Expression of synthetic Phytochelatin EC20 in E. Coli increases its biosorption capacity and cadmium resistance / Expressão de Fitoquelatina EC20 sintética em E. Coli aumenta sua capacidade de biossorção e resistência ao cádmio

In this study E. coli recombinant clones that express the EC20 synthetic phytochelatin intracellularly were constructed. The increasement of Cd2+ biosorption capacity, and, also, the increasement of resistance to this toxic metal were analyzed. A gene that encodes the synthetic phytochelatin EC20 wassynthesized in vitro. The EC20 synthetic gene was amplified by PCR, inserted into the DNA cloning vectors pBluescript®KS+ and pGEM®-TEasy, and also into the expression vectors pTE [pET-28(a)® derivative] and pGEX-T4-2®. The obtained recombinant plasmids were employed for genetic transformation of E. coli: pBsKS-EC20 and pGEM-EC20, they were introduced into DH10B and DH5α strains, similarly to pTE-EC20 and pGEX-EC20 that were introduced into BL21 strain. The EC20 expression was confirmed by SDS-PAGE analysis. The recombinant clones' resistances to Cd2+ were determined by MIC analyses. The MIC for Cd2+ of DH10B/pBKS-EC20 and DH10B/pGEM-EC20 were 2.5 mM (EC20 induced), and 0.312 mM (EC20 repressed);respectively, 16 and 2 times higher than the control DH10B/pBsKS (0.156 mM). The MIC for Cd2+of BL21/pTE-EC20 was 10.0 mM (EC20 induced) and 2.5 mM (EC20 repressed), compared with the control BL21/pTE which was only 1.25 mM. Analysis of ICP-AES showed that BL21/pGEX-EC20, after growth on the condition of EC20 expression, absorbed 37.5% of Cd2+, and even when cultured into the non-induction condition of EC20 expression, it absorbed 11.5%.These results allow the conclusion thatrecombinant E. coli clonesexpressing the synthetic phytochelatin EC20 show increased capacity for Cd2+ biosorption and enhanced resistance to this toxic ion.

Foram construídos clones recombinantes de E. coli que expressam intracelularmente a fitoquelatina sintética EC20. Foi analisado o aumento na capacidade de biossorção de Cd2+ e o aumento da resistência a este metal tóxico.Foi sintetizado in vitro um gene codificante da fitoquelatina sintética EC20. O gene EC20 sintético foi amplificado por PCR, inserido nos vetores de clonagem pBluescript®KS+ e pGEM®-TEasy, e nos vetores de expressão pTE [derivado de pET-28(a)®] e pGEX-T4-2®. Os plasmídeos recombinantes foram empregados na transformação genética de E. coli: pBsKS-EC20 e pGEM-EC20 foram introduzidos nas linhagens DH10B e DH5α; e, pTE-EC20 e pGEX-EC20 na linhagem BL21-DE3. A expressão EC20 foi analisada por SDS-PAGE. As resistências a Cd2+ dos clones recombinantes foram determinadas por análises de MIC.A MIC para Cd2+ de DH10B/pBsKS-EC20 e de DH10B/pGEM-EC20 foi 2,5 mM (EC20induzido) e 0,312 mM (EC20 reprimido); respectivamente, 16 e 2 vezes superiores às do controle DH10B/pBsKS (0,156 mM). A MIC para Cd2+ de BL21/pTE-EC20 foi 10,0 mM (EC20 induzido) e 2,5 mM (EC20 reprimido), comparado a do controle BL21/pTE que foi apenas 1,25 mM. A análise de ICP-AES mostrou que BL21/pGEX-EC20, após crescimento na condição de expressão de EC20, absorveu 37,5% de Cd2+e, mesmo quando cultivado na condição de não-indução de expressão EC20, absorveu 11,5% de Cd2+. Estes resultados permitem a conclusão de que os clones recombinantes de E. coli que expressam a fitoquelatina sintética EC20 apresentam aumento da capacidade de biossorção de Cd2+ e de resistência a este íon tóxico.

