Functional Analysis Of OsMT1e In Rice（Oryza Sativa）for Detoxification Of Excess Heavy Metals

With the development of industrialization and urbanization,the use of pesticides and fertilizers has escalated the concen about heavy metal contamination.Heavy metal pollution in soil has therefore necessitated urgent attention in countries around the world.Heavy metals in soil mainly include chromium,lead,mercury,arsenic,copper and cadmium.Cadmium（Cd）is one of the most important pollutants in agricultural soil.When plants are grown in the environment contaminted with Cd,it will cause damage to plant cells and reduce plant growth,and accumulate in the grains that seriously threaten human health.Metallothionein plays an important role in chelating metals,detoxifying metals and maintaining the homeostasis of metal ions in cells.In this study,we cloned the unidentified OsMT1e gene in rice and performed a functional analysis of it.Functional studies showed that OsMT1e can increase the tolerance of rice to Cd and Cu by chelating the metals.OsMT1e belongs to the metallothionein family.It has a highly conserved cysteine sequence at the N-terminus and C-terminus of the polypeptide chain,and has a Pfam Metallothio-2 domain.The qRT-PCR results showed that OsMT1e was highly expressed in the roots of rice during the whole growth cycle;OsMT1e was hardly expressed in other tissues except rice roots during the flowering period（14 weeks）.At the same time,the expression of OsMT1e was induced by high concentrations of Cd,Cu,Zn,Mn,and Fe,and the induction in root was prominent.Subcellular localization of tobacco leaf epidermal cells transformed with P35S-OsMT1e-GFP showed that OsMTle was abundantly expressed in the nucleus and cytoplasm.We transferred OsMT1e to yeast mutants ycf1,cup2,zrc1,pcr1,and ccc1,which are sensitive to Cd,Cu5 Zn,Mn,and Fe,respectively.We found that heterologous expression of OsMT1e can rescued the growth of yeast mutants ycfl and cup2 on medium containing excess Cd,Cu and accumulated more Cd and Cu in the cells,but heterologous expression of OsMT1e could not improve the tolerance of yeast mutants zrc1,pmr1,and ccc1 to Zn,Mn,and Fe,respectively.In order to further study the function of OsNf1e,we constructed OsMT1e RNAi and overexpressing transgenic lines.Under excess Cd and Cu stress,RNAi lines were more sensitive to Cd and Cu compared to the wild-type plants.The specific manifestations of RNAi lines include short plants height,reduced dry weight and the leaves are curled and yellowed.OsMT1e overexpressing lines increased the tolerance to Cd and Cu.Furthermore,When treated with excess Zn,Mn,and Fe,overexpressing OsMT1e in rice could not improve the tolerance to Zn,Mn,and Fe.At the same time,we analysis of the metal content of transgenic lines and wild-type,the results revealed that RNAi lines accumulated lower leves of Cu and Cd in shoot and root compared to WT,in contrast,overexpression lines recorded higher amont of Cd and Cu than WT.In summary,OsMTle can increase the tolerance of rice to Cd and Cu by chelating both metals.