新疆农业科学 ›› 2023, Vol. 60 ›› Issue (8): 2006-2012.DOI: 10.6048/j.issn.1001-4330.2023.08.022
收稿日期:
2022-11-05
出版日期:
2023-08-20
发布日期:
2023-08-14
通信作者:
曾幼玲(1971-),女,四川人,教授,博士生导师,研究方向植物的逆境生理和分子机制,(E-mail)zeng_ylxju@126.com作者简介:
毛晓菲(1995-),女,山西人,硕士研究生,研究方向为植物的逆境生理和分子机制,(E-mail)mao_xf@126.com
基金资助:
MAO Xiaofei(), HUANG Shiping, YIN Fangliu, ZENG Youling()
Received:
2022-11-05
Online:
2023-08-20
Published:
2023-08-14
Correspondence author:
ZENG Youling (1971- ), Female, Professor, Master adviser, Research direction is plant stress physiology and molecular mechanism, (E-mail)zeng_ylxju@126.comSupported by:
摘要:
【目的】研究盐穗木乙醛脱氢酶7B4基因(HcALDH7B4)在原核细胞中的耐盐抗旱功能,为耐盐抗旱育种提供候选基因。【方法】利用qRT-PCR检测高盐和干旱胁迫下HcALDH7B4基因的表达水平;利用原核表达重组大肠杆菌(pET-28a-HcALDH7B4)在盐旱胁迫下的菌落大小和生长活力分析其盐旱抗性。【结果】在600 mM NaCl处理和自然干旱条件下,盐穗木同化枝中HcALDH7B4被显著诱导。相较对照菌株(pET-28a),原核表达的重组大肠杆菌在500 mM NaCl、500 mM KCl和500 mM、800 mM Mannitol胁迫下表现出更好的生长活力。【结论】盐穗木HcALDH7B4是一个胁迫应答基因,可以提高重组菌盐旱胁迫的耐受性。
中图分类号:
毛晓菲, 黄世平, 银芳柳, 曾幼玲. 盐穗木HcALDH7B4基因的原核表达增强耐盐抗旱性[J]. 新疆农业科学, 2023, 60(8): 2006-2012.
MAO Xiaofei, HUANG Shiping, YIN Fangliu, ZENG Youling. Prokaryotic expression of HcALDH7B4 gene enhanced salt tolerance and drought resistance[J]. Xinjiang Agricultural Sciences, 2023, 60(8): 2006-2012.
Name | Primer | Primer sequence(5'-3') |
---|---|---|
qRT -PCR | RHcALDH7B4-P1 | GTGCTCCAACAACTCCATTG |
RHcALDH7B4-P2 | CCCTTGAACTGCCTGTGAAT | |
RHcactin-P1 | CCAAAGGCCAACAGAGAGAAGAT | |
RHcactin-P2 | TGAGACACACCATCACCAGAAT |
表1 引物
Tab.1 Primer sequence
Name | Primer | Primer sequence(5'-3') |
---|---|---|
qRT -PCR | RHcALDH7B4-P1 | GTGCTCCAACAACTCCATTG |
RHcALDH7B4-P2 | CCCTTGAACTGCCTGTGAAT | |
RHcactin-P1 | CCAAAGGCCAACAGAGAGAAGAT | |
RHcactin-P2 | TGAGACACACCATCACCAGAAT |
图1 盐穗木ALDH7B4蛋白特性预测 注:A、B分别为HcALDH7B4蛋白的疏水性分析和三级结构预测
Fig.1 Characteristics of predicted protein of ALDH7B4 of Halostachys caspica Note:A, B:The hydrophobicity analysis and tertiary structure prediction of HcALDH7B4
图2 盐穗木乙醛脱氢酶7B4与其他物种的系统发育树 注:HcALDH7B4由红色显示
Fig.2 Phylogenetic tree analysis of ALDH7B4 from Halostachys caspica and other species Note:HcALDH7B4 is shown in red
图3 HcALDH7B4基因在盐和干旱胁迫下的表达 注:不同浓度的NaCl胁迫(A);600 mmol/L NaCl胁迫(B);不同时期的干旱胁迫(C)
Fig.3 The relative expression of HcALDH7B4 under salt and drought treatment Note: Different concentrations of NaCl treatment(A); 600 mmol/L NaCl treatment(B); Drought treatment at different times(C)
图4 pET28a-HcALDH7B4重组蛋白的SDS-PAGE和Western blot 注: A: SDS-PAGE鉴定。M: 蛋白分子量Marker;1,3,5: 重组菌诱导前;2,4,6: 重组菌诱导后;7: 对照菌诱导前;8: 对照菌诱导后。B: Western blot分析。1,2,3: 重组菌诱导前;4,5,6: 重组菌诱导后。Total: 细胞总粗蛋白;Ins: 不溶性蛋白质;Sol: 可溶性蛋白
