一株耐盐碱柠檬酸杆菌在促进植物生长中的应用及其耐盐碱基因挖掘

A saline-alkali tolerant strain of Citrobacter: application in promoting plant growth and mining of saline-alkali tolerance genes

摘要:

【背景】耐盐碱促生细菌对改善盐碱胁迫下的作物生长和提高作物的盐碱耐受能力具有较好效果。【目的】获取兼具耐盐碱和促生功能的微生物资源，深入探究耐盐碱菌的生理生化特性及其耐盐碱分子机理，研究拟从宁夏回族自治区银川市盐碱地土壤中分离获得耐盐碱细菌。【方法】应用形态和显微观察、Biolog Gen Ⅲ微孔板测定和16S rRNA基因序列测定对菌株进行鉴定；利用细菌培养方法进行菌株耐盐碱性和促生效果分析；应用全基因组基因功能注释分析菌株基因组信息。【结果】菌株在pH 8.0-12.0及NaCl 0%-9%的LB培养基中均可生长，且在pH 8.5-9.5培养环境中降碱率可达到13%以上。菌株碳源利用能力显示菌株可利用54种碳源并对另外20种化学敏感性测试物质敏感。菌株YS-AT2属于柠檬酸杆菌属(Citrobacter)，其在盐碱胁迫下对植物的促生效应结果显示：(1) 菌株可在pH 9.5且含NaHCO₃的盐碱性环境中促进拟南芥野生型种子生长；(2) 在50-200 mmol/L NaCl或50-200 mmol/L NaHCO₃盐碱胁迫下促大豆种子萌发；(3) 在pH 8.2、土壤电导率(electricity conductivity, EC)为450 μs/cm土壤中大豆株高、鲜重、干重和根长可分别提高19.84%、18.75%、10.31%和32.58%。对菌株全基因组基因功能注释分析发现，菌株YS-AT2具有多个与嗜盐和嗜碱密切相关的基因，分析其耐盐碱机制可能是通过自身细胞对盐碱性物质的运输、对钠、钾离子的转运和调节及合成高浓度细胞物质而实现的。【结论】本研究解析了耐盐碱促生菌Citrobacter YS-AT2对拟南芥和大豆的促生效应，挖掘其中耐盐碱相关基因，不仅为开发具有耐盐碱促生功能的生物肥料提供理论依据和菌种资源，还为进一步揭示嗜盐碱细菌的分子机理奠定了理论基础。

Abstract:

[Background] Saline-alkali tolerant and plant growth-promoting bacteria are praised for the effects on improving the growth and enhancing the saline-alkali tolerance of crops under saline-alkali stress. [Objective] To acquire the microbial resources that possess both saline-alkali tolerance and growth-promoting effects and decipher the mechanism underlying saline-alkali tolerance, we aimed to isolate a bacterial strain from the saline soil in Yinchuan City, Ningxia Hui Autonomous Region, China. [Methods] The strain was identified by morphological and microscopic observation, Biolog Gen Ⅲ test, and 16S rRNA gene sequencing. The culture method was used to examine the saline-alkali tolerance and growth-promoting effect of the strain. The genome information of the strain was analyzed by functional gene annotation. [Results] Strain YS-AT2 could grow in the LB media with pH 8.0–12.0 and 0%–9% (W/V) NaCl, and showed the alkali reduction rate reaching 13% and above in the media at pH 8.5–9.5. Strain YS-AT2 could use 54 carbon sources and was sensitive to 20 chemical sensitivity test substances. According to 16S rRNA gene sequencing results, YS-AT2 belonged to the genus Citrobacter. strain YS-AT2 could promote the seed growth of Arabidopsis thaliana in a saline environment with NaHCO₃ (pH 9.5) and the germination of soybean seeds under 50–200 mmol/L NaCl or 50–200 mmol/L NaHCO₃ stress. Furthermore, it increased soybean plant height, fresh weight, dry weight, and root length by 19.84%, 18.75%, 10.31%, and 32.58%, respectively, in the soil with pH 8.2 and EC 450 μs/cm. Strain YS-AT2 carried multiple genes associated with saline-alkali tolerance. The saline-alkali tolerance of this strain was probably realized through the transport of saline-alkali substances, the transport and regulation of sodium and potassium ions, and the synthesis of high-concentration cell solutes. [Conclusion] We analyzed the growth-promoting effect of the saline-alkali tolerant strain Citrobacter YS-AT2 on A. thaliana and soybean and mined the genes associated with its saline-alkali tolerance. The findings not only provided a theoretical basis and strain resources for the development of biofertilizers with saline-alkali growth promoting function but also laid a theoretical foundation for deciphering the mechanism underlying the saline-alkali tolerance.