Biodiversity2013

«Biodiversity. Ecology. Adaptation. Evolution.» Odessa, 2013

BACTERIA AND YEASTS OF THE SYSTEMS OF THE

HYDROBIOLOGICAL CLEANING OF MARINE WATERS

Doroshenko Yu. V.

Institute of Biology of the Southern Seas, Sevastopol, Ukraine

E-mail: julia_doroshenko@mail.ru

The role and functions of periphyton microorganisms of technical construction

– hydrobiological system for cleaning of polluted waters placed in Oil Harbour (Sevastopol Bay, Black sea) are studied.

It’sshownthattotalquantityofheterotrophicbacteriainperiphytonchangesfrom 104 to107 cell/g.Thequantityofothergroupsvariesfrom10to106 cell/g.Thequantity of heterotrophic bacteria in area of hydrobiological system placing is decreased by 4 – 5 orders as compared with periphyton`s quantity. This fact has reflected the law of bacteria expansion in sea water.

Fromperyfiton155culturesofbacteriaareselected.Fromthoseculturesprevailed the bacteria Vibrio – 36 %, 22 % cultures Marinococcus, and 19 % – Pseudomonas, 16 % – Microbacterium.

Yeasts (66 cultures) from the periphyton systems of the hydrobiological cleaning arefirstselected.Thedominantrepresentativesspeciescompositionweredetermined accordingtomodernnomenclature:Candida lambica –26%,Candida krusei –24% and Rhodotorula mucilaginosa – 22%.

The sea yeasts ability to grow on media with high concentrations of the diesel fuel was evaluated.

LOW CONCENTRATIONS OF RHODIOLA ROSEA AQUEOUS EXTRACT

DEMONSTRATE STRESS-PROTECTIVEAND GEROPROTECTIVE

EFFECTS ON YEAST SACCHAROMYCES CEREVISIAE

Hryshuk Ch., Burdyliuk N., Izers’ka L., Bayliak M.

Vassyl Stefanyk Precarpathian National University, Ivano-Frankivsk, Ukraine

Е-mail: grishuck.khristina@yandex.ua

The extracts from Rhodiola rosea rhizome was reported to have numerous health benefits in humans and animals, including adaptogenic, antioxidant, and antiaging activities. Despite documentation of a variety of beneficial effects, molecular mechanisms of R. rosea action are not clear yet. In the present study, we estimated concentration-dependent effects of aqueous extracts from R. rosea rhizome, collected at high altitudes in Ukrainian Carpathians, on nonspecific stress resistance of “young” exponentiallygrowingS.cerevisiaecells,andlifespanof“old”stationaryyeastcultures, which are considered as a good model for studying biology of higher eukaryotes.

In the study, two strains of S. cerevisiae, YPH250 and W303-1A, were used. The cellsweregrowninYPDmedium.Fortheadaptationexperiments,exponential-phase cells were pretreated with different concentrations of R. rosea extract (1-20μl/ ml) for 2 h. Then cells were harvested and resuspended in 50 mM potassium phosphate buffer (pH 7.0), and exposed to 10 mM H2O2 and heat shock (40°C). The aqueous

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extraction was performed on the boiled water-bath in the ratio of 1:20 (comminuted dried rhizome material: distilled water) for 30 min. The viability of yeast cells by long-termcultivationwasdeterminedbymethylenebluestainingandbycountingthe number of colonies generated onto agar plates. Activity of antioxidant enzymes and level of oxidized proteins were measured by spectrophotometric methods.

Concentration-dependent effects of aqueous extract from R. rosea root on longterm survival and stress resistance of budding yeast Saccharomyces cerevisiae were studied. At low concentrations, R. rosea aqueous extract extended yeast chronological lifespan, enhanced oxidative stress resistance of stationary-phase cells and exponentially growing yeast cultures. At high concentrations, R. rosea extract sensitized yeast cells to stresses and shortened yeast lifespan. These biphasic concentration-responses describe a common hormetic phenomenon characterized by low-dose stimulation and high-dose inhibition. Yeast pretreatment with low doses of R. rosea extract enhanced yeast survival and prevented protein oxidation under H2O2-induced oxidative stress. Positive effect of R. rosea extract on yeast survival underheatshockexposurewasnotaccompaniedwithchangesinantioxidantenzyme activities and levels of oxidized proteins.

