欢迎访问作物学报,今天是

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)

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)。"

[1] Zhang L W, Xu Y, Zhang X T, Ma X K, Zhang L L, Liao Z Y, Zhang Q, Wan X B, Cheng Y, Zhang J S, Li D X, Zhang L M, Xu J T, Tao A F, Lin L H, Fang P P, Chen S, Qi R, Xu X M, Qi J M, Ming R. The genome of kenaf (Hibiscus cannabinus L.) provides insights into bast fibre and leaf shape biogenesis. Plant Biotechnol J, 2020,18:1796-1809.
doi: 10.1111/pbi.13341 pmid: 31975524
[2] Yang A, Dai X, Zhang W H. A R2R3-type MYB gene , OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J Exp Bot, 2012,63:2541-2556.
doi: 10.1093/jxb/err431 pmid: 22301384
[3] Duan Y J, Jiang Y Z, Ye S L, Karim A, Ling Z Y, He Y Q, Yang S Q, Luo K M. PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana. Plant Cell Rep, 2015,34:831-841.
doi: 10.1007/s00299-015-1745-5 pmid: 25627252
[4] Wang G, Zhang S, Ma X, Wang Y, Kong F, Meng Q. A stress-associated NAC transcription factor (SlNAC35) from tomato plays a positive role in biotic and abiotic stresses. Physiol Plant, 2016,158:45-64.
pmid: 26991441
[5] 田云, 卢向阳, 彭丽莎, 方俊. 植物WRKY转录因子结构特点及其生物学功能. 遗传, 2006,28:1607-1612.
Tian Y, Lu X Y, Peng L S, Fang J. The structure and function of plant WRKY transcription factors. Herditas, 2006,28:1607-1612 (in Chinese with English abstract).
[6] 史书婷, 龚小庆, 邹养军. WRKY转录因子与植物逆境响应. 中国生物化学与分子生物学报, 2017,33:674-680.
Shi S T, Gong X Q, Zou Y J. The role of WRKY transcription factors in biotic and abiotic stress responses in plants. Chin J Biochem Mol Biol, 2017,33:674-680 (in Chinese with English abstract).
[7] Wu J, Chen J B, Wang L F, Wang S M. Genome-wide investigation of WRKY transcription factors involved in terminal drought stress response in common bean. Front Plant Sci, 2017,8:380.
doi: 10.3389/fpls.2017.00380 pmid: 28386267
[8] 王岩岩, 张永兴, 郭葳, 代文君, 周新安, 矫永庆, 沈欣杰. 野生大豆转录因子GsWRKY57 基因的克隆与抗旱性功能分析. 中国油料作物学报, 2019,41:524-530.
Wang Y Y, Zhang Y X, Guo W, Dai W J, Zhou X A, Jiao Y Q, Shen X J. Cloning and function of GsWRKY57 transcription factors gene response to drought stress. Chin J Oil Crop Sci, 2019,41:524-530 (in Chinese with English abstract).
[9] 柯丹霞, 彭昆鹏, 夏远君, 朱玉莹, 张丹丹. 盐胁迫应答基因GmWRKY6的克隆及转基因百脉根的抗盐分析. 草业学报, 2018,27(8):95-106.
Ke D X, Peng K P, Xia Y J, Zhu Y Y, Zhang D D. Clonging of salt-stressed responsive gene GmWRKY6 and salt resistance analysis of transgenic Lotus japonicas. Acta Pratac Sci, 2018,27(8):95-106 (in Chinese with English abstract).
[10] 王玲, 刘峰, 戴明剑, 孙婷婷, 苏炜华, 王春风, 张旭, 毛花英, 苏亚春, 阙友雄. 甘蔗ScWRKY4基因的克隆与表达特征性分析. 作物学报, 2018,44:1367-1379.
Wang L, Liu F, Dai M J, Sun T T, Su W H, Wang C F, Zhang X, Mao H Y, Su Y C, Que Y X. Cloning and expression characteristic analysis of ScWRKY4 gene in sugarcane. Acta Agron Sin, 2018,44:1367-1379 (in Chinese with English abstract).
[11] Zhang Y, Yu H, Yang X, Li Q, Ling J, Wang H, Gu X, Huang S, Jiang W. CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA dependent manner. Plant Physiol Biochem, 2016,108:478-487.
doi: 10.1016/j.plaphy.2016.08.013 pmid: 27592172
[12] 王影, 邱文敏, 李鹤, 贺雪莲, 刘明英, 韩小娇, 曲同宝, 卓仁英. 东南景天SaWRKY7基因对镉胁迫的响应研究. 南京林业大学学报(自然科学版), 2019,43(3):59-66.
Wang Y, Qiu W M, Li H, He X L, Liu M Y, Han X J, Qu T B, Zhuo R Y. Research on the response of SaWRKY7 gene to cadmium stress in Sedum alfredii hance. J Nanjing For Univ (Nat Sci Edn), 2019,43(3):59-66 (in Chinese with English abstract).
[13] Li H, Li D F, Chen A G, et al. RNA-seq for comparative transcript profiling of kenaf under salinity stress. J Plant Res, 2017,130:365-372.
pmid: 27999968
