Cloning Of Metallothionein Genes From Arachis Hypogaea L. And Characterization Of AhMT2α

Heavy metal toxicity is one of the major problems in modern agriculture, and much attention has been given lately to heavy metal tolerance and detoxification mechanism of plants, cloning and characterization of heavy metal metabolism-related genes, and phytoremediation by which to repair soil and water polluted by heavy metals. Metallothioneins (MTs) are generally thought to role in heavy metal tolerance and detoxification, metal homeostasis, essential metal storage, participation in stress responses, developmental regulation, and ROS (reactive oxygen species) scavenging. A few cases have been reported that MT-overexpressing plants exhibited enhanced metal tolerance and accumulation. With peanut (Arachis. hypogaea L. cv. Luhua 14), we isolated 3 type 2 MT-encoding cDNAs using RT- PCR, and cloned 1 type 2, 1 type 3, and 2 type 4 MT-encoding cDNAs by construction of cDNA library and large scale sequencing. We selected AhMT2a (DQ178616) to characterize its functions primarily by detecting its expression patterns in the presence of different metals and many other stress conditions, determinating heavy metal tolerance and accumulation of E. coli and Arabidopsis thaliana overexpressing AhMT2a. The results were shown as follows: 1. Four type 2 MT-encoding genes have higher homology in the sequences of nucleotides and their deduced amino acids with 80 or 81 residues, AhMT2a and AhMT2b have an identical deduced amino acid sequence, and there is only one amino acid residue substitution between AhMT2a and AhMT2c. AhMT3a containing 66 amino acid residues has two cysteine-rich domains as those type 2 MTs. AhMT4a and AhMT4b encode 82 amino acids, in which of them 17 are Cys residues, and their protein sequences differ from other peanut MTs by having three cysteine-rich domains. 2. The results of atomic absorption spectrophotometry for determination of metal contents in peanut tissues treated for 15 d with 300μmol/L CdCl2, 200μmol/L ZnSO4 and 500μmol/L CuSO4, respectively, show that cadmium and copper accumulated mainly in roots, while the maximum zinc content was found in leaves. The content of cadmium in peanut roots is about five- and eight-fold of that in stems and leaves, respectively. The higher levels of copper and cadmium in roots corresponded well with significant enhancement of AhMT2a mRNA transcript in roots, suggesting that AhMT2a protein might detoxify copper and cadmium in roots mainly.3. We determined the spatial distribution of AhMT2a expression and its expression pattern in the presence of various stresses by northern blotting analysis. MTs are encoded by a small gene family, and there are at least four type 2 MT members in peanut, so we can't exclude the possibility of cross hybridization of other type 2 MT genes in northern analysis. The expression level in old leaves was much higher than that in young stems, a very weak hybridization signal was detected in young leaves, but no hybridization signal was detected in mature roots. The above results indicate less restricted tissue-specificity, and is not in accord with what has been generally found for type 2 plant MTs which show a higher expression in shoots than in roots.To investigate whether AhMT2a gene expression was regulated by heavy metals and other exogenous factors, northern blot analysis was performed. Treatments with 500μmol/L CuSO4 or 300μmol/L CdCl2 stimulated the expression of AhMT2a in roots and leaves, indicating that AhMT2a may function in the metabolism and detoxification of cadmium and copper, and maybe different MTs play specific roles in different metals. When treated with 120μmol/L ABA or 42℃for 6 h,mRNA accumulation of AhMT2a in leaves and roots increased. Seven mmol/L H2O2 induced the expression of AhMT2a gradually. Furthermore, the expression of AhMT2a gene in leaves can also be induced by 200 mmol/L NaCl when treated for 24 h, but mRNA accumulation in roots exhibited no obvious change. On the basis of above results, we propose that AhMT2a protein not only function in detoxifying heavy metals, but might also be involved in scavenging of ROS.4. In the present study, we constructed a prokaryotic expression vector, pET-AhMT2a, and transformed it into E. coli. The purified protein and total cell extracts were analyzed by 15% SDS-PAGE, and AhMT2a fusion protein showed an expected relative molecular mass of ¹1 kDa. E. coli expressing AhMT2a exhibited enhanced tolerance to cadmium, copper and lead significantly, and metal accumulation in bacteria was also higher than the control. Copper accumulation was approximately 5.7-fold greater than that in cells containing the empty vector pET28a(+), cadmium and lead accumulation by E. coli expressing recombinant AhMT2a was about 2-fold greater than that in control cells. These results indicate that AhMT2a might play significant roles in copper detoxification.5. We introduced pROK-AhMT2a into Arabidopsis thaliana and obtained 26 transformants. Seed germination of transgenic plants exhibited higher tolerance to cadmium and lead than the control, but its tolerance to copper was not obvious. When treated with Cd²⁺, Cu²⁺ and Pb²⁺, respectively, seedlings of transgenic plants showed increased tolerance than the control. Furthermore, we determined total chlorophyll content, MDA, and Fv/Fm in transgenic and wide type Arabidopsis plants treated with Cd. The results showed that the expression of AhMT2a gene in A. thaliana enhanced tolerance to Cd²⁺. Because there are more than 0.7% MT-encoding ESTs in the cDNA library constructed with peanut seeds collected from different developmental stages, we suppose that AhMT2a may also be related with the regulation of seed development.The innovations of this dissertation can be summarized as follows:1. It was the first time to isolate MT genes from peanut using RT-PCR and cDNA library. We obtained 3 types, 7 members of peanut MT-encoding cDNA.2. We characterized the functions of AhMT2a gene. Northern analysis showed that the expression of AhMT2a was induced by Cu²⁺, Cd²⁺, ABA, heat shock, H2O2, and NaCl, indicating that AhMT2a may be related with metal detoxification and scavenging of ROS.3. We constructed prokaryotic expression vector, and transformed it into E. coli. The transformants displayed higher tolerance to Pb²⁺,Cd²⁺, and Cu²⁺, and metal accumulations were also higher than that in control. Moreover, we introduced AhMT2a gene into A. thaliana, seed germination and seedling growth of transgenic lines of T3 showed enhanced tolerance to Pb²⁺, Cd²⁺, and Cu²⁺ than wide type plants.All results demonstrated that AhMT2a may be involved in heavy metal detoxification and ROS scavenging. This work will contribute to a better understanding of plant MTs and help to further analyze the functions of these genes.