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作物学报 ›› 2021, Vol. 47 ›› Issue (6): 1090-1099.doi: 10.3724/SP.J.1006.2021.04201

• 专题：主要麻类作物基因组学与遗传改良 • 上一篇    下一篇

红麻非生物逆境胁迫响应基因HCWRKY71表达分析及转化拟南芥

李辉1,2(), 李德芳2,*(), 邓勇2, 潘根2, 陈安国2, 赵立宁2, 唐慧娟2   

  1. 1湖南文理学院生命与环境科学学院, 湖南常德 415000
    2中国农业科学院麻类研究所, 湖南长沙 410205
  • 收稿日期:2019-12-19 接受日期:2020-10-14 出版日期:2021-06-12 网络出版日期:2020-09-08
  • 通讯作者: 李德芳
  • 作者简介:E-mail:guangjunmuzi@126.com
  • 基金资助:
    国家现代农业产业技术体系建设专项(CARS-19-E07);中国农业科学院科技创新工程一年生麻类育种项目(ASTIP-IBFC03);湖南省教育厅项目(18C0737);湖南文理学院博士科研启动项目(17BSQD13)

Expression analysis of abiotic stress response gene HcWRKY71 in kenaf and transformation of Arabidopsis

LI Hui1,2(), LI De-Fang2,*(), DENG Yong2, PAN Gen2, CHEN An-Guo2, ZHAO Li-Ning2, TANG Hui-Juan2   

  1. 1College of Life and Environment Science, Hunan University of Arts and Science, Changde 415000, Hunan, China
    2Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China
  • Received:2019-12-19 Accepted:2020-10-14 Published:2021-06-12 Published online:2020-09-08
  • Contact: LI De-Fang
  • Supported by:
    The China Agriculture Research System(CARS-19-E07);The Agricultural Science and Technology Innovation Program at the Chinese Academy of Agricultural Science(ASTIP-IBFC03);The Hunan Education Department Project(18C0737);The Doctoral Research Start-up Project of Hunan University of Arts and Sciences(17BSQD13)

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摘要：

WRKY转录因子在植物响应非生物逆境胁迫过程中具有重要的调控作用。本研究根据红麻转录组unigene序列(CL3883.Contig4), 设计引物进行PCR扩增, 经sanger测序获得全长为957 bp的HcWRKY71基因cDNA序列。该基因开放读码框为957 bp, 编码1个含有318个氨基酸的蛋白, 具有1个WRKY功能保守结构域, 属于II类WRKY转录因子。在盐胁迫下, HcWRKY71基因表达量随NaCl溶液浓度升高而增加; 在干旱胁迫下, HcWRKY71基因表达量随干旱胁迫时间的增加呈现先下降再上升然后再下降的趋势; 在重金属镉胁迫下, HcWRKY71基因表达量随CdCl2溶液浓度的增加而降低。表明该基因表达受盐、干旱和重金属镉胁迫的诱导。利用农杆菌介导花序浸染法将该基因转化拟南芥发现, HcWRKY71基因提高了转基因拟南芥幼苗的耐盐性, 这为进一步研究HcWRKY71基因的耐逆机制奠定了坚实的基础。

关键词: 红麻, 耐盐, 干旱, 重金属镉, HcWRKY71

Abstract:

WRKY transcription factor plays an important role in plant responded to abiotic stress. In this study, the unigene sequence (CL3883.Contig4) of the transcriptome in kenaf was used as a reference. Primers were designed for PCR amplification. The full length of HCWRKY71 gene was 957 bp obtained by the sanger sequencing. HCWRKY71 gene had an open reading frame length of 957 bp, and encoded a protein containing 318 amino acids, with a conserved functional domain of WRKY, which belonged to WRKY transcription factor II. Under the salt stress, its relative expression level increased with the increase of NaCl concentration; under the drought stress, with the extension of drought time, the relative expression of the HcWRKY71 gene first decreased, then increased and finally decreased; under the stress of heavy metal cadmium, its expression decreased with the increase of CdCl2 concentration, indicating that the expression of the gene was induced by salt, drought and heavy metal cadmium stress. The gene was transformed into Arabidopsis by Agrobacterium-mediated inflorescence impregnation. It was found that the HcWRKY71 gene improved the salt tolerance of transgenic Arabidopsis seedlings. This laid a solid foundation for further study of stress tolerance mechanism of HcWRKY71 gene.

Key words: kenaf, salt tolerance, drought, heavy metal cadmium, HcWRKY71

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Primer usage
HcWRKY71-F ATGTCGGATCATGGATTTA 基因克隆
HcWRKY71-R TCATGGTTCGTGTTTAAGGA Gene cloning
HcWRKY71-QF GGTGCGGAGGAATTAGTA 实时荧光定量PCR
HcWRKY71-QR ATGAAAGCAAATCGTGGT qRT-PCR
Actin-QF CAGGCAGTTCTTTCTTTGT 内参基因
Actin- QR ATCCTCCAATCCAGACACT Reference gene
PC1301-35S-HcWRKY71-GFP-F GAGAACACGGGGGACTGGTACCCGGGGATCCATGTCGGATCATGGATT 目的基因与表达载体的连接
Ligation of target gene with expression vector
PC1301-35S-HcWRKY71-GFP-R ACAGCTCCTCGCCCTTGCTCACCATGTCGACTGGTTCGTGTTTAAGGAA
HcWRKY71-PF AGATCAGAAGATGGTGGCG 转基因拟南PCR鉴定及实时荧光定量分析
HcWRKY71-PR TCGGATATGGGCTGTTCTT PCR identification and real-time fluorescence quantitative analysis of transgenic Arabidopsis

图1

HcWRKY71 cDNA全长琼脂糖凝胶电泳 M: 2K Plus II; 1: PCR产物。"

图2

HcWRKY71基因的核酸序列及其编码的氨基酸序列 红色框为WRKYGQK基序; 黑色框为C2H2基序(CX4CX23HXH)。"

附图1

红麻HcWRKY71蛋白的保守功能结构域预测"

附图2

HcWRKY71转录因子磷酸化位点的预测"

图3

红麻HcWRKY71与其他植物WRKY71蛋白氨基酸序列一致性比对 不同颜色代表不同氨基酸残基的保守性。蓝色表示氨基酸完全保守; 粉红色、青色、黄色分别表示氨基酸的保守性为75%以上、50%以上及33%以上; 白色表示氨基酸的保守性不足33%。"

图4

红麻HcWRKY71蛋白与其他植物WRKY71蛋白的系统进化树"

图5

HcWRKY71基因在盐、干旱和镉胁迫下的表达 A: 盐胁迫; B: 干旱胁迫; C: 镉胁迫。*和*分别表示在0.05和0.01水平上差异显著。误差线为每组处理的标准误差(n = 3)。"

图6

HcWRKY71基因在盐、干旱和镉胁迫下不同器官的表达 A: 盐胁迫; B: 干旱胁迫; C: 镉胁迫。*和*分别表示在0.05和0.01水平上差异显著。误差线为每组处理的标准误差(n = 3)。"

图7

转基因拟南芥鉴定 A: 潮霉素鉴定; B: PCR鉴定; C: 实时定量PCR鉴定。*和*分别表示在0.05和0.01水平上差异显著。误差线为每组处理的标准误差(n = 3)。"

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