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作物学报 ›› 2020, Vol. 46 ›› Issue (12): 1862-1869.doi: 10.3724/SP.J.1006.2020.04045

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定

陈杉彬(), 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红*()   

  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 / 北京市作物遗传改良重点实验室, 北京 100193
  • 收稿日期:2020-02-27 接受日期:2020-06-02 出版日期:2020-12-12 网络出版日期:2020-07-02
  • 通讯作者: 翟红
  • 基金资助:
    国家自然科学基金项目(31872878);国家重点研发计划项目(2018YFD1000700);国家重点研发计划项目(2018YFD1000704);国家现代农业产业技术体系建设专项(CARS-10)

Cloning of IbCAF1 and identification on tolerance to salt and drought stress in sweetpotato

Shan-Bin CHEN(), Si-Fan SUN, Nan NIE, Bing DU, Shao-Zhen HE, Qing-Chang LIU, Hong ZHAI*()   

  1. Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis and Utilization, Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China
  • Received:2020-02-27 Accepted:2020-06-02 Published:2020-12-12 Published online:2020-07-02
  • Contact: Hong ZHAI
  • Supported by:
    National Natural Science Foundation of China(31872878);National Key Research and Development Program of China(2018YFD1000700);National Key Research and Development Program of China(2018YFD1000704);China Agriculture Research System(CARS-10)

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摘要：

CAF1 (CCR4-associated factor 1)基因在植物发育、抗病等方面发挥着重要的作用。本研究根据前期获得的差异表达EST序列, 克隆得到甘薯IbCAF1基因。IbCAF1基因的开放阅读框(open reading frame, ORF)长度为846 bp, 编码281个氨基酸, 分子量为32.13 kD, 等电点为4.83。氨基酸序列比对和系统进化树分析表明, IbCAF1与甘薯近缘野生种Ipomoea triloba (2x)同源蛋白ItlCAF1有较高的同源性, 序列一致性为96.8%。IbCAF1基因受到NaCl、PEG、ABA和H2O2的诱导表达。利用根癌农杆菌介导法将IbCAF1基因转入烟草, 过表达IbCAF1基因显著提高了转基因烟草植株的耐盐性和抗旱性。在200 mmol L -1 NaCl和10% PEG-6000的胁迫下, IbCAF1基因的过表达显著上调了转基因烟草植株中活性氧清除系统和脯氨酸合成相关基因的表达, 增加了SOD活性、POD活性、脯氨酸含量, 降低了H2O2含量和丙二醛含量。表明IbCAF1基因能够提高转基因烟草植株的耐盐性和抗旱性。本研究为后续甘薯IbCAF1基因耐盐抗旱基因工程研究奠定了基础。

关键词: 甘薯, IbCAF1, 转基因烟草, 耐盐, 抗旱

Abstract:

CAF1 (CCR4-associated factor 1) gene plays an important role in plant development and disease resistance. In this study, the IbCAF1 gene of sweetpotato was cloned according to the EST sequence. The ORF of IbCAF1 was 846 bp, encoding 281 amino acids, with a molecular weight of 32.13 kD and an isoelectric point of 4.83. The results of amino acid sequence alignment and phylogenetic tree analysis showed that IbCAF1 had higher homology with ItlCAF1, a homologous protein of Ipomoea triloba (2x), and the homology was 96.8%. IbCAF1 gene was induced and expressed by NaCl, PEG, ABA, and H2O2. The IbCAF1 gene was transferred into tobacco by Agrobacterium tumefaciens mediated transformation. The overexpression of IbCAF1 gene significantly improved the salt and drought tolerance of transgenic tobacco plants. After 200 mmol L -1NaCl and 10% PEG-6000 treatments, the transgenic tobacco plants showed significant upregulation of the genes involved in ROS scavenging system and proline biosynthesis related genes, significant increase of SOD activity, POD activity and proline content and significant decrease of H2O2 and malondialdehyde contents. These results demonstrate that the IbCAF1 gene could improve salt and drought tolerance in transgenic tobacco. This study will lay a foundation on salt and drought tolerance gene engineering of IbCAF1 gene in sweetpotato for the following research.

Key words: sweetpotato, IbCAF1, transgenic tobacco, salt tolerance, drought tolerance

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
IbCAF1扩增引物
Primers for IbCAF1 amplification		 IbCAF1-F: ATGGGTGTACAAGAAGATGTTTTG
IbCAF1-R: CTAAAAAACTTCTAGTCCGTACAATACT
鉴定转基因引物
Primers for identifying transformants		 IbCAF1-OPF: GAGGCTTACGCAGCAGGTC
IbCAF1-OPR: TTATAATTCATACTCGTGGAAGCGC
实时定量PCR引物
Primers for real-time quantitative PCR		 IbActin-F: AGCAGCATGAAGATTAAGGTTGTAGCAC
IbActin-R: TGGAAAATTAGAAGCACTTCCTGTGAAC
Actin-F: GAGGAATGCAGATCTTCGTG
Actin-R: TCCTTGTCCTGGATCTTAGC
IbCAF1RT-F: TCAGCTACCTCATCGACGAC
IbCAF1RT-R: AGTCAACCCGAGCTGAATCA
SOD-F: CTATTACCGACAAGCAGATTCCTC
SOD-R: TACCACAAGCAACCCTTCCAC
APX-F: GATGTTCCCTTTCACCCTGG
APX-R: CAGATAGACCCATTTGCTTCACA
POD-F: TCCGGGAGCCACACCATTGG
POD-R: TGGTCGGAATTCAACAG
P5CS-F: TTGTGACACGGACTGATGGAA
P5CS-R: TATCTAAGCCGCTGACGACCA

图1

IbCAF1基因序列分析 A: IbCAF1蛋白序列分析; B: 甘薯CAF1蛋白和其他植物中的CAF1蛋白序列比对; C: 甘薯CAF1蛋白和其他植物中的CAF1蛋白的同源进化树分析。ItlCAF1: 三裂叶野牵牛(XP_031110715.1); InCAF1: 牵牛(XP_019199562.1); NtCAF1: 烟草(XP_016511744.1); SpCAF1: 番茄近缘野生种(XP_015079674.1); CaCAF1: 辣椒(NP_001312000.1); SlCAF1: 番茄(XP_004241342.1); StCAF1: 马铃薯(XP_006361099.1)。"

图2

IbCAF1基因在鲁薯3号中的表达分析 A: IbCAF1基因在鲁薯3号不同组织中的表达; B: 200 mmol L-1 NaCl、20% PEG-6000、100 μmol L-1 ABA和10 mmol L-1 H2O2分别处理不同时间后, 鲁薯3号中IbCAF1基因的表达分析。*与**分别表示在0.05和0.01水平下差异显著。"

图3

转基因烟草植株的IbCAF1基因的qRT-PCR分析 **表示在0.01水平下差异显著。"

图4

IbCAF1增强了转基因烟草植株的耐盐性和抗旱性 A: 转IbCAF1基因烟草植株和WT烟草植株在无胁迫或添加200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周; B~H: 在无胁迫、200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周的IbCAF1转基因烟草和WT烟草叶片的DAB染色(B)、NBT染色(C)、H2O2含量(D)、SOD活性(E)、POD活性(F)、脯氨酸含量(G)、丙二醛含量(H)。*与**分别表示在0.05和0.01水平下差异显著。"

图5

转基因植株及WT植株的抗逆相关基因的表达分析 *与**分别表示在0.05和0.01水平下差异显著。"

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