根际效应对双加氧酶基因多样性、表达和蛋白的影响及与多环芳烃降解的关系

With polycyclic aromatic hydrocarbons (PAHs) contaminated field soil and spiked soil as target soils. This project will carry out integrated phyto-microbial remediation of contaminated soils through compartmented rhizo-box experiments, conduct simulated rhizodeposition remediation by the application of root exudates or their components, and set up microcosms consisting of root exudate and cultured strains. Degenerative primers will be designed, quantitative PCR, reverse transcript-PCR, molecular cloning-sequencing, two dimensional electrophoresis and time of flight mass spectrometry will be used to analyze the rhizospheric effects on the characteristics of dioxygenase gene abundance, diversity, expression and related proteomic profiling. The relationship between PAH degradation in rhizosphere and microbial dioxygenase will also be investigated in this study. The results of the project can help to 1) clarify the effects of root exudate concentration and components on characteristics of dioxygenase gene abundance, diversity, and expression of microorganisms in agricultural soil, 2) identify dioxygenase genes, proteins, and microbial populations which play an important role in degradation of PAH in soil under rhizosphere effects, 3) understand the relationships between PAH degradation and characteristics of soil microbial dioxygenase gene abundance, diversity and expression under rhizosphere effects, 4) interpret plant and microorganism interactions in remediation of PAH contaminated soil. Base on these results, this project can provide support information for PAH degradation enhancement by the rhizospheric effect, and is of significance for further development of PAH contaminated soil remediation.

针对多环芳烃原污染及模拟农田土壤，应用分室根箱实验进行植物-微生物联合修复，应用植物根系分泌物及其组分进行模拟根际修复，并建立根系分泌物-纯培养菌微宇宙，设计针对多种双加氧酶的兼并引物，用qPCR、RT-qPCR、分子克隆测序、双向电泳及飞行时间质谱等手段分析根际效应对微生物双加氧酶多样性、表达及相关蛋白特征的影响机理，探讨多环芳烃根际降解与微生物双加氧酶的关系。项目成果可阐明根系分泌物浓度、组分对农田土壤微生物多种双加氧酶基因多样性数量、组成及表达特征变化规律的影响机理；揭示根际效应作用下对土壤多环芳烃降解起重要作用的双加氧酶基因、蛋白及微生物种类；建立根际效应作用下多环芳烃降解与土壤微生物双加氧酶数量、组成和表达特征间的相关联系；深入认识污染土壤修复中的植物-微生物作用关系。本项目可以为利用根际效应促进多环芳烃的降解提供依据，为多环芳烃污染土壤修复理论与技术的进一步发展奠定基础。

针对多环芳烃污染农田土壤，应用分室根箱实验进行植物-微生物联合修复，应用植物根系分泌物及其组分进行模拟根际修复，并建立根系分泌物-纯培养菌微宇宙，设计针对多种双加氧酶的兼并引物，用qPCR、RT-qPCR、分子克隆测序、双向电泳等手段分析根际效应对微生物双加氧酶多样性、表达及相关蛋白特征的影响机理，探讨多环芳烃根际降解与微生物双加氧酶的关系。成果表明1)三叶草根系分泌物使分枝杆菌加氧酶基因拷贝数明显增加，从而促进了分枝杆菌对多环芳烃的降解；2）大豆/玉米根系分泌物与分支杆菌共同作用，促进了多环芳烃的降解，根系分泌物改变了土壤细菌的群落结构；3）黑麦草根际效应促进了土壤中多环芳烃的降解，提高了细菌的多样性及丰度，60天后革兰氏阳性及阴性菌双氧酶基因表达量增加；4）高羊茅根际效应改变了土壤细菌的群落结构，促进了根基区根际区多环芳烃降解菌的生长；5）紫锥菊，高羊茅，火凤凰和苜蓿都有修复多环芳烃的潜能；在60，120和150天时多酚氧化酶 (火凤凰除外)、脱氢酶(火凤凰除外)和脲酶(紫花苜蓿除外)的酶促反应都比较显著；6）焦化厂土壤中3和4环多环芳烃具有明显的降解作用，焦化厂土壤与农田土壤的降解效果不同；Tenax-TA和固相微萃取都具有预测焦化厂土壤中多环芳烃的生物有效性的潜能，对于污染农田土壤Tenax-TA的预测效果比固相微萃取更精确。该研究成果深入认识了污染土壤修复中的植物-微生物作用关系，为利用根际效应促进多环芳烃的降解提供依据，为多环芳烃污染土壤修复理论与技术的进一步发展奠定基础。