Fig.4 SDS-PAGE and western blot detection for transettas protein containing pET-28a-HcALDH7B4 Note: A: SDS-PAGE identification.M: Protein molecular weight marker; 1,3,5: Un-induced recombinant E.coli Transetta:: pET28a-HcALDH7B4; 2,4,6: induced recombinant E.coli Transetta:: pET28a-HcALDH7B4; 7, Un-induced control Transetta:: pET28a; 8: induced control Transetta:: pET28a.B: Western blot analysis.1,2,3: Un-induced recombinant E.coli Transetta:: pET28a-HcALDH7B4; 4,5,6: induced recombinant E.coli Transetta:: pET28a-HcALDH7B4; Total: total crude protein of cells; Ins: insoluble protein; Sol: soluble protein
图6 盐旱胁迫下大肠杆菌菌株的生长检测 注:(A) 对照;(B) 500 mmol/L NaCl胁迫;(C) 500 mmol/L KCl胁迫;(D) 500 mmol/L Mannitol胁迫;(E) 800 mmmol/L Mannitol胁迫
Fig.6 Growth detection of E.coli strains under salt and drought stress Note: (A) Control; (B) 500 mmol/L NaCl stress; (C) 500 mmol/L KCl stress; (D) 500 mmol/L Mannitol stress; (E) 800 mmol/L Mannitol stress
[1] |
Bartels D. Targeting detoxification pathways: an efficient approach to obtain plants with multiple stress tolerance?[J]. Trends in Plant Science, 2001, 6(7):284-286.
DOI PMID |
[2] |
Chen X, Zeng Q, Wood A J. The stress-responsive Tortula ruralis gene ALDH21A1 describes a novel eukaryotic aldehyde dehydrogenase protein family[J]. Journal of Plant Physiology, 2002, 159(7):677-684.
DOI URL |
[3] |
Kibbe D S, Liu F, Wen T J, et al. Charaterization of the aldehyde dehydrogenase gene families of Zeamays and Arabidopsis[J]. Plant Molecular Biology, 2002, 48: 751-764.
DOI URL |
[4] |
Buchman C D, Hurley T D. Inhibition of the Aldehyde Dehydrogenase 1/2 Family by Psoralen and Coumarin Derivatives[J]. Journal of Medicinal Chemistry, 2017, 60(6): 2439-2455.
DOI PMID |
[5] |
Stiti N, Giarola V, Bartels D. From algae to vascular plants: The multistep evolutionary trajectory of the ALDH superfamily towards functional promiscuity and the emergence of structural characteristics[J]. Environmental and Experimental Botany, 2021, 185:104376.
DOI URL |
[6] |
Konĉitíková R, Vigouroux A, Kopeĉná M, et al. Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7[J]. Biochemical Journal, 2015, 468: 109-123.
DOI PMID |
[7] |
Zhao J Y, Missihoun T D, Dorothea B. The ATAF1 transcription factor is a key regulator of aldehyde dehydrogenase 7B4 (ALDH7B4) gene expression in Arabidopsis thaliana[J]. Planta, 2018, 248(4): 1017-1027.
DOI |
[8] |
Nakazono M, Tsuji H, Li Y, et al. Expression of a gene encoding mitochondrial aldehyde dehydrogenase in rice increases under submerged conditions[J]. Plant Physiology, 2000, 124(2):587-598.