POTENTIAL MECHANISMS OF SOIL NITRIC OXIDE PRODUCTION

Medinets S.1, Medinets V.1, Skiba U.2, Butterbach-Bahl K.3

1Odessa National I. I. Mechnikov University (ONU), Odessa, Ukraine 2Centre for Ecology and Hydrology (CEH), Edinburgh, UK 3IMK-IFU, KIT, Garmisch-Panterkirchen, Germany

E-mail: s.medinets@gmail.com

Nitric oxide is a precursor of tropospheric ozone (Cameides et al., 1994; Laville et al., 2011), which is one of the most important high reactive gaseous pollutant impacting human health and plant productivity (Staffelbach et al., 1997; Ludwig et al., 2001).

Revealing and figuring out possible NO production mechanisms in soils to mitigate nitric oxide influence on ozone formation in sensitive areas was the main goal of our study. Google Scholar andWeb of Knowledge interactive data bases were used for review investigation of recent studies.

Biological N transformation processes are usually considered as potential sourced of NO productions, at the same time chemically N transformation should not be neglected. Chemodenitrification is a non-enzymatic chemically transformation of nitrite or nitrate to different gas compounds, including NO (Skiba, 2008). It was shown(Zumft,1997;Skiba,2008)thatthisprocessnormallycouldoccuratpH<5in soilswithhighconcentrationoforganicmatterandatpresenceofiron,ammoniumor amines, whilst high NO2- concentration level (Ludwig et al., 2011) and temperature (Kesiketal.,2006)influencedonconversionrateaswell.HighratesofNOemission, associated with chemodenitrification, were reported by Cheng et al. (2004) for agricultural and Kesik et al. (2006) for temperate forest acidic soils. Dissimilatory

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nitrate reduction (DNRA) to ammonium is a microbial enzymatic process of nitrate transformation into ammonium via nitrite (Cole and Brown, 1980; Cole, 1990) and could be performed by different group of bacteria: obligatory anaerobes, facultative anaerobes and aerobes. It was demonstrated that DNRA was favored at intensively reduced and C-rich soils (Tiedje, 1988; Schmidt et al., 2011). Since N2O production was found during DNRA (Baggs, 2011), hypothetically nitric oxide could be produced as an obligatory precursor (Russow et al., 2009), but nothing was reported until present time. Urgent and intensively investigations of DNRA as a potential process for NO production/emission are needed to estimate rate of this process.

The detailed discussion of these findings point and demonstrate undiscovered, poorly investigated issues, regarding NO production, which urgently should be taken into account and investigated.

Authors gratefully acknowledge support from the projects “Effects of Climate Change onAir Pollution Impacts and Response Strategies for European Ecosystems” (ÉCLAIRE), funded under the EC 7th Framework Programme (Grant Agreement No. 282910), “Evaluation of Agriculture and Fires Impacts to Lower Dniester Ecosystems and Greenhouse Gases Emission intoAtmosphere” (No. 505), funded by the Ministry of Education and Science of Ukraine, and EU COST Action ES0804 - Advancing the integrated monitoring of trace gas exchange Between Biosphere and Atmosphere (ABBA).

THE ROLE OF NITRIFICATION AND DENITRIFICATION IN SOIL

NITRIC OXIDE PRODUCTION

Medinets S.1, Medinets V.1, Skiba U.2, Butterbach-Bahl K.3

1Odessa National I. I. Mechnikov University (ONU), Odessa, Ukraine 2Centre for Ecology and Hydrology (CEH), Edinburgh, UK 3IMK-IFU, KIT, Garmisch-Panterkirchen, Germany

E-mail: s.medinets@gmail.com

Nitric oxide is highly reactive compound in near ground atmosphere (Fowler et al.,2009)andisconsideredasthemainprecursoroftroposphericozoneinruralareas (Cameides et al., 1994; Laville et al., 2011).

The main purpose of our survey study is an understanding of NO production/ emission mechanisms in soils allow to develop the mitigation strategy for its reduction, leading to O3 level declining. Published data were obtained using Web of Knowledge and Google Scholar research article data bases.

