甜菜碱醛脱氢酶基因BvBADH2的克隆及在植物盐胁迫中的作用

doi:10.11686/cyxb2025207

摘要/Abstract

摘要：

甜菜作为我国北方盐碱地改良的先锋作物，其关键耐盐基因的挖掘对作物遗传改良具有重要理论价值。研究显示，甜菜碱醛脱氢酶基因家族BvBADHs通过生物合成甘氨酸甜菜碱可能在甜菜盐胁迫中发挥作用。本研究以耐盐甜菜品种‘甘糖7号’为材料，克隆该家族BvBADH2基因，利用农杆菌介导法构建过表达拟南芥株系，系统验证和解析其耐盐调控机制。基因克隆及生物信息学分析显示：BvBADH2 编码序列（CDS）全长1512 bp，编码503个氨基酸；预测蛋白相对分子量为54.78 kDa，等电点（pI）为5.37，不稳定指数为31.62；含有保守的乙醛脱氢酶结构域（Aldedh， PF00171），且启动子区域具有多种非生物胁迫响应的顺式作用元件。转基因拟南芥盐胁迫表型鉴定发现，150 mmol·L^-1 NaCl胁迫下，过量表达株系OE₁与OE₃的根长、鲜重和叶绿素含量较野生型分别显著增加129.1%、125.0%、104.8%和137.5%、126.3%、107.2% （P<0.01）。进一步研究揭示，OE₁/OE₃通过特异性积累甘氨酸甜菜碱、脯氨酸和可溶性糖等渗透相容性物质，有效维持细胞渗透势稳态；同时动态激活超氧化物歧化酶、过氧化物酶和过氧化氢酶活性，显著抑制活性氧积累和缓解膜脂过氧化损伤。上述结果表明，BvBADH2通过“渗透-抗氧化协同调控网络”赋予植物耐盐性，首次在模式植物中验证其功能保守性，为作物耐盐分子设计育种提供了关键酶基因资源。

关键词: 甜菜碱醛脱氢酶, 盐胁迫, 渗透调节, 氧化损伤, 抗氧化系统

Abstract:

Sugar beet （Beta vulgaris） is used as a pioneer crop for saline-alkali soil remediation in northern China. In this context， there is significant theoretical value in investigating crop genetic improvement through the identification of key salt-tolerance genes. Studies have shown that the betaine aldehyde dehydrogenase gene family BvBADHs may play a role in beet salt stress through facilitating glycine betaine biosynthetis. In this study， BvBADH2 was cloned from the salt-tolerant sugar beet cultivar ‘Gantang 7’ and BvBADH2-overexpressing Arabidopsis thaliana lines were generated by Agrobacterium-mediated transformation to systematically analyze and confirm its salt-tolerance mechanisms. Gene cloning and bioinformatics analysis showed that the full length of the BvBADH2 coding sequence （CDS） is 1512 bp， encoding 503 amino acids. The relative molecular weight of the predicted protein is 54.78 kDa， the isoelectric point is 5.37， and the instability index is 31.62. It contains a conserved aldehyde dehydrogenase domain （Aldedh， PF00171）， and the promoter region has a variety of abiotic stress-responsive cis-acting elements. In the salt-stress tolerant phenotype of transgenic A. thaliana under 150 mmol·L^-1 NaCl stress， the root length， fresh weight and chlorophyll content of the higher expression transgenic lines OE₁ and OE₃ were significantly increased by 129.1%， 125.0%， 104.8% and 137.5%， 126.3%， 107.2%， respectively， compared with the wild type （P<0.01）. Further studies revealed that BvBADHs overexpression of transgenic plants effectively maintained cell osmotic potential homeostasis by specifically accumulating osmotic protective substances such as glycine betaine， proline， and soluble sugar. Concurrently， dynamic activation of antioxidant enzyme systems-superoxide dismutase， peroxidase， and catalase-significantly reduced reactive oxygen species accumulation and alleviated membrane lipid peroxidation. These findings indicate that BvBADH2 confers salt tolerance through an “osmotic-antioxidant synergistic regulatory network”， marking the first functional validation of this gene’s conservation in model plants. This study provides critical insight for the development of enzymatic gene resources aimed at enhancing crop salt tolerance through molecular design breeding.

Key words: betaine aldehyde dehydrogenase, salt stress, osmotic adjustment, oxidative damage, antioxidant system

魏明, 吴欣蕊, 吴璇, 李昊, 伍国强, 张伟杰, 程子义. 甜菜碱醛脱氢酶基因BvBADH2的克隆及在植物盐胁迫中的作用[J]. 草业学报, 2026, 35(5): 185-195.

