降解邻苯二甲酸酯的植物内生细菌筛选及其降解机理研究

Phthalic acid esters (PAEs) is one class of ubiquitous environmental organic pollutants of concern. Till now, no study has reported the PAE-degrading endophytic bacteria. Our previous study indicated that uptake and accumulation of PAEs by different cultivars of crops (e.g., rice, Chinese flowering cabbage, maize) were species-specific and cultivar-specific. This variation may result from plant uptake and transportation, plant metabolism, and degradation by endophytic bacteria. The former two topics have been studied to some degree. However, very few study has reported about PAE-degrading endophytic bacteria and their effects on the accumulation variation of cultivars. In this project, high/low PAE-accumulating genotypic cultivars screened previously by our group and plants growing in the areas contaminated by PAEs will be selected as materials, and PAEs in these samples will be extracted and measured by gas chromatography coupled with mass spectrometry (GC/MS). The objectives of this project are to isolate and identify PAE-degrading endophytic bacterial strains from these materials above using selective enrichment cultures and plate-screened. Endophytic bacterial strains will be identified by morphology, physiological and biochemical characteristics, and 16SrDNA gene-base phylogenetic alalyses, and analyzed their community diversity using high-throughput sequencing technology and biological information. Then, two endophytic bacterial strains with higher PAH degradation rates will be selected to investigate PAE degradation kenitics and its possible degradation pathways. The PAE degradation-associated gene will be studied and screened by construction of genomic library. The results will be helpful for guaranteeing the safety of agricultural products.

邻苯二甲酸酯(PAEs，俗称塑化剂)是一类广受关注的环境有机污染物。但目前国内外尚未有关于具有PAEs降解性能的植物内生细菌报道。我们前期研究发现农作物(水稻、菜心等)对PAEs的吸收累积存在作物种类差异和品种(基因型)差异。该差异可能由植物吸收运移、植物自身代谢作用、植物内生细菌降解导致。目前国内外对前二者进行了一定的研究，但有关植物内生细菌降解有机污染物进而导致品种吸收累积差异的研究鲜见报道。本项目拟以前期筛选获得PAEs高、低累积作物品种和PAEs污染区植物为材料，分离筛选具有PAEs降解性能的植物内生细菌，采用高通量测序并结合生物信息学分析其生物多样性，研究其生物学特性、PAEs降解动力学及降解途径，通过构建基因组文库研究内生细菌的降解基因，阐明其降解机理，为保障农产品质量安全提供科学依据。

邻苯二甲酸酯(PAEs，俗称塑化剂)是一类广受关注的环境有机污染物。但目前国内外尚未有关于具有PAEs 降解性能的植物内生细菌报道。本项目从植物中分离筛选具有PAEs降解性能的植物内生细菌，研究其生物学特性、PAEs 降解动力学及降解途径，通过全因组和转录组预测相关降解基因，并进行降解基因验证。主要研究结果如下：. 配制PAEs污染土壤种植PAEs高、低累积作物（选取六种供试植物品种），结果表明，作物内生菌对DBP和DEHP的降解率为11-64%。在某污染水渠边采集植物，从根茎叶中提取出内生菌，发现植物中的内生菌对DBP有80%左右、对DEHP有40%左右的降解率。选用污染水渠植物中提取的内生菌作为本研究的菌株，该菌株经16S rDNA鉴定为为枯草芽孢杆菌（Bacillus subtilis）。. 对降解菌进行革兰氏染色、接触酶反应、氧化酶测定、淀粉水解试验、脲酶测定，发现革兰氏染色呈阴性，其余呈阳性。该菌具有分泌 IAA的能力、不具备固氮能力、解磷活性不强、具有解钾能力。该菌对DBP和DEHP降解的最佳条件为：温度为30℃，pH为7.0，初始浓度为100 mg/L。降解菌对DBP的降解速率为0.825d-1，半衰期为0.84天，对DEHP的降解速率为0.1901d-1，半衰期为3.65天。. DBP降解途径：DBP的一个酯键断开形成MBP，然后另外一个酯键断开进一步水解成PA，PA失去一个羧基然后形成BA。最后BA进过一系列水解反应生成H2O和CO2。随着降解时间的增加，MBP、PA、BA可被该菌彻底降解。DEHP降解途径：DEHP中的一个酯键断开然后形成MEHP，然后另外一个酯键断开进一步水解成PA，最后PA经过一系列水解反应，生成水和氧气。. 该菌对铜、锌、镉、汞的耐受浓度分别为75、 30、 20、 25mg/L，对氯霉素、庆大霉素、盐酸四环素、利福平的耐受浓度分别为 4、 0.25、 8、16 mg/L。. 对植物内生菌进行了全基因组测序和转录组分析，预测出两个降解基因（N-1A002471和N-1A003551），降解验证表明：N-1A002471和N-1A003551基因表达的蛋白对DBP的降解率分别为35%、39%。本研究为PAEs污染土壤的植物、微生物修复及保障农产品质量安全提供了重要科学依据。