The basic soil biological N transformation processes could be considered as potentialsourcedofNOproductions.NitrificationisstepwiseconversionofNH4+ via hydroxylamine(HA)intoNO2- andtofinalproduct–NO3- (Zumft,1997;Wrageetal., 2001).NOproductionisconsideredasanintermediateinastepofHAtransformation into NO2- (Hooper and Terry, 1979; Ludwig et al., 2001). Nitrification is affected by NH4+ availability, soil O2 level, soil moisture content, pH and temperature (e. g. Zumftetal.,1997;Ludwigetal.,2001).SignificanceofnitrificationforNOemission wasshownbymanyresearchers(e.g.GascheandPapen,1999;VentereaandRolson,

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2000;Luoetal.,2012)forvariousecosystems.Denitrificationisabiologicalstepwise reduction of NO3- into NO2-, NO, N2O and N2 (Zumft, 1997; Skiba, 2008). Classical (heterothrophic) denitrification is attributed to facultative aerobes organisms (including bacteria, archaea and fungi), which under O2 depletion can switch to anaerobic respiration (Hayatsu et al., 2008; Skiba, 2008). Nitrifier denitrification is a process, when ammonia oxidizing bacteria at low O2 condition reduce NO2- to NO, N2O and N2 (Wrage et al., 2001; Skiba, 2008). Heterothrophic denitrification is attributed to facultative aerobes organisms under O2 stress (Hayatsu et al., 2008; Skiba, 2008). Nitrifier denitrification is a process, when ammonia oxidizing bacteria under low O2 condition reduce NO2- to gaseous N compounds (Wrage et al., 2001; Skiba, 2008). Denitrification is controlled by soil moisture content, soil temperature, N-NO3- availability, soil properties and management practice (Zumft et al., 1997; Skiba, 2008). This process associated with high NO production as an obligatory intermediate in a step from nitrate to nitrous oxide (Skiba et al., 2008; Russow et al., 2009), but not related with high NO emission, that was explained by ‘diffusion limitation’hypothesis (Firestone and Davidson, 1989; Skiba et al., 1997; Russow et al., 2009) when up to 100% of nitric oxide produced under anaerobic condition are trapped and converted into N2O.

Authors gratefully acknowledge support from the projects “Effects of Climate Change onAir Pollution Impacts and Response Strategies for European Ecosystems” (ÉCLAIRE), funded under the EC 7th Framework Programme (Grant Agreement No. 282910), and “Evaluation of Agriculture and Fires Impacts to Lower Dniester Ecosystems and Greenhouse Gases Emission intoAtmosphere” (No. 505), funded by the Ministry of Education and Science of Ukraine.

THE INFLUENCE OF AMINO ACIDS AND MONOSACCHARIDES ON

BACILLUS THURINGIENSIS ІМV В-7324 FIBRINOLYTIC PEPTIDASE

STABILITY

Nidialkova N. A., Matseliukh О. V.

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of

Ukraine, Kyiv

E-mail: Nidialkova@gmail.com

Thermostability is the enzyme property which depends on composition of hydrophilic and hydrophobic amino acids, carbohydrates, ionic interactions, presence of metal and disulfide bridges. The biotechnology and engineering enzymology require carrying out many enzymatic processes at the strict conditions: high temperature, presence of organic additions etc. It was shown that the purified fibrinolytic peptidase of Bacillus thuringiensis ІМV В-7324 is stable at 20-60 °C during 2 h. The aim of this study was investigation the influence of amino acids and monosaccharides which were observed in the fibrinolytic peptidase on its stability.

It is known that hydrophobic amino acids are required to stabilization of protein structure. It was shown that the fibrinolytic peptidase B. thuringiensis ІМV В-7324

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consists ~ 70% of hydrophobic amino acids such as glycine (19.32 %), alanine (6.3 %), phenylalanine (1.94 %), leucine (6.27 %) and serine (6.34 %). Probably they form areas in the structure of protein molecule which avoid interaction with molecules of water. B. thuringiensis ІМV В-7324 fibrinolytic enzyme contains threonine (17.06 %) – hydrophilic amino acid that can involve in the forming of tertiary protein structure. By phenol-sulfuric acid method (Dubois, 1956) and high liquid pressure chromatography it was established that the fibrinolytic peptidase contains carbohydrates (1.9 %): D-mannose and D-glucose.

