MERCURY DETOXIFICATION USING GENETIC ENGINEERED NICOTIANA TABACUM

摘要

Toxic metal contamination of soil, aqueous waste streams and groundwater poses a major environmental and human health problem, all of which are still in need for effective and affordable remediation methods.Phytoremediation is a low cost alternative solution to soil contamination compared with traditional removal and/or disposal techniques. It has been proposed to remove excess metals from soils. Plants with both high metal uptake and high biomass production are needed to extract metals from soils within a reasonable time frame. One of the phytoremediation technologies is the phytovolatilization, whereby the contaminant is not primarily accumulated in above-ground tissues, but is instead transformed by the plant into the atmosphere. Plant by genetic modifications for improving metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. The detoxification of highly toxic organmercurial compounds and subsequent volatilization of elemental mercury is a unique example for the successful phytoremediation based on genetic engineering approach. For mercury removal techniques we constructed a dicistronic construct containing the bacterial mercury detoxification genes merA and merB under the control of the Arabidopsis Actin2 promoter and terminator. The merA/merB dicistronic construct is made typically with merB sequence in the first position, merA sequence in the second position and the Human eukaryotic initiation factor 4G (elF4G) IRES sequence in the intercistronic region which allows the ribosome to bind and translate the merA gene. We confirmed the presence of the three gene sequences by restriction analysis of small scale preparation of plasmid DNA. The resulted construct was introduced to Agrobacterium competent cells using heat shock transformation method. In the mean time, we germinated tobacco (Nicotiana tabacum var. Gold leaf) seeds on MS media and infected leaf discs of 6-8 weeks tobacco seedlings with Agrobacterium containing Ti plasmid harboring merA/merB dicistronic construct. We confirmed the integration of the merA/merB genes in the plant genome by amplifying merB gene using polymerase chain reaction (PCR) technique. The DNA samples isolated from tobacco wild type (WT) or transformed seedlings are used as a template and the merB specific primers {merBRcaSI and merB207A) were used in this experiment. The results showed that about 90% of tobacco seedlings were carrying the mer genes. These seedlings were allowed to grow in the green house to produce seeds. Seeds were collected from wild type and transgenic lines and tested for mercury resistance. The results showed that transgenic plants are resistant to both Phenyl mercuric acetate (PMA) and HgCl_2 The root length and dry weight of wild and transgenic seedlings growing on both media amended with mercury compounds and media without mercury (control) were scored. The results showed that the root lengths and dry weight of the transgenic lines are significantly higher by 60 and 17-folds, respectively, compared to wild type. The results showed clear evidence that the transgenic plants are resistant to both organic and inorganic mercury compounds and can be used to clean up mercury contaminated sites.