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

几十年来，植物修复被认为是连续降解多氯联苯等难降解氯代芳香族化合物的一种经济有效的方法。已知几种细菌可降解多氯联苯和其他氯化芳香族化合物。编码富营养化Ralstonia (Ralstonia eutropha) NH9中氯儿茶酚二氧合酶的cbnA基因和编码氯戊酸环异构酶的cbb基因催化了许多细菌降解多氯联苯中间化合物氯儿茶酚的重要步骤。它们裂解3-氯儿茶酚的芳香环，产生毒性还原的2-氯戊酸盐和粘内酯。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拟南芥和杂交杨树（Populus tremula x tremuloides）中。分离出转基因系进行后续分析。PCR和Western blot分析表明，这两个基因在拟南芥和杨树细胞中均有整合和表达。采用高效液相色谱法定量检测转基因杨树细胞中氯儿茶酚二氧化酶的活性。转基因拟南芥对氯儿茶酚和甘露酸盐具有抗性。结果表明，cna基因和cbnB基因在杨树细胞和拟南芥细胞中成功表达。由于污染土壤中的污染物主要存在于土壤中，因此引入植物细胞的基因应该在根组织中表达。为了分离根特异性表达的强启动子，我们从桉树中克隆了一个编码磷酸转运蛋白的基因，Northern blot分析表明其表达是根特异性的。

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.