It was found that the certain amino acids effect on stabilization of the enzyme. So alanine and phenylalanine increased fibrinolytic activity (in 1.5 and 2.0 times, respectively) at 70 °С during 2 h of incubation. However, the presence of lysine stabilized the fibrinolytic activity only during a half of hour.Addition of D-mannose and D-glucose contributes of enzyme stability at 70 °С during 2 h.

Thusthehydrophobicandhydrophilicaminoacidsandmonosaccharides(glucose and mannose) which were found in the structure of the B. thuringiensis ІМV В-7324 fibrinolytic peptidase can be used for the increased of enzyme thermostability. The established fact is useful for applying of enzyme in industrial processes.

CATALASE ACTIVITY OF СORYNEBACTERIUM VARIABILE

UKMAC-717 UNDERADAPTATION TO LINCOMYCIN

Pastyrya A., Furtat I.

National University of “Kyiv-Mohyla Academy”

Е-mail: Luminous_Flower@mail.ru, furtat.im@gmail.com

The emergence of multi-resistant forms of microorganisms takes place due to theirwideuseinmedicineandfoodindustryandcausesanxietybecauseantibiotics are able to change biochemical properties and metabolism of bacteria. Moreover, antibiotics could mediate oxidative stress in bacteria, and catalase, as one of the major antioxidant enzymes could take part in the stress response.The principle aim of this research was to study the catalase activity (CA) of strain Сorynebacterium variabile UKMAc-717T and its variant adapted to lincomycin (varLIN).

In this paper we compared the CA of strain С. variabile UKM Ac-717T and its variant (varLIN). Adaptation to lincomycin was started with minimal inhibiting concentration(MIC=1.25μg/ml),graduallyincreasingtheconcentrationofantibiotic in the culture medium. It was shown that С. variabile was quickly adapted to lincomycin, as it was able to grow at concentrations higher than 50 μg/ml after 20 passages. It was also ascertained that С. variabile varLIN adapted to relatively low concentration of lincomycin, further showed the ability to grow at concentration of 1500 μg/ml of this antibiotic. That’s why, adaptation to low concentrations of antibiotics can lead to the formation of bacterial resistance to higher concentrations, andthereforethebehaviorofsuchresistantstrainscanbeunpredictable.Afterthat,we determined concentrations of lincomycin that inhibited growth of wild strain and an adapted variantLIN for 50 and 70 %. Upon studying the catalase activity of wild strain

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werechosenconcentrationsof1.5and3μg/ml,whichinhibitedvitalactivityfor47.3 and 67.2 %. In case of the variantLIN these concentrations were 600 and 1200 μg/ ml which inhibited vital activity for 48.6 and 68.3 % respectively. Stress conditions were modeled by introducing to the culture medium lincomycin in the chosen concentrations and incubated for 14 h of growth of both strains that corresponds to the middle of the exponential growth phase. Catalase activity of cultures was determined spectrophotometrically by the degradarion of hydrogen peroxide (λ 240 nm) in a standardized suspension of intact cells (1×109 cl/ml) at 90, 180, 360 min and 24 h after the addition of lincomycin, as the generation time ofCorynebacterium spp. is 90-180 minutes. The data has showed that the presence of lincomycin in the culturemediumdidnotaffectthecatalaseactivityofwildstrainanditsvariantvarLIN. Therewasnostatisticallysignificantdifference(α=0.01)betweentheratesofCAof both cultures under normal cultivation conditions and in the presence of lincomycin. Thus, the CAvalue of the wild strain within 24 h after lincomycin adding were 1354 and 1311 μmol/min•109cells (at concentrations of 1.5 and 3 μg/ml, respectively), and practically did not differ from control (CA = 1333 μmol/min • 109cells). In the adapted variant rates of CA were slightly lower in comparison with the wild type (CA = 1213 μmol/min •109cells), nevertheless there was no difference between the control and in the presence of lincomycin (CA=1199 and 1269 μmol/min•109cells at concentrations of 600 and 1200 μg/ml, respectively).

THE OPERATIVE BIOMONITORING MICROBIOLOGICAL PARAMETERS OF WATER AND SEDIMENT IN THE SIMULATE EXPERIMENTS

Starosyla Iev.

