDREB1A, NtKAT2, and NtNHX1) after salt and drought treatments. These results indicated that NtNAC080 could be play a negative role in response to salt and drought stresses by regulating the activity of antioxidant enzymes and the relative expression level of stress-related genes.

Key words: tobacco, NtNAC080, ROS, the relative expression analysis, abiotic stresses

表1

用于qRT-PCR分析的引物序列"

基因名称
Gene name		 基因编号
Accession number		 正向引物序列
Forward primer (5°-3°)		 反向引物序列
Reverse primer (5°-3°)
NtActin XM_016618658 ACCTCTATGGCAACATTGTGCTCAG CTGGGAGCCAAAGCGGTGATT
NtNAC080 XM_016596133 TTCACGACTATTTGATGACAATGCTA TATTGGTCATTGTGTTTTGGTTGTT
NtDREB1A XM_016627803 TAACCCCAAGAAGCGAGCAG TAGCCGCCATTTCTGCAGAA
NtKAT2 XM_016602939 CCGCGCAAATCCAGAAGATG TGGCCCTCCATTTGTTTGGT
NtERF5 XM_016584910 TGGATGAAGAGCCAAGCCAT GAATCTCCGCCGCGAATTTC
NtNHX1 XM_016578291 CCCCCACAGAGGCAGTTAAG GCGCCAGTATCTATGCACCT
NtSOS1 XM_016611193 TCGCTTTGCTTGTTCTTGGC ACCGCCAACAGAAGATCAGG
AtActin NM_001338359 TGTGCCAATCTACGAGGGTTT TTTCCCGCTCTGCTGTTGT
AtDREB1A NM_118680 TTTCAAACCGCTGAGATGGC AAGCCGAGTCAGCGAAATTG
AtKAT2 NM_001341273 TTCGCCTTTGATGTCTGCTC TCTCAAGCCTTGCAAACAGC
AtERF5 NM_124094 CGCAGCTGAAAACACCAAAC AATTTCCCCCACGGTCTTTG
AtNHX1 NM_122597 GGTGCCATATTTGCTGCAAC ATCGCGTTGAAGACCACAAC
AtSOS1 NM_126259 ACCAATGAAACTGCGTGGTG ATGCAGCGAGTGATTGTTGC

图1

NtNAC080表达模式分析 LB: 侧芽; R: 根; S: 茎; UL: 上部叶; ML: 中部叶; LL: 下部叶; AP: 顶芽; F: 花。*表示在0.05概率水平差异显著。"

图2

烟草ntnac080敲除植株在盐胁迫下表型分析 A: ntnac080敲除系和K326烟草植株在盐胁迫下的表型。B: 幼苗在含0、50、100、150和200 mmol L-1 NaCl的MS培养基上生长2周的根长统计。C~E: 盐处理前后ntnac080敲除系和K326烟草叶片中抗氧化酶活性(SOD、CAT和POD)分析。F~H: 盐处理前后ntnac080敲除系和K326烟草叶片中MDA含量、可溶性蛋白含量以及脯氨酸含量。*表示在0.05概率水平差异显著。"

图3

烟草ntnac080敲除株系和K326在干旱胁迫下表型分析 A: ntnac080敲除系烟草植株和K326在干旱胁迫下的表型。B~D: 干旱处理前后ntnac080敲除系和K326烟草叶片中抗氧化酶活性(SOD、CAT和POD)分析。E: 干旱处理前后ntnac080敲除系和K326烟草叶片中MDA含量。*表示在0.05概率水平差异显著。"

图4

NtNAC080过表达降低了转基因拟南芥植株的耐盐性和抗旱性 A: NtNAC080基因在转基因拟南芥中的表达量分析。B~C: NtNAC080-OE在盐胁迫下的萌发率。D: NBT染色。E: 测定不同基因型植物离体叶片中H2O2的积累。F~H: 盐胁迫处理后NtNAC080-OE过表达系和WT的POD、SOD、CAT活性。I: NtNAC080过表达系(OE3和OE6)和WT在干旱胁迫下的表型。J: 干旱胁迫后NtNAC080过表达系(OE3和OE6)和WT的存活率。K: NtNAC080过表达系(OE3和OE6)和WT离体叶片失水率。*表示在0.05概率水平差异显著。"

图5

干旱和盐胁迫应答基因在烟草敲除株系中的表达模式 *表示在0.05概率水平差异显著。* means significant difference at the 0.05 probability level."

图6

干旱和盐胁迫应答基因在拟南芥过表达株系中的表达模式 *表示在0.05概率水平差异显著。"

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