PMID |
[9] | Xu X, Guo R, Cheng C, et al. Overexpression of ALDH2B8, an aldehyde dehydrogenase gene from grapevine, sustains Arabidopsis growth upon salt stress and protects plants against oxidative stress[J]. Plant Cell Tissue & Organ Culture, 2013, 114(2): 187-196. |
[10] |
Huang W, Ma X, Wang Q, et al. Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays)[J]. Plant Molecular Biology, 2008, 68:451-463.
DOI PMID |
[11] | 黄世平, 戴玲玲, 宋策, 等. 盐穗木醛脱氢酶基因HcALDH7A1原核表达载体的构建及蛋白诱导表达与优化[J]. 新疆农业科学, 2015, 52(8): 1510-1516. |
HUANG Shiping, DAI Lingling, SONG Ce, et al. The Vector Construction and Prokaryotic Expression of Halostachys caspica Aldehyde Dehydrogenase Gene(HcALDH7A1)in E coli[J]. Xinjiang Agricultural Sciences, 2015, 52(8): 1510-1516. | |
[12] | 周涛, 王娟, 胡佳蕙, 等. 番茄转录因子基因SlWRKY6的克隆与原核表达分析[J]. 西北植物学报, 2020, 40(11):1824-1832. |
ZHOU Tao, WANG Juan, HU Jiahui, et al. Cloning and prokaryotic expression analysis of a WRKY transcription factor gene SlWRKY6 in solanumly copersicum[J]. Acta Botanica Boreali-Occidentalia Sinica, 2020, 40(11):1824-1832. | |
[13] |
Kirch H H, Bartels D, Wei Y L, et al. The ALDH gene superfamily of Arabidopsis[J]. Trends in Plant Science, 2004, 9(8): 371-377.
DOI URL |
[14] |
Liu F, Schnable P S. Functional specialization of maize mitochondrial aldehyde dehydrogenases[J]. Plant Physiology, 2002, 130(4): 1657-1674.
PMID |
[15] | Xia A, Duan F Y, Guo S, et al. Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses[J]. Journal of Integrative Agriculture, 2014, 9: 1900-1908. |
[16] |
Gao C X, Han B. Evolutionary and expression study of the aldehyde dehydrogenase (ALDH) gene superfamily in rice (Oryza sativa)[J]. Gene, 2009, 431(1): 86-94.
DOI URL |
[17] |
Rodrigues S M, Andrade M O, Gomes A P S, et al. Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress[J]. Journal of Experimental Botany, 2006, 57(9): 1909-1918.
PMID |
[18] |
Shin J H, Kim S R, An G. Rice aldehyde dehydrogenase7 is needed for seed maturation and viability[J]. Plant Physiology, 2009, 149(2): 905-915.
DOI URL |
[19] | 陈加敏, 朱承慧. 高粱耐逆基因SbALDH7的克隆与表达分析[J]. 中国农学报, 2013, 29(12): 62-68. |
CHEN Jiamin, ZHU Chenghui. Isolation and expressing analysis of a stress tolerance gene SbALDH7 in Sorghum bicolor[J]. Chinese Agricultural Science Bulletin, 2013, 29(12): 62-68. | |
[20] |
Kirch H H, Schlingensiepen S, Kotchoni S, et al. Detailed expression analysis of selected genes of the aldehyde dehydrogenase (ALDH) gene superfamily in Arabidopsis thaliana[J]. Plant Molecular Biology, 2005, 57(3): 315-332.
DOI URL |
[21] |
Gautam R, Ahmed I, Shukla P, et al. Genome-wide characterization of ALDH Superfamily in Brassica rapa and enhancement of stress tolerance in heterologous hosts by BrALDH7B2 expression[J]. Scientific Reports, 2019, 9(1): 7012-7025.
DOI |
[22] |
Gautam R, Rajesh K M, Woch N, et al. Ectopic expression of BrALDH7B2 gene encoding an antiquitin from Brassica rapa confers tolerance to abiotic stresses and improves photosynthetic performance under salt stress in tobacco[J]. Environmental and Experimental Botany, 2020, 180:104223.
DOI URL |
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