Expression, Metal-ion Tolerance Of Rice Metallothionein Gene In Escherichia Coli And Molecular Cloning Of Metallothionein Gene Family In Rice

Metallothioneins (MTs) is a low molecular weight, cysteine-rich, and heavy metal-binding protein. MTs are extensively distributed in eukaryotes including the vertebrates, invertebrates, plants, and microorganisms, as well as in some prokaryotes. The widespread occurrence of MTs in nature suggests that they serve an important biological function, although their precise role remains to be investigated further. Most attention has centered on detoxification of heavy metals by sequestering excess amounts of certain metal ions. MTs have been shown to associate with high affinity with many metal ions, notably cadmium, zinc and copper, all of which are significant environmental pollutants. Therefore, the expression of metallothionein may provide a generic strategy for enhancing tolerance and accumulation of toxic heavy metal ions and may therefore find application in the engineering of bioremediating organisms.In order to examine the metal-binding characteristics of rice metallothionein, an Escherichia coli strain BL21 was used to express ricMT and its two putative functional domains (a and P fragment). A constructed expression plasmid, pGEX-4T-l, in which rice metallothionein and its two potential domains were inserted as a fusion to the carboxyl terminal of Glutathione-S-Transferase (GST) respectively, was transformed BL21. The fusion protein was highly soluble in the cytoplasm of E.coli. Enhanced tolerance to cadmium was observed in the transformed cell with ricMT and its P domain (ricMTp), while enhanced tolerance to zinc was found in the transformed cell with its a domain (ricMTa); When grown in the presence of copper, cells expressing ricMT, ricMTa and ricMTp all showed increased viability compared with control cells; On the other hand, when grown in the presence of lead, only cell transformed with ricMT showed increased viability.The results of atomic absorption spectrum analysis of all metal ions accumulation in these cells consisted with their tolerance to certain metal ions. It was demonstrated mat rice metallothionein functioned for cadmium, zinc, copper and lead detoxification in E. coli.Besides, RT-PCR method was used to clone the cDNAs of rice MT gene family. With the bioinformatics methods, we attempt to find putative essential functional motif, the related transcription factors and crucial clues for a revised classification method. Here we have cloned and classified the metallothionein gene family of rice, which is important to the analysis of the expression pattern by Northern blot in the future.