МикроРНК и регуляция экспрессии генов Cu/Zn-СОД в растении Thellungiella salsuginea при действии различных концентраций меди

Исследование посвящено изучению роли микроРНК в посттранскрипционной регуляции генов Cu/Zn-СОД и гена CCS в растении Thellungiella salsuginea при действии различных концентраций меди. Объектом исследований являлись растения Thellungiella salsuginea. Оценку содержания белка CSD1 и CCS1 проводили с помощью вестерн-блот анализа, уровень экспрессии других генов проводили методом ОТ-ПЦР с ген-специфичными праймерами. Показано, что в зависимости от концентрации меди в питательной среде в растениях прослеживается реципрокный характер взаимоотношений между экспрессией miR398 и экспрессией генов CSD1 CCS1. Сделано предположение, что повышенное содержание меди в питательной среде ингибирует процессинг белка Cu/Zn-СОД за счет нарушения структуры активного центра ионами меди.

MicroRNA and regulation of gene expression of Cu/Zn-SOD in Thellungiella salsuginea plants under effect of the different concentrations of copper

One of the important element of posttranscriptional regulation of gene activity is a mechanism for silencing genes, due to RNA interference, which is associated with expression of small RNAs. In Arabidopsis thaliana L. plants found two major classes of small RNAs in size 21-23 base pair involved in the suppression of gene expression: siRNA (small interfering) and miRNA (microRNA). There is increasing evidence that under stress conditions changes as the expression of miRNA and expression of they targets genes and the activity of miRNA-protein complexes. The purpose of present study was to examine the role of microRNAs in posttranscriptional regulation of genes Cu/Zn-SOD and CCS gene in a plant Thellungiella salsuginea under the influence of different concentrations of copper, given that copper is a trace element essential for plant nutrition and is a member of the cofactors of plastocyanin, Cu/Zn- SOD, cytochrome c and laccase. Object of research is plants Thellungiella salsuginea, Pallas were grown in water culture in a medium of Johnson. At the age of 6 weeks the plants were divided into groups and subjected to treatment CuSO4 (0,1 and 100 μM). Experiments were performed in three biological and three analytical replications. Isolation of total RNA was performed TRIzol® reagent. Level of expression of miRNAs was assessed by Northern blot hybridization. Evaluation of protein content CSD1 and CCS1 performed using Western blot analysis by standard methods Laemmli (1970). The level of expression of genes other studied was performed by RT-PCR with gene-specific primers. Several studies have shown that in the conditions of a copper lack decrease content of the protein Cu/Zn-SOD and its enzymatic activity. It was also shown that in A. thaliana and other higher plants, various concentrations of copper regulate expression of mRNA CCS1, encoding a chaperone copper, delivering Cu to apoprotein different isoenzymes of Cu/Zn-SOD. As have shown Nozern-blot hybridization of total RNA Th. salsuginea, at entering 100 μM CuSO4 in a nutrient medium occurred almost full inhibition of an expression miR398. In experiments with absence of copper in a nutrient medium observed increase expression of miR398. The study of CSD1 expression showed that after 24 hours absence of copper in the growth medium caused a decrease expression of this gene. When the copper concentration was 100 μM in all parts of the plant Th. salsuginea observed increase in mRNA CSD1. However, western blot analysis with specific forms for the cytosolic Cu/Zn- SOD antibody revealed that this protein was determined only in the leaves of plants at all studied concentrations of copper in the medium. This give evidence that the synthesis of the protein may be exercised only in the leaves. At high concentrations of copper (100 μM) in the leaves was shown enhance of CSD1 expression, however, increase of the protein content CSD1 didn`t happened. On the other hand, the study of gene expression CCS1 copper chaperone for SOD in leaves and roots showed that the amount of mRNA of this gene increased in the leaves at a concentration of CuSO4 1 and 100 μM, in the absence of copper as expression of this gene significantly decreased in the leaves and roots. CCS1 protein found only in the leaves in the control plants (0,25 μM CuSO4), as well as 1 μM CuSO4 in the nutrient medium. In terms of different concentrations of copper in the growth medium of plants observed reciprocal relationship between the expression of miR398 and genes expression CSD1 and CCS1. We can assume that under conditions of different concentrations of copper in the growth medium, there is a miR398-mediated regulation of genes CSD1 and CCS1. It is likely that the increase due to miR398 regulation of mRNA CCS1 in the absence of copper in the growth medium reduces the number of copper delivered to the protein CSD1 and, consequently, reduced its contents in the leaves. Probably, in the absence of copper in the nutrient medium may be coming redistribution of copper ions between Cu/Zn-SOD and copper-containing proteins other important, for example, such as plastocyanin.