Ming WEI, Xin-rui WU, Xuan WU, Hao LI, Guo-qiang WU, Wei-jie ZHANG, Zi-yi CHENG. Cloning of the betaine aldehyde dehydrogenase family BvBADH2 gene and its role in plant salt tolerance[J]. Acta Prataculturae Sinica, 2026, 35(5): 185-195.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物名称Name of primer	序列Sequence （5′-3′）	用途Purpose
BvBADH2 F₁	ATGGCGATCCCAATACCT	基因克隆 Gene cloning
BvBADH2 R₁	CAGTTTTGAGGGAGACTTGTAC	基因克隆 Gene cloning
BvBADH2 F₂	gaggacacgctcgagATGGCGATCCCAATACCT	载体构建 Vector construction
BvBADH2 R₂	tttgtaatccccgggCAGTTTTGAGGGAGACTTGTAC	载体构建 Vector construction
BvBADH2 qRT F	GTCCTGTTGTCAGCAAGGGA	实时定量PCR Quantitative real time PCR
BvBADH2 qRT R	GCATGGACGTGGAGACATCA
AtACTIN qRT F	AGATCCAGGACAAGGAAGGTATTC
AtACTIN qRT R	CGCAGGACCAAGTGAAGAGTAG
35S Pro F	GACGCACAATCCCACTATCC	转基因植株鉴定 Identification of the transgene plants
BvBADH2 R₃	CTCGTCATCGGAACTAAATG	转基因植株鉴定 Identification of the transgene plants

图1 BvBADH2分子克隆及编码蛋白序列分析A：BvBADH2基因扩增产物，大小1512 bp；B：BvBADH2全长编码序列分析。M：DL2000 DNA分子量标准；N：阴性对照；P：PCR产物；红色加粗氨基酸序列为乙醛脱氢酶保守结构域。下同。A： BvBADH2 gene amplification product， size 1512 bp. B： Analysis of the full-length coding sequence of BvBADH2. M： DNA marker DL2000； N： Negative control； P： PCR product. Red crude amino acid sequence is aldehyde dehydrogenases conserved domain. The same below.

Fig.1 Molecular cloning and protein sequence analysis of BvBADH2

图2 BvBADH2过表达载体构建与转基因植株鉴定A：双元载体T-DNA区域示意图Schematic diagram of the T-DNA region of the binary vector； MAS Ter：甘露碱合成酶终止子Mannopine synthase terminator； BlpR：类细菌素肽调控蛋白Bacteriocin-like peptide regulatory protein； MAS Pro：甘露碱合成酶启动子Mannopine synthase promoter； 35S Pro：花椰菜花叶病毒35S启动子Cauliflower mosaic virus 35S promoter； OCS Ter：章鱼碱合成酶终止子Octopine synthase terminator. B：转基因株系PCR鉴定PCR identification of transgenic lines； 1~5： 5个独立的过表达株系OE1~5 Five independent overexpression lines OE1-5；N：阴性对照（野生型植株DNA） Negative control （wild-type plant DNA）； P：阳性对照（质粒DNA） Positive control （plasmid DNA）； C：转基因株系BvBADH2表达量的qRT-PCR检测qRT-PCR analysis of BvBADH2 relative expression levels in different transgenic lines； WT：野生型Wild type. 不同小写字母代表不同株系间差异达到显著水平（P<0.05）。Different lowercase letters indicate significant differences among the lines （P<0.05）.

Fig.2 Construction of the BvBADH2 overexpression vector and identification of transgenic plants

图3 过表达植株的盐胁迫表型分析不同小写字母表示对照与处理组不同株系间差异达到显著水平（P<0.05），下同。Different lowercase letters indicate significant differences among controls and treatments within different lines （P<0.05）， the same below.

Fig.3 Phenotypic analysis of overexpression plants under salt stress

图4 过表达植株盐胁迫生理生化分析

Fig.4 Physiological and biochemical analysis of overexpression plants under salt stress

图5 过表达植株盐胁迫诱导的抗氧化酶活性分析

Fig.5 Analysis of salt stress-induced antioxidant enzyme activities in overexpression plants

图6 过表达植株中盐胁迫诱导的活性氧积累分析

Fig.6 Analysis of salt stress-induced reactive oxygen species accumulation in overexpression plants

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