菱形节杆菌BFL-3分离鉴定及耐盐碱机理研究

doi:10.3969/j.issn.1674-7968.2023.07.015

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摘要耐盐碱微生物资源能促进土壤养分循环,提高植物保水耐盐能力,因此在盐碱土改良领域有重要应用意义。课题组前期分离得到一株耐盐碱细菌菌株 BFL-3,为明确该菌株的遗传进化关系、耐盐碱程度及机理,本研究对 BFL-3 进行了形态学观察和 16S rRNA 序列分析,采用光密度法研究了 BFL-3 在不同pH及 NaCl胁迫下的生长特性。利用二代测序技术对 BFL-3 进行全基因组测序分析,并对其进行比对注释,通过 qPCR 验证 BFL-3 在含 5% NaCl、pH 9 的培养基中耐盐碱相关基因的表达。结果显示,菌株 BFL-3 为菱形节杆菌(Arthrobacter rhombi),其对 NaCl 的耐受范围为 0.5%~10%,对 pH 的耐受范围为 5~10; 全基因组测序分析最终获得 35 条支架(Scaffold)序列,总序列长度为 3 476 199 bp,G+C 摩尔百分含量为69.15%,编码基因数量为 3 170 个,含有 tRNA 基因 50 个,rRNA 基因 7 个。基因注释显示,菌株 BFL-3 在氨基酸和碳水化合物代谢途径富集大量基因,同时在离子转运蛋白相关途径富集一定数量的基因,表明 BFL-3 可能采用相容溶质积累和离子平衡两种策略来响应盐碱胁迫。qPCR 验证显示,BFL-3 在培养基含5% NaCl、pH 值9的条件下,海藻糖合成酶 (trehalose synthase, treS)、二氨基丁酸 -2- 酮戊二酸转氨酶 (diaminobutyrate-2-oxoglutarate transaminase, ectB)、谷氨酸脱氢酶 (glutamate dehydrogenase, gdhA)、甜菜碱/L-脯氨酸 ABC 转运透性酶(glycine betaine/L-proline ABC transporter permease, proW)、Trk系统钾离子转运蛋白(Trk system potassium transporter, trkA)、胆碱 ABC 转运透性酶(choline ABC transporter permease, opuBD)表达量均上调,分别是对照的 11.0、6.2、6.0、13.1、7.4 和 6.3 倍(P<0.01),说明菌株 BFL-3 通过在胞内合成积累海藻糖、四氢嘧啶、谷氨酸等相容性物质并调控离子平衡来缓解盐碱胁迫的压力。本研究结果明确了 BFL-3 的耐盐碱特性,初步探索了该菌株的盐碱耐受机理,为进一步利用该菌株修复盐碱地提供科学依据。

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关键词 ：菱形节杆菌BFL-3, 耐盐碱性, 全基因组测序

Abstract：The resources of saline-alkaline tolerant microorganisms can promote soil nutrient circulation, enhance the water reservation and salinity tolerance ability of the plant, they have important application in remediation of saline-alkaline soil. Previously, a saline-alkaline resistant bacterial strain BFL-3 was isolated, the purpose of this study was to clarify the genetic evolution relationship, saline-alkaline tolerance and mechanism of the strain. The strain BFL-3 was identified by morphology and 16S rRNA sequence analysis. The growth characteristics of strain BFL-3 under different NaCl content and pH stresses were determined by optical density measurement. Besides, the whole genome of strain BFL-3 was sequenced, followed by gene function annotation and bioinformatics analysis. qPCR was applied to verify the expression of genes related to saline-alkaline tolerance of BFL-3 in medium containing 5% NaCl and pH 9. The results showed that the strain BFL-3 was identified as Arthrobacter rhombi, it could tolerant 0.5%~10% of NaCl, the tolerance range of pH value was 5~10. The strain contained 35 scaffolds, the total sequence length was 3 476 199 bp, G+C mol% was 69.15%, coding 3 170 genes, including 50 tRNA genes and 7 rRNA genes. Gene annotation showed that strain BFL-3 was enriched in a large number of genes in amino acid and carbohydrate metabolism pathways, and a certain number of genes in ion transporter related pathways, suggesting that the strain adopts 2 strategies of solute accumulation and ion balance in response to saline-alkaline stress. When the NaCl content was 5% and the pH value was 9, the expression level of trehalose synthase gene (treS), diaminobutyrate-2-oxoglutarate transaminase gene (ectB), glutamate dehydrogenase gene (gdhA), glycine betaine/L-proline ABC transporter permease gene (proW), Trk system potassium transporter gene (trkA) and choline ABC transporter permease gene (opuBD) were up-regulated, 11.0, 6.2, 6.0, 13.1, 7.4 and 6.3 times of the control group, respectively (P<0.01). These results indicated that strain BFL-3 could alleviate the saline-alkaline stress through the synthesis and accumulation of cytocompatible substances (such as trehalose, ectoine and glutamate) and regulating ion balance in the cell. This study clarified the saline-alkaline tolerance characteristics of BFL-3, explored its saline-alkaline tolerance mechanism, provided scientific basis for the further application of this strain in remediation of saline-alkaline soil.

Key words： Arthrobacter rhombi BFL-3 Saline-alkaline tolerance Whole genome sequencing

收稿日期: 2022-07-27

基金资助:陕西省科学院青年联合培养项目(2021K-14)；国家自然科学基金(31800100)；陕西省科技厅重点研发项目(2021NY-046)；西安市科技计划项目(20NYYF0028)

通讯作者: *baiyn@xab.ac.cn

引用本文:

卢雨欣, 颜宏, 白亚妮, 梁建强, 冯志珍. 菱形节杆菌BFL-3分离鉴定及耐盐碱机理研究[J]. 农业生物技术学报, 2023, 31(7): 1501-1512.
LU Yu-Xin,YAN Hong,BAI Ya-Ni, ,LIANG Jian-Qiang,FENG Zhi-Zhen,. Isolation, Identification and Saline-alkaline Tolerance Mechanism Study of Arthrobacter rhombi Strain BFL-3. 农业生物技术学报, 2023, 31(7): 1501-1512.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.07.015 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I7/1501

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