盐穗木HcALDH7B4基因的原核表达增强耐盐抗旱性

doi:10.6048/j.issn.1001-4330.2023.08.022

摘要/Abstract

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

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

中图分类号:

S188

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.

图/表 7

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5 重组大肠杆菌菌株的盐旱抗性

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

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

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