Selenium toxicity resistance gene isolated from puccinella distans

Abstract

The invention relates to a gene with 85% similarity to thiocyanate methyltransferase 2 mRNA, which provides resistance to selenium (Se) toxicity found in split salt grass (Puccinellia distans), a hyperaccumulator plant, to compensate for selenium deficiency in organic nutritional supplements or to provide resistance to selenium in agricultural production. The cDNA that is the subject of the invention provides resistance to toxic doses of selenium and has a nucleotide sequence of SEQ ID NO:1. The gene that is the subject of the invention is determined by transferring cDNAs obtained from root tissues of Puccinellia distans into (S. cerevisiae) cells. Yeast (S. cerevisiae) cells expressing this gene can be used in organic nutritional supplements to compensate for selenium deficiency, and thanks to this gene, agricultural productivity is increased by ensuring that plants are resistant to the toxic effects of selenium.

Description

[0001] SELENIUM TOXICITY RESISTANCE GENE ISOLATED FROM PUCCINELLIA DISTANS

[0002] Technical Field of the Invention

[0003] The invention relates to a gene with 85% similarity to thiocyanate methyltransferase 2 mRNA, which provides resistance to selenium (Se) toxicity found in split salt grass (Puccinellia distans), a hyperaccumulator plant, to compensate for selenium deficiency in organic nutritional supplements or to provide resistance to selenium in agricultural production. Said gene is determined by transferring cDNAs obtained from root tissues of Puccinellia distans into yeast (S. cerevisiae) cells.

[0004] State of the Art

[0005] Efficiency and sustainability in agricultural production are affected by multiple stress factors, including low or high temperature, lack or excess water, high salinity, heavy metals and ultraviolet radiation. The fact that heavy metals are a stress factor in plants is due to the contamination of the soil during activities such as mining, and when the contamination level exceeds the threshold concentration, heavy metals have a toxic effect and affect plant development by participating in metabolic reactions and acting as micronutrients [1].

[0006] Selenium (Se), a metalloid, is present in all natural materials on earth, including rocks, soils, water, air and biological tissues. Although it is an essential element for humans and animals, excessive intake of Se causes diabetes and hypertension in humans [2], as well as the emergence of some types of cancer such as pancreatic and skin cancer, and also causes an increase in reactive superoxides (ROS) such as hydrogen peroxide and disruption of cell redox homeostasis in plants. Ultimately, excess selenium, which is essential for living things, causes toxicity in all living things. Toxicity of metals / metalloids such as Se generally occurs as a result of intensive industrial and mining activities. In addition, selenium can cause environmental pollution in the form of "selenate" or "selenite" in agricultural and industrial wastewater as a result of the use of fossil fuels. Selenium, which is found in high amounts in soils called selenium, has a toxic effect, preventing the cultivation of many crop plants and negatively affecting agricultural production [3]. In the state of the art, washing process is applied in order to reduce high amounts of selenium concentration in the soil, and optimisation studies are carried out by changing parameters such as soil washing technique, process time, temperature and pH. However, considering that even the use of water for production activities is not sufficient throughout the world, its use is not an effective method for improving the soil. Therefore, there are other methods to ensure that agricultural activities are sustainable without decreasing productivity in areas where irrigation facilities are limited. In order to purify soils from metals / metalloids such as Se, the use of hyperaccumulator or accumulator plants that can accumulate these metals / metalloids in high amounts is an effective method, and these plants are used in phytoremediation studies to reduce metals / metalloids that cause environmental pollution to their less toxic forms [4] These plants can accumulate high amounts of metals / metolloids in their tissues (above or below ground) and do not show any signs of toxicity [5]. For this reason, investigating the genes associated with resistance to metal / metolloid stress in hyperaccumulator plants in order to develop phytoremediation applications is a very popular research topic and is becoming increasingly widespread and agricultural productivity and sustainability are positively affected by transformant organisms obtained as a result of transferring genes obtained from hyperaccu ulator species to different organisms for the purpose of purifying metals / metalloids found in soil or water. Yeast cells, which are organisms developed for use in this context, take up metal ions in soils contaminated with metals and metalloids through their cell membranes. Ingested metal ions can be reduced, oxidised or turned into complex structures that cannot be dissolved in soil, depending on the metabolic pathways of the cells. In this way, elements found in toxic amounts in soil, water, etc. can be removed [6]. At the same time, yeast cells are easy to manipulate and propagate, have high adaptability, and are economically cheap, making these cells preferred. Therefore, in phytoremediation studies, hyperaccumulator plants, which have previously been determined to be resistant to a certain metal / metalloid and can accumulate these metals / metalloids in high amounts, are used.

[0007] Although high amounts of selenium in the soil causes toxic effects on plants and other living things, its deficiency also causes some problems. Moderate selenium deficiency in the human body causes myodegenerative diseases such as muscle weakness, low selenium level causes symptoms such as depressed mood, anxiety and confusion [7], The selenium level required for the body can also be achieved by enriching microorganisms with certain trace elements and using it as an organic nutritional supplement. In this way, organisms can directly take the inorganic form of selenium with low availability, such as sodium selenite, from the nutrient medium and convert it into forms with high availability, and by means of this transformation, the nutrients are improved in terms of element ratio.

