Metabolic engineering of photorespiration by transforming multiple genes into chloroplasts of rice and potato
Boran Shen, Zhen Yao, Han He, Xinxiang Peng*
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
*Corresponding author: Tel: +86-20-85282023 or 85281870 Fax: +86-20-85282023;Email：firstname.lastname@example.org
Environmental stresses, such as drought and high temperature, are increasingly limiting factors for rice production, thus generating resistant engineering plants is of practical significance. Our project is to create a shortcut pathway for photorespiration inside the chloroplast of rice, and such modification would lead to a direct release of the photorespiratory CO2 within the chloroplast thereby forming a novel CO2 concentrating mechanism. Three genes, i. e. glycolate dehydrogenase (GDH) from Synecocystis sp, glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) from E.coli, were fused to the rice rbcS chloroplast targeting peptide (TP) at N-term and cloned into a multiple gene expression vector pYL1305. The constructed plasmid was then transformed into rice and potato. Eight independent lines of rice were obtained, and RT-PCR analysis showed that the three genes were appreciably expressed at the mRNA level. However, for the time being the proteins and enzymatic activities failed to be detected. By using the protoplast transient expression technology, fusion proteins, i. e. GFP-TP, TP-GDH-GFP, TP-GCL-GFP, TP-TSR-GFP, were respectively transformed into rice leaf protoplasts. Fluorescence observation demonstrated that, while GFP-TP could be well localized into the chloroplast, the other fusion genes were expressed mainly outside the chloroplast. The results implicate that rice may be able to prevent heterologous proteins from getting into the chloroplast. We are currently focusing on understanding the possible mechanisms.
Key Words: multigene transformation; photorespiration; chloroplasts; rice; potato