石油降解菌HX-2耐盐机制及甜菜碱转运蛋白基因的研究

Salt tolerance mechanism and betaine transport genes of a petroleum-degrading strain HX-2

摘要:

【背景】修复石油烃污染的高盐水体及土壤是具有挑战性的，因此探究石油烃降解菌株的耐盐机制尤为重要。【目的】对石油降解菌HX-2的耐盐机制及与耐盐性相关的基因进行研究。【方法】通过GC分析菌株HX-2在不同石油加入量及高盐条件下的烃降解情况；利用电导率仪及原子吸收光谱对细胞内离子含量进行分析；比较外源添加甜菜碱前后对胞外多糖(extracellular polysaccharide，EPS)及高盐土壤中石油降解情况的影响；最后对耐盐相关基因进行了qPCR分析研究。【结果】石油降解菌Rhodococcus sp. HX-2可以对10 000?100 000 mg/L的石油进行降解，3 d降解率均达到70%以上，并可在1%?10% NaCl存在下降解石油，在6% NaCl浓度下仍有43.8%的降解率。对HX-2菌株耐盐机制的研究表明，细胞内阳离子浓度随着盐浓度的变化没有显著差异，而积累相容性物质甜菜碱并促进EPS的合成才是石油降解菌HX-2的耐盐机制。同时，扫描电镜结果表明，外源甜菜碱的添加通过刺激EPS的合成提高菌株的耐盐性。由HX-2菌株得到4种甜菜碱转运蛋白基因H0、H1、H3、H5和1种甜菜碱合成相关基因BetB。对菌株HX-2的基因转录分析表明，NaCl、甜菜碱诱导H0、H1、H3和H5的表达；在甜菜碱与NaCl共存时，基因转录水平达到最大值。【结论】Rhodococcus sp. HX-2具有在盐渍化环境中修复烃类污染物的应用潜力。

Abstract:

[Background] It is challenging to remediate water and soil contaminated by petroleum hydrocarbons under high salt conditions. Therefore, it is important to explore the salt tolerance of petroleum hydrocarbon-degrading strains. [Objective] Salt tolerance and relevant genes of petroleum-degrading strain HX-2 were studied. [Methods] The hydrocarbon-degrading strain HX-2 under different petroleum amount and high salinity conditions was analyzed by GC. The intracellular ion content was analyzed by conductivity meter and atomic absorption spectroscopy. The effects of exogenous betaine on extracellular polysaccharide and petroleum degradation in high salinity soils were compared before and after adding betaine; finally, the related genes for salt tolerance were analyzed by qPCR. [Results] The petroleum-degrading strain Rhodococcus sp. HX-2 degraded 10 000?100 000 mg/L of petroleum. After three days of cultivation, the degradation rate of petroleum reached more than 70%, and it degraded petroleum in the presence of 1%?10% NaCl. At 6% salt concentration, the degradation rate was still 43.8%. Studies on the salt tolerance mechanism showed that there was no significant difference in intracellular cation concentration with the change of salt concentration. Accumulating betaine compatible substances and promoting the synthesis of extracellular polysaccharide were the salt tolerance mechanisms of petroleum hydrocarbon-degrading strain HX-2. Meanwhile, the results of scanning electron microscopy showed that the addition of exogenous betaine could improve the salt tolerance of the strain by stimulating the synthesis of EPS. Four betaine transporter genes H0, H1, H3, H5 and a betaine synthesis related gene BetB were obtained from HX-2 strain. The analysis of gene transcription showed that NaCl and betaine induced the expression of H0, H1, H3 and H5. When betaine coexisted with NaCl, the level of gene transcription reached the maximum. [Conclusion] Rhodococcus sp. HX-2 has potential application in remediation of hydrocarbon pollutants in saline environment.