Institute Hydrobiology, Kiev, Ukraine

E-mail: jenya_star@ukr.net

The microbiological samples of water and sediment served from systems (microcosm) since community aquatic invertebrates. The community invertebrates

Dreissena polymorpha (Pallas), Dreissena bugensis (Andrusov), Dikerogammarus haemobaphes (Eichwald) and Chaetogammarus ischnus (Stebbing) pick upped at river Dnepr. The invertebrates placed to biotechnological complex in waterpool by volume 400 l3, at the temperature of the water 20±0.5оС. Provender consisted of the larva’s сhironomus, chlorella’s paste, yeast.

The number of bacteria consisted of ecology-trophy group, cells with active electronic-transport system in the water and sediment, decomposition organic matter atthewaterinsimulateexperimentsincecommunityaquaticinvertebratesfunctioning studied. The aim of the researches was interactions microbiological parameters with process of vital activity hydrobionts, temperature and organic matter.

For period of the operative biomonitoring number of microorganisms consisted of ecology-trophy group (eurtophic bacteria) in water in system varied over a wide range from 1.0 to 48.4 thous.cell/ml (average 13.4±3.1 thous.cell/ml, n=19).

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The percent of the cells with active electronic-transport system in the water of the microcosm was 8.4−99.9% (average 72.5±16.9%). For it period in sediment of the simulateexperimentnumbereurtophicbacteriawas2.2―93.5thous.cell/mg(average 30.1±7.0 thous.cell/mg); the part cells with active electronic-transport system in sediment was 7.5−99.8% (average 75.8±17.4%). The decomposition organic matter in microcosm was 0.03−0.31 mgC/ml day (0.10±0.02 mgC/ml day).

During the whole operative biomonitoring noted fluctuations of the microbiological parameters in the water and sediment of the experiment. The decomposition organic matter had significant amplitude of the fluctuation since trend of the gradual increase. The particularities of the change to number bacteria and metabolic activity microorganism in microcosm stipulated by difference to rates of the passing of the processes to vital activity aquatic invertebrates and the fluctuations of temperature. The fall of the temperature of the water till 14−150С slowed down food need invertebrates and had brought about accumulation organic matterinsystem.Thefurtherincreasingofthetemperatureofthewateractuatedboth feeding hydrobionts and processes mineralized of organic matter.After salvaging of its abundance in system of the number bacteria quickly became stabilized.

CATALASE ACTIVITY OF DESULFUROMONAS ACETOXIDANS

BACTERIUM UNDER THE INFLUENCE OF TRANSITION METAL

SALTS

Vasyliv O. M., Hnatush S. O., Tsap O. R., Novitska K. B.

Ivan Franko National University of Lviv, Lviv, Ukraine

E-mail: oresta.vasyliv@gmail.com

Application of ecological biotechnologies is one of the current state-of-the-art directions in reduction of toxic metal ions environmental pollution. Investigations of effective biological mechanisms of harmful compounds detoxification by various microorganisms is one of the ways of it’s development. Sulfurand sulfate-reducing bacteria possess a unique potential of spontaneous removal of metal ions and hydrogensulfideaccordingtotheprocessofmetalsulfidesprecipitation.Thisprocess represents an effective mechanism of biological remediation of high concentrations of heavy metals in the environment. Aerobes and facultative anaerobes possess an antioxidant defensive system activity that protects them against toxic compounds and oxygen influence. One of the most important components of it is catalase, which causes thetwo-electronH2O2 cleavage withO2 andH2O production.Catalaseactivity has been observed among the several species of obligate anaerobic sulfate-reducing bacteria of Desulfovibrio genus. Despite that it isn’t investigated enough in sulfurreducing bacteria, such as Desulfuromonas acetoxidans.

Thus,theaimofourworkwastoinvestigatetheinfluenceofvariousconcentrations of FeSO4, FeCl3 and MnCl2 on catalase activity of D. acetoxidans bacterial cells.

D. acetoxidans bacteria were cultivated in the modified Postgaite C medium during four days under the anaerobic conditions with addition of 0.5-2.5 mM

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of FeSO4, FeCl3 and MnCl2 into their growth medium. Control samples didn’t contain any investigated metal compounds. Catalase activity was measured spectrophotometrically (wavelength 410 nm) by the quantity of cleavaged H2O2.