[14] Tan X L, Fan Z Q, Li L L, Wu Y, Kuang J F, Lu W J, Chen J Y. Molecular characterization of a leaf senescence-related transcription factor BrWRKY75 of Chinese flowering cabbage. Hortic Plant J, 2017,2:272-278.
[15] Wang C, Deng P, Chen L, Wang X, Ma H, Hu W, Yao N, Feng Y, Chai R, Yang G, He G. A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco. PLoS One, 2013,8:e65120.
doi: 10.1371/journal.pone.0065120 pmid: 23762295
[16] 禹阳, 贾赵东, 马佩勇, 郭小丁, 谢一芝, 边小峰. WRKY 转录因子在植物抗病反应中的功能研究进展. 分子植物育种, 2018,16:7009-7020.
Yu Y, Jia Z D, Ma P Y, Guo X D, Xie Y Z, Bian X F. Research progress on the role of WRKY transcription factors in plant defense. Mol Plant Breed, 2018,16:7009-7020 (in Chinese with English abstract).
[17] Guo D S, Zhang J Z, Wang X L, Han X, Wei B Y, Wang J Q, Li B X, Yu H, Huang Q P, Gu H Y, Qu L J, Qin G J. The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulation RAX genes in Arabidopsis. Plant Cell, 2015,27:3112-3127.
pmid: 26578700
[18] Guo D S, Qin G J. EXB1/WRKY71 transcription factor regulates both shoot branching and responses to abiotic stresses. Plant Signal Behav, 2016,11:e1150404.
[19] Yu Y C, Liu Z H, Wang L, Kim S G, Seo P J, Qiao M, Wang N, Li S, Cao X, Park C M, Xiang F. WRKY71 accelerates flowering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana. Plant J, 2016,85:96-106.
doi: 10.1111/tpj.13092 pmid: 26643131
[20] Qin Z, Lyu H J, Zhu X L, Chen M, Quan T Y, Wang M C, Xia G M. Ectopic expression of a wheat WRKY transcription factor gene TaWRKY71-1 results in hyponastic leaves in Arabidopsis thaliana. PLoS One, 2013,8:e63033.
doi: 10.1371/journal.pone.0063033 pmid: 23671653
[21] 邹克琴, 汪得凯, 胡东维, 李素芳, 陈振煌. 水稻WRK转录因子OsWRKY71的cDNA分离和表达分析. 浙江农业学报, 2014,26:1405-1411.
Zou K Q, Wang D K, Hu D W, Li S F, Chen Z H. Isolation and expression analysis of OsWRKY71 transcription factors in rice. Acta Agric Zhejianggensis, 2014,26:1405-1411 (in Chinese with English abstract).
[22] Yu Y C, Wang L, Chen J C, Liu Z H, Park C M, Xiang F X. WRKY71 acts antagonistically against salt-delayed flowering in Arabidopsis thaliana. Plant Cell Physiol, 2018,59:414-422.
doi: 10.1093/pcp/pcx201 pmid: 29272465
[23] Kim C Y, Vo K T X, Nguyen C D, Jeong D H, Lee S K, Kumar M, Kim S R, Park S H, Kim J K, Jeon J S. Functional analysis of a cold-responsive rice WRKY gene, OsWRKY71. Plant Biotechnol Rep, 2016,10:13-23.
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 周文期, 强晓霞, 王森, 江静雯, 卫万荣. 水稻OsLPL2/PIR基因抗旱耐盐机制研究[J]. 作物学报, 2022, 48(6): 1401-1415.
[3] 李祎君, 吕厚荃. 气候变化背景下农业气象灾害对东北地区春玉米产量影响[J]. 作物学报, 2022, 48(6): 1537-1545.
[4] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[5] 王霞, 尹晓雨, 于晓明, 刘晓丹. 干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响[J]. 作物学报, 2022, 48(5): 1191-1198.
[6] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[7] 胡亮亮, 王素华, 王丽侠, 程须珍, 陈红霖. 绿豆种质资源苗期耐盐性鉴定及耐盐种质筛选[J]. 作物学报, 2022, 48(2): 367-379.
[8] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[9] 曹亮, 杜昕, 于高波, 金喜军, 张明聪, 任春元, 王孟雪, 张玉先. 外源褪黑素对干旱胁迫下绥农26大豆鼓粒期叶片碳氮代谢调控的途径分析[J]. 作物学报, 2021, 47(9): 1779-1790.
[10] 张明聪, 何松榆, 秦彬, 王孟雪, 金喜军, 任春元, 吴耀坤, 张玉先. 外源褪黑素对干旱胁迫下春大豆品种绥农26形态、光合生理及产量的影响[J]. 作物学报, 2021, 47(9): 1791-1805.
[11] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[12] 李洁, 付惠, 姚晓华, 吴昆仑. 不同耐旱性青稞叶片差异蛋白分析[J]. 作物学报, 2021, 47(7): 1248-1258.
[13] 李增强, 丁鑫超, 卢海, 胡亚丽, 岳娇, 黄震, 莫良玉, 陈立, 陈涛, 陈鹏. 铅胁迫下红麻生理特性及DNA甲基化分析[J]. 作物学报, 2021, 47(6): 1031-1042.
[14] 周步进, 李刚, 金刚, 周瑞阳, 刘冬梅, 汤丹峰, 廖小芳, 刘一丁, 赵艳红, 王颐宁. 利用红麻HcPDIL5-2a非全长基因创制雄性不育新种质[J]. 作物学报, 2021, 47(6): 1043-1053.
[15] 李鹏程, 毕真真, 孙超, 秦天元, 梁文君, 王一好, 许德蓉, 刘玉汇, 张俊莲, 白江平. DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘[J]. 作物学报, 2021, 47(4): 599-612.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!