[0008] Split salt grass (Puccinellia distans), which is included in the state of the art and is a selenium hyperaccumulator plant, is a monocotyledonous plant belonging to the Poaceae family that generally grows in salty and alkaline soils, and although it can tolerate toxic amounts of boron and selenium [8], it is not known which gene(s) this tolerance originates from, and accordingly, phytoremediation applications made by transformation of this gene and its use as an organic food additive are not included.

[0009] Due to reasons such as the limitations and inadequacies of the solutions in the current technique, the amount of selenium in the soil being at toxic levels and the plants being negatively affected by this, the methods used in the current technique to reduce the amount of selenium in the soil not being effective, and the emergence of disease symptoms in the living body due to selenium deficiency in foods, it has become necessary to make improvements in this area.

[0010] Brief Description and Aims of the Invention

[0011] In the invention, a gene with 85% similarity to thiocyanate methyltransferase 2 mRNA, which provides resistance to selenium (Se) toxicity found in split salt grass (Puccinellia distans), a hyperaccumulator plant, to compensate for selenium deficiency in organic nutritional supplements or to provide resistance to selenium in agricultural production is described. Said gene is determined by transferring cDNAs obtained from root cells of Puccinellia distans into yeast (S. cerevisiae) cells. S. cerevisiae cells expressing this gene can be used in organic nutritional supplements to compensate for selenium deficiency, and thanks to this gene, agricultural productivity is increased by ensuring that plants are resistant to the toxic effects of selenium. The cDNA that is the subject of the invention provides resistance to toxic doses of selenium and has a nucleotide sequence of SEQ ID NO:1 . The aim of the invention is to ensure that plants growing in soils with high selenium content gain tolerance to selenium. By identifying the gene that provides resistance to selenium in S. cerevisiae cells and transferring said gene to plants, plants become resistant to selenium.

[0012] Another aim of the invention is to eliminate selenium deficiency in case the selenium taken from food is insufficient for living beings. By expressing the gene that is the subject of the invention in S. cerevisiae (yeast) cells and adding these cells to organic nutrients, the selenium deficiency in the food content is eliminated.

[0013] Description of Drawings

[0014] Figure 1. Serial dilution of yeast cells carrying the gene with the nucleotide sequence of SEQ ID NO:1 , which is the subject of the invention, in YNB medium containing different concentrations of sodium selenite.

[0015] Figure 2. Change in the expression levels of the gene with the nucleotide sequence of SEQ ID NO:1 in P. distans root samples with 0, 6, 12, 24 and 48 hours of stress (80 mg / L sodium selenite) application.

[0016] Figure 3. Changing expression levels as a result of applying 1 , 3, 7 and 10 mM sodium selenite stress to yeast cells carrying the gene with the empty vector 0 mM and the gene with the nucleotide sequence of SEQ ID NO:1 .

[0017] Detailed Description of the Invention

[0018] The invention relates to a gene with 85% similarity to thiocyanate methyltransferase 2 mRNA, which provides resistance to selenium (Se) toxicity found in split salt grass (Puccinellia distans), a hyperaccumulator plant, to compensate for selenium deficiency in organic nutritional supplements or to provide resistance to selenium in agricultural production. The cDNA that is the subject of the invention provides resistance to toxic doses of selenium and has a nucleotide sequence of SEQ ID NO:1 .

[0019] The gene that is the subject of the invention is determined by transferring cDNAs obtained from root tissues of Puccinellia distans into (S. cerevisiae) cells. S. cerevisiae cells expressing this gene can be used in organic nutritional supplements to compensate for selenium deficiency, and thanks to this gene, agricultural productivity is increased by ensuring that plants are resistant to the toxic effects of selenium.

[0020] Plasmids containing P. distans root cDNAs were transferred into yeast (S. cerevisiae) cells. To determine selenium-resistant cells, 150 pl of yeast cells carrying cDNAs were taken and spread on YNB medium containing 15 mM Na2SeO3. As a negative control, yeast cells transformed with empty vector without cDNA were also subjected to the same procedure. Petri dishes were incubated at 30 °C and colony formations were monitored daily and compared with the negative control.

[0021] Selected resistant colonies were grown in liquid YNB medium at 30 °C with shaking at 225 rpm for 24 hours. After the cell densities were diluted to OD600 = 0.2 in YNB medium, 5 pl of yeast cells from each colony were transferred to YNB medium containing 0 - 50 mM Na2SeO3. Colony development was observed for 5 days in petri dishes incubated at 30°C. At the end of these 5 days, the colony growing in the medium containing 15 mM sodium selenite, which is the toxic level for the wild-type yeast cells, was selected and serial dilutions were made with wild type cells from this colony at different concentrations, respectively (10’1,10’2,1 O’3, 10’4), as shown in Figure 1. In Figure 1 , the upper row, where colony formation occurs less or not at all, comprises cells containing empty vector, the ones below that row are colonies containing SEQ ID NO: 1 , which is the subject of the invention, and the amounts of sodium selenite in each medium are stated in Table 1.

