利用转基因杨树修复高分子量多环芳烃污染土壤的研究

Polycyclic aromatic hydrocarbons (PAHs) are widespread toxic contaminants in the environment. With the development of industrialization, the polycyclic aromatic hydrocarbons in a substantial increase in the soil. There are also significant negtively correlated between the benzene ring number and biodegradation, high molecular weight polycyclic aromatic hydrocarbons (HMW)-PAHs are mainly PAH in soil. In view of existing situation that high molecular weight polycyclic aromatic hydrocarbons can not be rapid degradatied, plants were used to enhance absorption and degradation ability in this project. In order to broaden the plants to remedy a range of polycyclic aromatic hydrocarbons in contaminated soil and accelerate the absorption efficiency, the root secretion expression vector of human cytochrome P450 gene and the intracellular expression vector of UDP-glucoside glycosyltransferase gene were constructed. We cloned Mycobacterium dioxygenase gene which could degrade high molecular weight polycyclic aromatic hydrocarbons, dihydrodiol dehydrogenase gene of the benzene ring cleavage and benzene lyase gene. All the codons in these genes were optimized and preferentially designed for plants. Construction of a fusion gene expression vector by foot-and-mouth disease virus 2A fusion was completed. So plants can degrade polycyclic aromatic hydrocarbons directly. In order to achieve deep soil remediation, two kinds of expression vectors were introduced into poplar by co-transformation method. PAHs degradation ability by transgenic poplar in enclosed environment and open soil environment was studied. Degradation products of polycyclic aromatic hydrocarbons in plant were analyzed. The remediation effect of soil and pollution degree was discussed.

多环芳烃是一种广泛存在于环境中的有毒污染物，随着工业化发展，土壤中多环芳烃含量大幅度增加。由于苯环数与生物可降解性呈负相关关系，土壤中多环芳烃以高环为主。本项目针对土壤中高分子量多环芳烃不能快速降解的难题，利用基因工程加强植物对它们的吸收和降解能力。首先构建人细胞色素P450基因根分泌表达和UDP-葡萄糖苷糖基转移酶基因胞内表达载体，拓宽植物对多环芳烃污染土壤修复范围，加快吸收效率；然后克隆能够降解高分子量多环芳烃分枝杆菌的双加氧酶系统基因，苯环裂解所需的二氢二醇脱氢酶和苯环裂解酶基因，将这些基因按植物偏爱密码改造，并通过口蹄疫病毒2A区融合，构建融合基因表达载体，使植物能直接降解多环芳烃。为了实现深层土壤修复，将两种表达载体通过共转化方法转化杨树。研究转基因杨树在封闭环境与开放土壤环境下对多环芳烃的降解能力，分析多环芳烃在植物中的降解产物，探讨土壤性质、污染程度对修复的影响。

多环芳烃是一种广泛存在于环境中的有毒污染物，随着工业化发展，土壤中多环芳烃含量大幅度增加。本项目针对土壤中高分子量多环芳烃不能快速降解的难题，利用基因工程加强植物对它们的吸收和降解能力。首先构建了人细胞色素P450基因根分泌表达和UDP-葡萄糖苷糖基转移酶基因胞内表达载体，拓宽植物对多环芳烃污染土壤修复范围，加快吸收效率；然后克隆了能够降解高分子量多环芳烃分枝杆菌的双加氧酶系统基因，苯环裂解所需的二氢二醇脱氢酶和苯环裂解酶基因，并将这些基因按植物偏爱密码改造，并通过口蹄疫病毒2A区融合，构建融合基因表达载体，使植物能直接降解多环芳烃。分别将表达载体电击转化农杆菌GV3101和EHA105。通过蘸花法遗传转化拟南芥获得15株以上的转基因拟南芥。通过农杆菌介导转化杨树，分别获得20株以上的转基因杨树。转基因技术的主要目的是通过优良基因实现对植物的定向改良。但由于技术上的限制和对基因功能认识的不足,转基因植物常常表现一些生长发育异常的性状。原因可能与基因的类型、结构以及外源基因在基因组中整合的位点、调控下游的靶基因种类等多种因素有关。今后有必要进行更深入的研究。而本研究将人单加氧酶基因CYP1A1和UDP-葡萄糖苷糖基转移酶基因PtUGT72B1成功转化拟南芥和杨树。双价基因转化植物的生长情况明显好于只转化CYP1A1的植物，耐多环芳烃的能力得到了改善。从以上试验中可以总结出以下几点：1）转入CYP1A1植株能够有效分解高分子量多环芳烃；2）只转化UDP-葡萄糖苷糖基转移酶PtUGT72B1的植株不能直接转运高分子量多环芳烃，使转基因植株体内多环芳烃的含量与非转基因植株相当。3）转入CYP1A1和PtUGT72B1双价基因后，双价基因转化植株的生长情况明显好于只转化CYP1A1的植株。本项目发表SCI论文2篇，培养博士和硕士3名。