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作物学报 ›› 2020, Vol. 46 ›› Issue (11): 1649-1658.doi: 10.3724/SP.J.1006.2020.04051

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

甘薯抗旱相关基因IbNAC72的克隆与功能分析

张欢1(), 杨乃科1, 商丽丽2, 高晓茹1, 刘庆昌1, 翟红1, 高少培1, 何绍贞1,*()   

  1. 1 中国农业大学农业农村部甘薯生物学与生物技术重点实验室 / 教育部作物杂种优势研究与利用重点实验室, 北京 100193
    2 山东省烟台市农业科学研究院, 山东烟台 265500
  • 收稿日期:2020-02-29 接受日期:2020-07-02 出版日期:2020-11-12 网络出版日期:2020-07-13
  • 通讯作者: 何绍贞
  • 作者简介:E-mail:zhanghuan1111@cau.edu.cn, Tel: 010-62732559
  • 基金资助:
    本研究由国家重点研发计划项目(2018YFD1000700);本研究由国家重点研发计划项目(2018YFD1000704);北京粮经创新团队项目(BAIC09-2020);国家现代产业技术体系建设专项(CARS-10,甘薯)

Cloning and functional analysis of a drought tolerance-related gene IbNAC72 in sweet potato

ZHANG Huan1(), YANG Nai-Ke1, SHANG Li-Li2, GAO Xiao-Ru1, LIU Qing-Chang1, ZHAI Hong1, GAO Shao-Pei1, HE Shao-Zhen1,*()   

  1. 1 Key Laboratory of Sweet Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis & Utilization and Joint Laboratory for International Cooperation in Crop Molecular Breeding, Ministry of Education, China Agricultural University, Beijing 100193, China
    2 Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong, China
  • Received:2020-02-29 Accepted:2020-07-02 Published:2020-11-12 Published online:2020-07-13
  • Contact: Shao-Zhen HE
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2018YFD1000700);This study was supported by the National Key Research and Development Program of China(2018YFD1000704);the Beijing Food Crops Innovation Consortium Program(BAIC09-2020);the China Agriculture Research System(CARS-10,甘薯)

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摘要：

NAC (NAM/ATAF/CUC, NAC)转录因子是植物中特有的转录调控因子, 在植物体的生长发育与逆境胁迫响应等多种生理过程中起着重要的调控作用。本研究利用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)从甘薯品种栗子香中克隆到甘薯抗旱相关基因IbNAC72, 该基因cDNA长为1319 bp, 开放阅读框(open reading frame, ORF)为1008 bp, 编码335个氨基酸, 分子量为37.4 kD, 等电点为8.76, 基因组全长为1199 bp, 含有3个外显子、2个内含子。多重序列比对和系统进化树分析显示, IbNAC72基因与牵牛花中同源基因的亲缘关系最近。实时荧光定量PCR分析表明, IbNAC72基因在甘薯叶片中的表达量显著高于在茎段和根系中; 同时IbNAC72基因受到干旱和盐胁迫显著诱导表达。利用农杆菌介导法转化烟草, 获得过表达IbNAC72基因的转基因烟草植株。对IbNAC72转基因烟草植株的抗旱性分析表明, 在长时间干旱条件下, 转基因植株的根系更加发达, SOD活性显著提高, MDA的含量显著降低, 抗旱性显著提高。本研究表明, IbNAC72是与抗旱性密切相关的基因, 为深入探究IbNAC72抗旱的分子机制提供了基础。

关键词: 甘薯, NAC转录因子, IbNAC72, 过表达, 抗旱

Abstract:

NAC (NAM/ATAF/CUC) is a plant-specific transcription factor family, which plays an important role in plant growth, development and stress responses. In this study, we cloned IbNAC72, a drought tolerance-related gene from sweet potato [Ipomoea batatas (L.) Lam.] variety Lizixiang by RACE method. The IbNAC72 cDNA of 1319 bp in length, had an open reading frame (ORF) of 1008 bp, and encoded a 335 amino acids polypeptide, with a molecular weight of 37.4 kD and an isoelectric point (pI) of 8.76. The genomic DNA of IbNAC72 gene was 1199 bp and was deduced to contain 3 exons and 2 introns. Sequence alignment and phylogenetic analysis revealed that IbNAC72 had a close relationship with the predicted protein products of Ipomoea nil. RT-qPCR analysis showed that IbNAC72 was expressed at the highest level in the leaves of sweet potato, and it was strongly induced by PEG-6000 and NaCl, respectively. IbNAC72 was transformed into tobacco via Agrobacterium-mediated transformation. Its overexpression significantly enhanced drought tolerance in the transgenic tobacco plants. Under drought stress, transgenic plants developed stronger root system; the SOD activity was significantly increased whereas the MDA content was significantly decreased in transgenic plants compared to those of wild type plants. This study showed that IbNAC72 gene was closely related to drought tolerance, providing a basis for in-depth study on the drought tolerance molecular mechanism of IbNAC72 in sweet potato.