[0022] Table 1. Amounts of sodium selenite in media

[0023] While yeast cells containing the empty vector failed to survive in YNB medium containing 3 mM sodium selenite (Figure 1 , C) and increasing sodium selenite concentrations, it is observed that yeast cells carrying the gene with the nucleotide sequence of SEQ ID NO:1 continue to survive in YNB media containing 50 mM sodium selenite. Based on these results, it was determined that the selected colony gained tolerance to increased selenium concentration compared to wild-type yeast cells, and it was concluded that the cDNA contained in these colonies was probably the key gene playing a role in selenium tolerance. RT-qPCR (real-time polymerase chain reaction) analyses were performed to examine the relationship between the expression level of the gene, which was determined to be tolerance to selenium in yeast cells, and selenium in plant cells. For these analyses, sterilised P. distans seeds were planted in MS medium and then allowed to grow under the same conditions. Two-month-old plants were transferred to hydroponic systems containing Hoagland solution. After ten days of acclimation and growth period, P. distans plants were exposed to stress with Hoagland's solution containing 80 pg / L sodium selenite. Three replicate plant samples harvested after 0 (Control), 6, 12, 24 and 48 hours of stress application were ground with liquid nitrogen and used in RNA isolation. Root total RNA isolation of plants was carried out using a plant RNA isolation kit. RNA amounts were determined by spectrophotometer. The samples were cleaned with DNAse enzyme (endonuclease) application and were made ready for RT-qPCR. The concentrations of RNA samples prepared by applying DNAse were measured with a spectrophotometer. RNA samples from each stress hour were diluted to a concentration of 5 ng / pl to be used in the RT- qPCR reaction. Then, the RT-qPCR master mix and other reaction components were thawed at room temperature and placed on ice (Primers with nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 and actin gene primers with nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5 were used as internal control). After complete dissolution, all components were mixed. Reaction mixtures were prepared as mixes, excluding sample RNAs. Then, 5 pl of the mix was distributed for each sample in 3 replicates. 10 ng of RNA was added to each replicate. RT-qPCR was performed on the samples prepared on ice by following the cycle protocol: 95 °C 60 seconds selfdenaturation, 95 °C 15 seconds denaturation, 60 °C 30 seconds extension, 60-95 °C 70 seconds melting curve, a total of 30 cycles to be completed. Primers with SEQ ID NO: 2 and SEQ ID NO: 3 nucleotide sequences designed for the determined genes were used and actin gene primers with SEQ ID NO: 4 and SEQ ID NO: 5 nucleotide sequences were used as an internal control. Gene expressions were determined using the ddCT method at (control), 6, 12, 24 and 48 hours (Figure 2). In Figure 2, gene expressions of each sample are compared with the unstressed control. It is seen that at the 48thhour, the expression levels of the gene with the nucleotide sequence SEQ ID NO:1 increased 38-fold compared to the control. RNA isolation was performed to determine the expression levels of the gene with the nucleotide sequence of SEQ ID NO:1 in yeast cells, and yeast cells containing the gene with the nucleotide sequence of SEQ ID NO:1 and yeast cells containing the empty vector were grown in 20 ml of YPD-containing medium until the morning. Then, the amount of cells was measured at ODeoo with a spectrophotometer. Initial cell amounts were diluted in YPD media to ODeoo = 0.2 and final concentrations of 1 mM, 3 mM, 7 mM and 10 mM sodium selenite were added. As a control group, yeast cells containing empty vector without any stress (0 mM) were grown under the same conditions. Then, the cells and the control group, which were stressed for 3 hours in a mixer at 250 rpm and 30 °C, were harvested. RNA isolation was performed from the obtained yeast cells. Primers with the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 designed for the determined genes and primers with the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7 were used as internal control. Gene expressions were determined using the ddCT method at (control), 1 mM, 3 mM, 7 mM and 10 mM sodium selenite stresses (Figure 3). In Figure 3, sodium selenite stress was not applied to the control (0 mM). By applying increasing concentrations of stress to yeast cells carrying the gene with the nucleotide sequence of SEQ ID NO:1 , their changing expressions were compared. Expression of said gene increases as sodium selenite concentration increases compared to control cells. The sequence of the cDNA (SEQ ID NO: 1 ) subject to the invention was obtained by sequencing.

[0024] REFERENCES

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Claims

CLAIMS1. A cDNA of Puccinellia distans that provides resistance to toxic doses of selenium in transformed eukayotes, especially plants, having the SEQ ID NO:1 nucleotide sequence.

2. A vector comprising cDNA according to Claims 1 .

3. Eukaryote comprising a vector according to Claim 2.

4. Use of the vector-containing eukaryote according to claim 3 as an organic nutritional supplement to correct selenium deficiency.

5. A eukaryote according to Claim 3, wherein said eukaryote is a plant or yeast.

6. Use of cDNA according to claim 1 in plant improvement to increase tolerance to selenium toxicity in transformed eukaryotes.

7. A eukaryote according to Claim 6 in which cDNA is transformed, wherein said eukaryote is a plant or yeast.

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