Copper stress induces antioxidant responses and accumulation of sugars and phytochelatins in Antarctic Colobanthus quitensis (Kunth) Bartl

BACKGROUND: In field, C. quitensis Is subjected to many abiotic extreme environmental conditions, such as low temperatures, high UV-B, salinity and reduced water potentials, but not metal or metalloid high concentrations in soil, however, other members of Caryophyllaceae family have tolerance to high concentrations of metals, this is the case of Silene genre. In this work, we hypothesize that C. quitensis have the same mechanisms of Silene to tolerate metals, involving accumulation and induction of antioxidant systems, sugar accumulation and the induction of thiols such as phytochelatins to tolerate. RESULTS: The results showing an effective antioxidant defensive machinery involving non-enzymatic antioxidants such as phenolics, GSH and ascorbic acid, in another hand, GSH-related oligomers (phytochelatins) and sugars was induced as a defensive mechanism. CONCLUSIONS: Colobanthus quitensis exhibits certain mechanisms to tolerate copper in vitro demonstrating its plasticity to tolerate several abiotic stress conditions.

An Overview of Carcinogenic Heavy Metal: Molecular Toxicity Mechanism and Prevention

Almost all heavy metals are serious toxicants as carcinogens. However, due to their chemical and physiological properties, heavy metals are useful in industrial areas including alloy, smelting and production of commercial products. Such applications increase the opportunity for heavy metal exposure. Waste from industrial processes is also a major source of environmental contamination and accumulation in the human body. Arsenic, cadmium, chromium, and nickel are classified as group 1 carcinogens by the International Agency for Research on Cancer, and are utilized commercially. In this review, we used molecular pathway analysis to understand the toxicity and carcinogenic mechanisms of these metals. Our analyzed data showed that above-mentioned metallic substances induce oxidative stress, DNA damage, and cell death processes, resulting in increase the risk of cancer and cancer-related diseases. Thus, we might think phytochelatin molecules and antioxidative phytochemical substances are helpful for prevention of heavy metal-induced cancer.

Heavy metal absorption, transportation and accumulation mechanisms in hyperaccumulator Thlaspi caerulescens / 生物工程学报

Thlaspi caerulescens, the famous model plant of heavy-metal hyperaccumulator, can uptake and accumulate large amount of heavy metals in its above-ground part of the plants. However, the very low biomass in Thlaspi caerulescens makes this plant unfit for direct application in phytoremediation. In recent years, there are many reports about the physiological and molecular characterization of Thlaspi caerulescens under heavy metals stresses, including absorption, transport and intracellular detoxification processes (e.g., chelation and compartmentation). Research teams have conducted many studies of chelators in plants, such as organ acid, amino acid, phytochelatins, metallothioneins and nicotianamine, and so on. Several transport protein families, such as Zinc Regulated Protein, Cation Diffusion Facilitator, Natural Resistance and Macrophage Protein and Heavy Metal ATPase, play important role in short/long distance transport in the plant. In this review, we summarize the current knowledge of the physiological and molecular mechanisms of heavy metals accumulation in Thlaspi caerulescens, with particular emphasis on the roles of transporters and chelatins in modulating plant heave-metal-stress responses.

Arsenic accumulation in root and shoot vis-a-vis its effects on growth and level of phytochelatins in seedlings of Cicer arietinum L.

Arsenic (As) contamination of water and soil has become a subject of prime interest due to its direct effect on human health through drinking water and food. In present study two varieties (CSG-8962 and C-235) of chickpea, Cicer arietinum L., which is a major supplementary food in many parts of India and a valuable source of protein, has been selected to estimate the level of arsenate in root and shoot of five day old seedlings vis-à-vis effect of arsenate on seedling growth and induction of thiols including glutathione (GSH) and phytochelatins (PCs) and their homologues. Both varieties accumulated arsenate to similar levels and most of the metalloid was confined to roots, only about 2.5% was translocated to shoot. Plant growth was also not affected significantly in both the varieties. Arsenate exposure significantly induced the levels of thiols including PCs and homophytochelatins (hPCs). The induction of thiols was much higher in roots than shoots and was greater in var C-235 between the two tested ones. Thus, both varieties tolerated and detoxified arsenic through chelation with GSH, PCs and hPCs, primarily in roots, however var C-235 performed better

Fly-ash induced synthesis of phytochelatins in chickpea (Cicer arietinum L.) plants.

Phytochelatins and related metabolites (cysteine and GSH) were found to be induced in the shoots of two varieties of Cicer arietinum viz., CSG-8962 and C-235 grown under different amendments of fly-ash with garden soil and press mud. Cysteine, GSH, PCs and its speciation were found in higher concentrations in amended fly-ash than in the control 100% soil. Two species of metal binding peptides i.e., PC2 and PC4 were found in both varieties and in amendments, however, their concentration varied depending upon the fly-ash concentrations in both amendments. Further, var. CSG-8962 was found more tolerant than var. C-235 because of higher concentrations of PCs and related metabolites.