Key words: sweet potato, NAC transcription factor, IbNAC72, overexpression, drought tolerance

表1

IbNAC72基因克隆和功能分析所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Function
NAC-1 CGGAAAGTTGGGATCAAGAA 3′-RACE扩增
NAC-2 CAGGAAGACCGGAAGTTCAA 3′-RACE amplification
NAC-3 TTGAACTTCCGGTCTTCCTG 5′-RACE扩增
NAC-4 ATTCGCCCTGCTAGGAAGAT 5′-RACE amplification
NAC-F ATGGGTGTGAAAGATATGGACC ORF、DNA序列扩增
NAC-R TTACTGCCTGAACGCTAAGCTAC ORF and DNA sequence amplification
NAC-QF TCTTCAACAAGACGACAAGTCAAG RT-qPCR检测
NAC-QR AGGAAAACTCAATGCCATTG RT-qPCR detection
Actin-F AGCAGCATGAAGATTAAGGTTGTAGCAC 甘薯内参基因检测
Actin-R TGGAAAATTAGAAGCACTTCCTGTGAAC Sweet potato internal control
NAC-OE-F (Xba I) GCTCTAGAATGGGTGTGAAAGATATGGACC 过表达载体构建
NAC-OE-R (Sac I) CGAGCTCTTACTGCCTGAACGCTAAGCTAC Construction of overexpression vector

图1

IbNAC72基因的序列分析 A: 不同物种中NAC72同源蛋白序列的多重比较; 红色线标注为a~e 5个亚结构域。B: 不同物种中NAC72蛋白序列的同源进化树分析。C: IbNAC72与拟南芥AtNAC72的基因组结构比较; 方框表示外显子, 直线表示内含子。"

图2

IbNAC72基因的表达分析 A: IbNAC72基因在栗子香试管苗不同组织中的表达分析; B: IbNAC72基因在栗子香大田植株不同组织中的表达分析; C: IbNAC72基因在100 mmol L-1 NaCl胁迫处理下的表达分析; D: IbNAC72基因在20% PEG-6000胁迫处理下的表达分析。数据表示为平均值 ± SD (n = 3)。采用单因素方差分析法对数据进行统计分析, 柱上不同的小写字母代表柱值在0.05水平差异显著。"

图3

IbNAC72基因过表达载体的构建与鉴定 A: IbNAC72基因ORF的PCR扩增; B: 过表达载体pC3301- 121-IbNAC72的酶切鉴定。"

图4

IbNAC72转基因烟草植株的获得及鉴定 A~D: IbNAC72基因转化烟草的转化、筛选和再生过程; E~I: 转基因烟草植株叶片、茎段和根系的GUS染色呈阳性, WT呈阴性; H: IbNAC72转基因烟草植株的PCR分析; I: 转基因烟草植株IbNAC72基因的表达分析。M: DL2000 marker; W: 水作为阴性对照; P: 质粒pCAMBIA3301-121-IbNAC72作为阳性对照; C: WT作为阴性对照; WT: 野生型烟草植株; T1~T3: 过表达IbNAC72烟草株系。数据表示为平均值 ± SD (n = 3)。** 表示用Student’s t-test在0.01水平差异显著。"

图5

IbNAC72基因的过表达提高了转基因烟草植株的抗旱性 A: 干旱胁迫4周后, 转基因烟草和野生型烟草植株的生长状态比较; B: 干旱胁迫4周后, 转基因烟草和野生型烟草植株株高的比较分析; C: 干旱胁迫4周后, 转基因烟草和野生型烟草植株根系的生长状态比较; D: 干旱胁迫4周后, 转基因烟草和野生型烟草植株根系鲜重的比较分析。数据表示为平均值 ± SD (n = 3)。** 表示用Student’s t-test在0.01水平差异显著。缩写同图4。"

图6

干旱胁迫对转基因植株中SOD活性和MDA积累量的影响 A: 干旱胁迫4周后, 转基因烟草和野生型烟草植株的超氧化物歧化酶(SOD)活性; B: 干旱胁迫4周后, 转基因烟草和野生型烟草植株的丙二醛(MDA)含量。数据表示为means ± SD (n = 3)。** 表示用Student’s t-test在0.01水平差异显著。缩写同图4。"

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