Poster: Phytoremediation

Abs # 978: Molecular breeding of transgenic plants expressing chlorocatechol dioxigenase gene

Presenter:	Ono, Toshiro , to817@aqua.ocn.ne.jp
Authors	Ono, Toshiro  (A)   Ohmiya, Yasunori  (B)   Kimura, Tetsuya  (A)   Ogawa, Naoto  (C)   Miyashita, Kiyotaka  (C)   Sakka, Kazuo  (A)   Ohmiya, Kunio  (A)
Affiliations:	(A): Faculty of Bioresources, Mie university (B): Forest Tree Breeding Center (C): National Institute for Agro Environmental Sciences

Phytoremediation is considered to be one of the cost-effective ways for successive degradation of enrichmental pollutants such as chlorinated aromatic compounds which are resistant to degradation for decades. Chlorocatechols are key intermediates in microbial degradation pathway of these chlorinated aromatic compounds.Degradation of chlorcatechols is a rate limiting step of chlorinated aromatic compounds.Ralstonia eutropha NH9 has the cbnA encoding gene chlorocatechol dioxigenase which catalyzes the cleavage of aromatic ring of 3- chlorocatechol to produce toxically reduced 2-chloromuconate.In this study, we constructed a binary vector pCAMBIA-E7131-cbnA transcribing cbnA 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 chromosomes was confirmed by PCR and positive transgenic lines of rice and poplar were analyzed further. Accumulation of CbnA protein was visualized by Western blotting and chlorocatechol dioxigenase activity was quantitatively detected by HPLC assay. When using rice and poplar transgenic calli were incubated 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. These results indicated that cbnA was efficiently expressed in the transgenic rice and poplar.