Maximal catalase activity of D. acetoxidans bacteria was observed under the influence of 0.5 mM of FeCl3 and 1.0 mM of MnCl2 respectively on the fourth day andundertheadditionof1.5mMofFeSO4ontheseconddayofbacterialcultivation. Increasing of metal salt concentrations in the growth medium caused gradual inhibition of bacterial catalase activity. It was higher by two times under the addition of FeCl3 in comparison with the influence of MnCl2 and FeSO4. Ferric iron chloride belong to strong reactive oxygen species that could result in increasing of hydrogen peroxide generation under it’s influence on D. acetoxidans and respective enhance of their catalase activity in comparison with the influence of other investigated metals.

Practical application of D. acetoxidans for electricity generation in microbial fuel cell with FeCl3 usage as terminal electron acceptor and it’s simultaneous detoxification was determined.

Секція 6. Генетика і біотехнологія

АНАЛІЗ ГЕНЕТИЧНОЇ МІНЛИВОСТІ ГЕНОТИПІВ РИЖІЮ ПОСІВНОГО CAMELINA SATIVA L. CRANTZ ЗА ДОПОМОГОЮ ISSRАНАЛІЗУ

Баєр Г.Я1., Пірко Я.В1., Шевченко Ю. О.2, Рахметов Д.Б3., Ємець А.І1.

1Інститут харчової біотехнології та геноміки НАН України, Київ, Україна 2Київський національний університет імені Тараса Шевченко ННЦ «Інститут біології», Київ, Україна 3Національний ботанічний сад ім. М.М. Гришка НАН України, Київ, Україна

E-mail: galinabayer@mail.ru

Зважаючинавеликийпотенціалрижіюпосівного(Camelina sativa L.Crantz) якбіоенергетичноїрослини,селекціяновихтазбільшенняпосівіввжеіснуючих високопродуктивних форм і сортів цієї культури є актуальним завданням. На сьогоднішнійденьвУкраїнівНаціональномуботанічномусадуім.М.М.Гришка НАН України створено цінний генофонд рижію, визначено перспективні сорти та форми для виробництва біопалив. Відомо, що встановлення молекулярногенетичного поліморфізму за допомогою ДНК-маркерів є сучасним підходом для вирішення багатьох теоретичних і практичних проблем селекції рослин. Тому метою роботи було оцінити можливості використання ISSR-аналізу для генотипування перспективних з точки зору виробництва біопалив сортів та селекційних форм Camelina sativa.

Для з’ясування генетичних особливостей 12 генотипів рижію посівного було використано чотири праймери: ISSR-3 (CTCTCTCTCTCTCTCTTG), ISSR-4 (CACACACACAGT), ISSR-16 (AGAGAGAGAGAGAGGT) та ISSR-18

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(ACTGACTGACTGACTG). Генетичні дистанції Нея-Лі (Nei & Li, 1979) між зразками розраховували за допомогою програми TREES (Календарь, 1994). У подальшому вони були використані для кластеризації досліджуваних генотипів

(UPGMA-метод).

У результаті проведеного аналізу було виявлено 291 локус (амплікон), 27,6% з яких виявилися поліморфними. Більшість фрагментів мали довжину в межах 300-900 п. о. Значення генетичних дистанцій між дослідженими генотипами варіювало в межах від 0,0445 до 0,7985. В цілому досліджувані зразкидоволічіткорізнилисяміжсобою,щозасвідчуєнепоганідиференціюючі властивості використаних маркерів. У результаті проведеної кластеризації високопродуктивнігенотипирижіюзгрупувалисяводинкластер.Такимчином, отримані результати ISSR-аналізу підтверджують можливість використання цього виду аналізу як ефективного експрес-методу для генотипування, дослідження генетичного поліморфізму та для оцінки диференціації перспективних селекційних форм рижію.

Studying of genetic variability of false flax Camelina sativa L. Crantz by ISSRanalysis

Bayer G.Ya., Pirko Ya.V., Shevchenko Yu.O., Rakhmetov D. B., Yemets А.І.

Using ISSR-analysis the genetic variability and differentiation of C. sativa genotypes were studied. The level of genetic polymorphism and genetic distances between patterns were estimated. It was shown that ISSR-analysis is the useful method for fingerprinting and determination of relationships between different C. sativa genotypes.

