Prokaryotic expression of HcALDH7B4 gene enhanced salt tolerance and drought resistance

doi:10.6048/j.issn.1001-4330.2023.08.022

Abstract

Abstract:

【Objective】 To study the salt tolerance and drought resistance function of Halostachys caspica aldehyde dehydrogenase gene(HcALDH7B4) in prokaryotic cells in the hope of providing possible candidate genes for salt tolerance and drought resistance breeding.【Methods】 qRT-PCR was used to detect the expression level of HcALDH7B4 gene under high salt and drought stress.The colony size and growth activity of prokaryotic expression recombinant E.coli (PET-28A-HCALDH7B4) under salt and drought stress were used to analyze its salt and drought resistance.【Results】 The results of qRT-PCR showed that HcALDH7B4 was significantly induced in the assimilated branches under 600 mmol/L NaCl treatment and natural drought condition and compared with the control strain (PET-28A), the recombinant E.coli expressed by prokaryotic expression showed better growth activity under 500 mmol/L NaCl, 500 mmol/L KCl and 500 mmol/L and 800 mmol/L Mannitol stress.【Conclusion】 HcALDH7B4 is a stress response gene, and can improve the tolerance of recombinant strains to salt and drought stress.

Key words: Halostachys caspica; HcALDH7B4; prokaryotic expression; salt and drought tolerance

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。

关键词: 盐穗木, HcALDH7B4, 原核表达, 耐盐耐旱

CLC Number:

S188

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.

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.

Figures/Tables 7

Tab.1 Primer sequence

Name	Primer	Primer sequence(5'-3')
qRT -PCR	RHcALDH7B4-P1	GTGCTCCAACAACTCCATTG
	RHcALDH7B4-P2	CCCTTGAACTGCCTGTGAAT
	RHcactin-P1	CCAAAGGCCAACAGAGAGAAGAT
	RHcactin-P2	TGAGACACACCATCACCAGAAT

Fig.1 Characteristics of predicted protein of ALDH7B4 of Halostachys caspica Note:A, B:The hydrophobicity analysis and tertiary structure prediction of HcALDH7B4

Fig.2 Phylogenetic tree analysis of ALDH7B4 from Halostachys caspica and other species Note:HcALDH7B4 is shown in red

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)

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

Fig.5 The analysis of recombinant E.coli strains resistance under drought and salt stress

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

References 22

[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