Expression of multiple genes encoding bacterial enzymes for phytoremediation in transgenic tree

植物修复被认为是连续降解氯代芳香族化合物(如多氯联苯)的经济有效的方法之一，这些化合物几十年来一直不能降解。已知有几种细菌可以降解多氯联苯和其他氯化芳香化合物。编码氯代儿茶酚二氧酶的cbnA基因和编码富营养罗氏菌NH9的氯代肉豆酸环异构酶的cbnB基因，催化了许多细菌降解多氯联苯中间体氯辛二酚的重要步骤。它们裂解3-氯代儿茶酚的芳香环，生成有毒还原的2-氯代丁酸和木香内酯。本研究在含有增强子的CaMV 35S启动子的控制下，构建了转录CBNA和cbnB基因的双元载体pCAMBIA-E7131-CBNA-cbnB，并通过农杆菌介导法转化拟南芥和杂交杨。转基因株系被分离出来用于随后的分析。聚合酶链式反应和蛋白质印迹分析表明，这两个基因在拟南芥和杨树细胞中均得到整合和表达。用高效液相色谱法对转基因杨树细胞中的氯代儿茶酚二加氧酶活性进行了定量检测。转基因拟南芥表现出对氯代儿茶酚和芥子酸的抗性。结果表明，CBNA基因和cbnB基因在杨树细胞和拟南芥中得到了成功的表达，对于污染土壤中污染物的植物修复，由于大多数污染化学物质都在土壤中，因此导入植物细胞的基因应该在根组织中表达。为了筛选根特异表达的强启动子，我们克隆了桉树磷酸转运蛋白的编码基因，Northern印迹分析表明其表达是根特异的。

Phytoremediation is considered to be one of the cost-effective ways for successive degradation of chlorinated aromatic compounds such as PCB which are resistant to degradation for decades. Several bacteria are known to degrade PCB and other chlorinated aromatic compound. The cbnA gen encoding chlorocatechol dioxigenase and the cbnB gene encoding chloromuconate cycloisomerase from Ralstonia eutropha NH9, catalyze the important steps for degradation of chlorocatechols which is intermediate compounds from PCBs by many bacteria. They 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 Arabidopsis and hybrid poplar (Populus tremula x tremuloides) by Agrobacterium mediated transformation. Transgenic lines were isolated for subsequent analyses. PCR and Western blot analysis indicated that both genes were integrated and expressed in Arabidopsis and poplar cells. Chlorocatechol dioxigenase activity was quantitatively detected by HPLC assay in transgenic poplar cells. Transgenic Arabidopsis showed resistance to chlorocatechol and muconate. The results showed that the cbnA gene and cbnB gene were successfully expressed in poplar cells and Arabidopsis..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 isolate strong promoters for root specific expression, we cloned the gene encoding a phosphate transporter from Eucalyptus and Northern blot analysis indicated that its expression is root specific.