ПОЛИМОРФИЗМ ГЕНА AMPD1 ПО ЛОКУСУ RS17602729 У СПОРТСМЕНОВ-ФУТБОЛИСТОВ

Бахчеван Е.Л.1,2, Чеботарь С.В.1

1Одесский национальный университет имени И.И. Мечникова, Одесса, Украина 2Молекулярно-генетическая лаборатория ООО «Гермедтех», Одесса, Украина

E-mail: r4t@list.ru

Молекулярная генетика спорта – новое направление исследований в генетике человека, центральной идеей которого является представление о том, что индивидуальные отличия в степени развития тех или иных физических и психических качеств во многом обусловлены полиморфизмом ДНК. В связи с этим, актуальным становится поиск молекулярно-генетических маркеров предрасположенности к различным видам спорта.

Одним из таких молекулярно-генетических маркеров является Gln12 аллель гена АМФ-дезаминазы (AMPD1), хромосомная локализация 1p13. AMPD1 кодирует М-изоформу АМФ-дезаминазы в скелетных мышцах. Считается, что основная причина недостатка АМФ-дезаминазы у человека

– это однонуклеотидная замена цитозина на тимин в 34 нуклеотиде (C34T) кодирующей последовательности в локусе rs17602729, который находится во

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втором экзоне, в результате чего глутаминовый кодон превращается в стопкодон (нонсенс-мутация в 12-м кодоне) (Norman et al., 1998). При данной мутации происходит блокирование синтеза цепи белка.

Целью работы было установление частот аллелей локуса rs17602729 гена AMPD1 у молодёжного состава команды профессиональных спортсменовфутболистов, имеющих спортивный разряд «кандидат в мастера спорта», и контрольной группы, не имеющей спортивного разряда. ДНК выделяли из проб буккального эпителия по методу Деллапорта (Дрейпер, Скотт 1991). Амплификацию проводили с аллель-специфичными праймерами, рекомендованнымифирмой«Литех»(Россия).ПродуктыПЦРфракционировали в 3% агарозном геле.

Проанализированы 14 образцов ДНК спортсменов-футболистов и 15 образцов ДНК контрольной группы. В группе футболистов преобладал С-аллель – встречался в 93% исследованных образцов ДНК, в контрольной группе его частота составила – 83%. Частота Т-аллеля в контрольной группе составила 17%, а в группе спортсменов-футболистов была 7 %. Распределение генотипов в группе футболистов наблюдалось следующее: СС – 93 %, СТ – 0 %, ТТ – 7 %. В контрольной группе распределение генотипов: СС – 67 %, СТ – 33 %, ТТ – 0 %. Поскольку критерий Хи-квадрат фактический - 6,36 при df=2 (p=0,05), то различия по частоте встречаемости генотипов между выборками достоверны.

Влитературе дискутируется вопрос о связи полиморфизма по гену AMPD1 (С34Т) с проявлением выносливости. Согласно Rubio et al. (2005) частота мутантного Т-аллеля AMPD1 значительно ниже у стайеров по сравнению

сконтрольной выборкой. Однако по данным исследований Федотовской (2006) различий по частоте мутантного аллеля при сравнении 207 российских спортсменов с контрольной группой выявлено не было.

Внаших исследованиях отмечена разница по частоте встречаемости генотипов у спортсменов-футболистов и контрольной группы. Для аргументированного подтверждения связи полиморфизма С34Т по локусу AMPD1 с предрасположенностью к занятиям спортом со смешанным типом, нагрузки выборка тестируемых образцов будет увеличена.

The polymorphism of gene AMPD1 in locus rs17602729 among professional football players

Bakhchevan H., Chebotar S.

The aim of the work was to determine the frequencies of alleles of gene AMPD1 atlocusrs17602729intheteamofprofessionalfootballplayersandthecontrolgroup without any sports category.According to our results, allele C dominates in football player’s’group - 93% compared to 83% in the control group and T-allele was more frequent in the control group (17% versus 7%). The distribution of genotypes in a group of players was as follows: CC - 93%, CT - 0%, TT - 7%. In the control group, the distribution of genotypes were: CC - 67%, CT - 33% TT - 0%.

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