基因aotM与trmD调控根瘤菌盐碱耐受性、抗氧化酶活性及细胞膜氧化损伤

doi:10.13560/j.cnki.biotech.bull.1985.2025-1045

摘要/Abstract

摘要：

目的野生型菌株根瘤菌Rhizobium yanglingense CCBAU 01603经连续传代得到进化菌株Alk_55，旨在探明Alk_55基因组上aotM和trmD发生SNPs变化导致耐盐碱能力大幅增强的原因。方法基于已构建的基因删除突变株△aotM和△trmD，探究基因aotM、trmD对根瘤菌耐盐碱性、细胞膜损伤、胞内抗氧化酶活性及活性氧含量的调控作用。结果与野生型菌株相比，突变株△aotM和△trmD耐盐、碱能力较强，且在一定浓度范围内对盐碱敏感性降低；MDA含量显著降低表明细胞膜脂质过氧化物损伤程度较小；胞内超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性显著升高，活性氧（ROS）含量显著降低。结论基因aotM、trmD对根瘤菌盐碱耐受性、胞内抗氧化酶活性及细胞膜氧化应激损伤具有较强的调控作用。

关键词: 根瘤菌, aotM, trmD, 耐盐碱能力, MDA含量, 抗氧化酶

Abstract:

Objective The evolved strain Alk_55 was obtained from Rhizobium yanglingense CCBAU 01603 through continuous subculturing. This work aims to investigate the underlying reasons for the significant enhancement of salt-alkali tolerance caused by SNP changes in aotM and trmD in the Alk_55 genome. Method The regulation of salt-alkali tolerance, cell membrane damage, intracellular antioxidant enzyme activity, and reactive oxygen species (ROS) content in rhizobia were assessed, based on two mutants of rhizobia, △aotM and △trmD, created by deleting genes aotM and trmD respectively. Result Mutant △aotM and △trmD had stronger salt-alkali tolerance and reduced sensitivity to salt-alkali stress within a certain concentration range, compared with the wild-type strain. Significantly lower malondialdehyde (MDA) content indicated less lipid peroxidative damage to the cell membrane. Intracellular superoxide dismutase (SOD) and catalase (CAT) activities markedly increased, whereas ROS content significantly decreased. Conclusion Genes aotM and trmD strongly regulate salt-alkali tolerance, intracellular antioxidase activity, and oxidative stress-induced cell membrane damage in rhizobia.

Key words: Rhizobium, aotM, trmD, salt-alkali tolerance, MDA content, antioxidase

刘君良, 袁晓霞, 乔美玲, 杨冰洁, 于秀敏, 穆莎茉莉, 冀照君. 基因aotM与trmD调控根瘤菌盐碱耐受性、抗氧化酶活性及细胞膜氧化损伤[J]. 生物技术通报, 2026, 42(8): 323-330.

LIU Jun-liang, YUAN Xiao-xia, QIAO Mei-ling, YANG Bing-jie, YU Xiu-min, MU Sha-mo-li, JI Zhao-jun. Genes aotM and trmD Regulate Salt-alkali Tolerance， Antioxidase Activity， and Cell Membrane Oxidative Damage in Rhizobia[J]. Biotechnology Bulletin, 2026, 42(8): 323-330.

图/表 6

图1 进化菌株Alk_55的aotM，trmD基因序列比对分析及保守性A：trmD基因及氨基酸序列比对结果；B：aotM基因及氨基酸序列比对结果；C：基于trmD基因的系统发育树；D：基于aotM基因的系统发育树

Fig. 1 Alignment analysis and conservation of aotM and trmD gene sequences in evolved isolates Alk_55A: Comparison of gene and amino acid sequences of trmD. B: Comparison of gene and amino acid sequences of aotM. C: Phylogenetic tree of trmD gene. D: Phylogenetic tree of aotM gene

图2 根瘤菌在含不同浓度NaCl的YMA液体培养基中生长情况A：不含NaCl；B：含0.5%NaCl；C：含0.6%NaCl；D：含0.7%NaCl；*P<0.05，**P<0.01，***P<0.001，****P<0.000 1，下同

Fig. 2 Growth of rhizobia in YMA liquid medium with different concentrations of NaClA: No NaCl. B: Containing 0.5% NaCl. C: Containing 0.6% NaCl. D: Containing 0.7% NaCl; *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. The same below

图3 菌株在不同pH值的YMA液体培养基中生长情况A：pH值为8.12的YMA液体培养基（含KOH母液100 μL）；B：pH值为8.90的YMA液体培养基（含KOH母液200 μL）

Fig. 3 Growth of rhizobia in YMA liquid medium with different pH valuesA: YMA broth with pH value of 8.12 (containing 100 μL KOH stock solution). B: YMA broth with pH value 8.90 (containing 200 μL KOH stock solution)

图4 根瘤菌抗氧化酶活性A：过氧化氢酶（CAT）活性；B：超氧化物歧化酶（SOD）活性

Fig. 4 Antioxidant enzyme activity of rhizobiaA: Catalase (CAT) activity. B: Superoxide dismutase (SOD) activity

图5 根瘤菌细胞膜MDA含量

Fig. 5 MDA contents in the membrane of rhizobia

图6 根瘤菌胞内ROS积累量

Fig. 6 Intracellular ROS accumulation in rhizobia

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