Degradation of phenolic compounds by transgenic trees

利用转基因树木作为植物修复手段，被认为是连续降解二恶英等难降解氯代芳香族化合物的经济有效途径之一。细菌基因cbnA编码氯儿茶酚二氧化酶，cbb编码嗜酸Ralstonia eutropha NH9氯多酚环异构酶，催化氯儿茶酚降解的重要步骤之一，裂解3-氯儿茶酚的芳香环，生成毒性还原的2-氯多酚酸酯和多酚内酯。本研究在CaMV 35S启动子和增强子的控制下构建了转录cbnA和cbnB基因的双载体pCAMBIA-E7131-cbnA-cbnB，并通过农杆菌介导的转化将其引入水稻和杂交杨树（Populus tremula x tremuloides）中。聚合酶链反应（PCR）证实了该基因整合到它们的染色体上，并分离出转基因系进行后续分析。Western blotting检测cbnA积累和cbb翻译，HPLC检测氯儿茶酚二氧化酶活性，GC含量高达65%。当杨树转基因愈伤组织在1 mM的3-氯儿茶酚中孵育2 h时，降解产物2-氯戊酸酯峰信号增加，3-氯儿茶酚底物峰信号降低。结果表明，cbnA基因和cbnB基因在水稻和杨树中成功表达。这些转基因植物将用于环境污染物的植物修复，如氯代芳香族化合物。由于受污染的化学物质大多存在于土壤中，因此植物对污染土壤中的污染物进行修复时，引入植物细胞的基因必须在根组织中表达。为了在根组织中表达基因，需要有强启动子来促进根的特异性表达。为了分离这些类型的启动子，我们选择了拟南芥的PHT1启动子。水稻和拟南芥中PHT1启动子的GUS报告子分析表明，该启动子可用于单子叶和双子叶植物根组织中基因的表达。少

Utilization of transgenic trees as phytoremediation is considered to be one of the cost-effective ways for successive degradation of chlorinated aromatic compounds such as dioxins which are resistant to degradation for decades. Bacterial genes cbnA encoding chlorocatechol dioxigenase and cbnB encoding chloromuconate cycloisomerase from Ralstonia eutropha NH9, catalyzes one of the important steps for degradation of chlorocatechols and cleaves the aromatic ring of 3-chlorocatechol to produce toxically reduced 2-chloromuconate and muconolactone.In this study, we constructed a binary vector pCAMBIA-E7131-cbnA-cbnB transcribing cbnA and cbnB gene under the control of CaMV 35S promoter with enhancers, and introduced to rice and hybrid poplar (Populus tremula x tremuloides) by Agrobacterium mediated transformation. Integration of the gene to their chlomosomes was confirmed by PCR and the transgenic lines were isolated for subsequent analyses. Accumulation of cbnA and cbnB translation was visu … More alized by Western bloting and chlorocatechol dioxigenase activity was quantitatively detected by HPLC assay despite its high GC content (65%). When poplar transgenic calli was incubated for 2 hrs in the presence of 1 mM of 3-chlorocatechol, increase of a degradation product 2-chloromuconate peak signal was observed in parallel with reduced peak of 3-chlorocatechol substrate. The results showed that the cbnA gene and cbnB gene were successfully expressed in rice and poplar tree. These kinds of transgenic plants will be used for phytoremediation of environmental pollutants such as chlorinated aromatic compounds.For phytoremediation of pollutants in contaminated soil, the genes intorduced into plant cells should be expressed in root tissues since most of the contaminated chemicals are in soil. To express the genes in root tissues, strong promoters for root specific expression are necessary. To isolate these kinds of promoters, we selected the PHT1 promoter of Arabidopsis thaliana. GUS reporter analysis of PHT1 promoter in rice and Arabidopsis indicated that this promoter is useful for gene expression in root tissues both in monocot and dicot plants. Less