| Heavy metal pollution has become a serious problem affecting human health. Phytoremediation technology has been widely accepted, which consists of two important elements:mechanism of hyperaccumulation and the feasibility assessment of the application of phytoremediation on the contaminated sites. Mechanisms of hyperaccumulation investigate the absorption, transport and transformation of heavy metal pollutants, including the molecular and biochemical research on cloning key genes and analysizing their functions in hyperaccumulation; while the other element is to find out how to apply the findings to practice, such as construction of transgenic plants from well-known functional genes to apply in phytoremediation. This research consists of two parts:the first part foucused on cloning a gene TcOPT3from a hyperaccumulator Thlaspi caerulescens and investigated its founciton of heavy metals transportation; and the second part evaluating the feasibility in using the transgenic tobaaco to express a citrate synthase for heavy metal pytoremediation.Thlaspi caerulescens is a naturally selected heavy metal hyperaccumulator that not only can tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation of heavy metals is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. We cloned and characterized a novel gene TcOPT3of OPT family from Thlaspi caerulescens. TcOPT3was primarily expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3expressed in the plant vascular systems, especially in the pericycle cells may be involved in the long-distance transportation of the metals. Sub-cellular localization showed that TcOPT3was a plasma membrane-localized protein. The expression of TcOPT3was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Furthermore, heterogonous expression of TcOPT3by mutant yeast (Saccharomy cescerevisiae) complementation experiments demonstrated that TcOPT3could transport Fe2+and Zn2+. Moreover, expression of TcOPT3in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). Our data demonstrated that TcOPT3might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate.This is the first report showing that TcOPT3may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.Citrate acids, which are abundant in plants, serve as potential chelators for heavy metal detoxification by forming complexes with the pollutants in vitro. The role of citrates in plant resistance to Pb was investigated using a transgenic tobacco (Nicotiana tabacum) line, CS42, overexpressing a rice (Oryza sativa L.) citrate synthase (CS) gene, OsCS1. The CS42plant showed enhanced resistance to Pb treatment both in agar and hydroponic media. Compared with the EV30plant that was transformed with empty vectors, the CS42plant had a higher CS activity (both with and without Pb treatment) and released more citrates from roots. An exogenous supply of citrate to the wild-type tobacco also enhanced plant resistance to Pb. When cultured in a hydroponic system, the Pb concentration of the CS42plant decreased in the roots, but increased in the shoots. Hence, the translocation rate of Pb increased from root to shoot. Overall, CS gene overexpression improved plant resistance to Pb stress by increasing the root citrate efflux and root-to-shoot Pb translocation. These traits may be potentially useful in the phytoremediation of a Pb-polluted environment. |
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