| Metallothioneins are a kind of widely distributed and low molecular weight cysteine-rich proteins,which were found to play an important role in heavy metal tolerance.Tartary buckwheat(Fagopyrum tataricum)is an important medicinal and edible crop and shows tolerance to barren,heavy metals and other environmental stress.But up to now,there was no report about the mechanism of heavy metal tolerance,and the expression regulation and function of metallothionein in tartary buckwheat.In this study,a cDNA fragment corresponding to the type 2 metallothionein(FtMT2)coding region was isolated from F.tataricum leaf.FtMT2-overexpressed Escherichia coli,S.cerevisiae and transgenic Arabidopsis thaliana were constructed in order to investigate the function of FtMT2.The hydroxyl free radical removal ability of the recombinant FtMT2 protein was also tested.In order to clarify the response of FtMT2 to Cu2+ stress,the promoter sequence of FtMT2 was cloned and its 5′ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F.tararicum.Functional characterization FtMT2 promoter provided the regulatory mechanism for establishment the method for copper contamination monitoring and underlying basis for further revealing the molecular mechanisms of tartary buckwheat to heavy metal resistance.The following results and conclusions have been made in this study:(1)Three exons contained 62,84 and 91 coding bases,comparing to the sequence of F.tararicum MT2 cDNA(GenBank No.KY643823),and two introns were consisted of 185 and 96 bp.All the intron borders started and ended with the consensus GT and AG splicing signals.Amino acid sequence analysis showed that the FtMT2 had 14 cysteine(17.9 %)residues amongst 78 amino acids,with a calculated molecular mass of 7.87 kD and contained the cysteine-rich domains with the presence of C-C,C-X-C,and C-X-X-C(X was other amino acids other than cysteine)motifs at amino-terminus and C-X-C motifs,and also had a highly conserved domain ‘MSCCGGNCGCGS’.Sequence and homology analysis showed that the FtMT2 protein sequence shared high homology with other plant type 2 MT-like proteins,and clustered with the Sesbania drummond.(2)The results of growth curve and plate colony analysis showed that the overproduction of recombinant FtMT2 improves the tolerance on Cu2+ in the Escherichia coli cells and Cd2+ in the Saccharomyces cerevisiae,respectively.(3)The recombinant Ft MT2 showed a hydroxyl radical scavenging activity with a IC50 of 46.5 μg/m L.(4)2.1 kb upstream sequence of FtMT2 was isolated by genome walking.There are some cis-acting elements involved in stress resistance in the promoter region predicted by bioinformacics,including ABRE(methyl jasmonate responsive element),HSE(heat stress responsive element),and three light response elements(‘ATCT-motif’,‘TCT-motif’ and ‘GATA-motif’).Especially,there are three MRE-like sequence elements ‘5′-TGCAG-3′’,highly conserved in the promoter sequence of animal metallothionein gene.(5)The 2.1 kb fragment and its 5′ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F.tararicum.The promoter activities of all thefragments were up-regulated and the full length promoter showed strongest inducibility in response to Cu2+ treatment.2.1 kb promoter activities were up-regulated for 6.38 fold,1.6 kb 6.15 fold,1.4 kb 3.01 fold and 0.98 kb 1.81 fold,indicated that the predicted metal response element may have a dose effect,and there are other important metal responsive elements in the promoter sequence that are responsive to the treatment of Cu2+.The plant expression vector of 1.6 kb promoter and MRE mutant-gus reporter gene was successfully constructed,and the T1 transgenic Arabidopsis plants were screened,which laid a foundation for determining the metal response elements on FtMT2 promoter sequence.At the same time,this experiment provided a report system for heavy metal detection.(6)The pCAMBIA3301-bar plasmid containing the gene encoding FtMT2 under the control of a CaMV35 S promoter was employed for Agrobacterium-mediated transformation of A.thaliana inflorescence by using a modified floral-dip method.The basta-resistant plants were then confirmed by PCR detection,which laid the foundation for futher study of the biological function of Ft MT2 in plants.The transient expression of GFP protein was carried out by Agrobacterium tumefaciens injection,using tartary buckwheat leaves as materials.In this experiment,it is proved that it is feasible to use the method of for instantaneous expression in tartary buckwheat,which provides a new way to study the transient expression of buckwheat gene. |
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