INCREASING SMALL REGULATORY RNAs WITH IMMUNOMODULATING PROPERTIES UNDER GROWTH REGULATORS ACTION IN PLANT CELLS V. . Tsygankova1, Ya.

B. Blume2, T. R. Stefanovska3, S. P. Ponomarenko4

1 2

Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, Kyiv Institute of food biotechnology and genomics, NAAS of Ukraine, Kyiv 3 National University of Life and Environmental Science of Ukraine, Kyiv 4 National Enterprise Interdepartmental Science & Technology Center "Agrobiotech" of NAS and MES of Ukraine, Kyiv Abstracts Our laboratory experiments demonstrated that application of the PGR enhances sugar beet and rape plant resistance against nematode infection. This resistance is gained through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the nematode. As a result, translation of mRNA of the nematode is blocked and leads to the death of parasite larvae. Over the past 15 years, much attention has been paid to isolate the small regulatory RNAs from eukaryotic cells and to determine their biological role in RNA interference (RNAi), a term referring to post-transcriptional gene silencing (PTGS) phenomena found in all eukaryotes (including plants, animals and fungi) [1, 2, 21]. Gene silencing was first demonstrated for the free-living nematode, Caenorhabditis elegans, and the underlying mechanism of RNAi has subsequently been studied in depth for this nematode [3]. The impact of this work was recognized in 2006 by the award of the Nobel Prize in Physiology or Medicine to Andrew Fire and Craig Mello [4]. Gene silencing mediated by either degradation or translation arrest of target RNA has roles in adaptive protection against viruses, genome defense against mobile DNA elements and developmental regulation of gene expression. These silencing systems involve processing of two type small regulatory RNAs: microRNA (miRNA) and short interfering RNA (siRNA) [6, 23].
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The miRNA is generated from miRNA precursor by two rounds of endoribonuclease cleavage by RNase III-like enzymes: pre-miRNA of ~70 nucleotides (nt) is initially processed by RNase III endoribonuclease named Drosha and is hairpin primary transcript from one strand of distinct genomic loci [10]. Then pre-miRNA is exported to the cytoplasm, where mature single-stranded ~21-22-nt miRNA is generated after processing of pre-miRNA by RNase III endoribonuclease named Dicer and incorporated into micro-ribonucleoproteins (miRNPs) [10, 13]. Another type of siRNA of ~22-24-nt is generated from longer double-stranded RNA (dsRNA) molecules through their cleavage by RNase III endoribonuclease named Dicer into short single-stranded (ss) siRNA [6]. Then one part of (ss) siRNA is used for silencing of target mRNA, but other part (ss) siRNA acts as a primer on complementary mRNA leading to the production of new dsRNA with the help of RNA-dependent RNA polymerase (RdRP). It is proposed that siRNA originates from longer transcripts derived from repetitive sequences such as transposons and transgenes. The si/miRNA and the anti-sense complementary structure to mRNA function in a dual role [1, 6, 23]. The roles are: 1) together with site-specific multisubunit RNase, referred to as RNA-induced silencing complex (RISC), si/miRNA determines an age period of each mRNA molecule by destroying aberrant and defective mRNA structures that can appear faultily in the cells by specifically mRNA degradation (cleavage) or their translation arrest (silencing) and 2) si/miRNA provides protection against pathogens and parasites. In both cases, these biological effects are achieved through specifically binding si/miRNA to complementary sequences to target different classes of own plant cell mRNA (which exhibited increased expression level at specialized feeding cells in infected roots and involves plant developmental processes) [7, 16] or to pathogen mRNA [8, 9, 14] or parasite mRNA [1, 23] with high degree of homology. In animal and plant cells, si/miRNA act in different ways: si/miRNA of animals binds to the 3'-

untranslated regions (3'-UTR) or the open reading frame (ORF) of target mRNA, whereas si/miRNA of plants binds to the coding regions of target mRNA [15, 22]. With respect to protection against pathogens and parasites, the number of si/miRNA molecules produced in plant cells against these antagonists in response to a mass infection is not sufficient to provide effective protection. There are two approaches to increase the number of si/miRNA in response to pathogen or parasite attacks [1, 5, 14]. These are either to put a number of additional si/miRNA genes in cells through genetic transformation or to activate the expression of si/miRNA synthesis cellular genes themselves by certain specific inductors. Recently, in laboratory and field studies, we found that the plant growth regulators (PGRs) significantly increased plant resistance to viral pathogens, nematodes and insect herbivores [19, 20]. Thus, we further explored the possibility of enhancement by PGRs of sugar beet plant immune-protective properties through increasing synthesis of small regulatory si/miRNA and, as a result, to achieve increased plant resistance to the plant-parasitic nematode, Heterodera schachtii. The materials and methods In our experiments the sugar beet and rape plants infected by the cyst sugar beet nematode, Heterodera shcachtii Shmidt were used. Experimental plants were treated by new plant growth regulators (PGRs) Biolan, Biogen and Radostimsuper, created in the Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, jointly with National Enterprise Interdepartmental Science & Technology Center "Agrobiotech" of NAS and MES of Ukraine. These polycomponent PGRs contain complex biologically active substances (mixture of amino acids, carbohydrates, fat acids, polysaccharides, plant hormones and microelements), produced in vitro culture by symbiotic micromycete isolated out of ginseng root system and aversectines complex antiparasitic antibiotics, that are metabolites produced by soil micromycete Streptomyces avermitilis [19].

Experimental parameters: Field experiments were conducted in 2010 at Uladovo-Lyulynetska experimental breeding station (Vinnitsa region). The laboratory experiments are fulfilled in the Department of Entomology and Phytopathology of the Institute of Bioenergy Crops and Sugar Beet NAS Ukraine and have a natural background infestation of the sugar beet nematode. The 15 experimental plots of 13.5 m2 were established in a complete randomized block design. The soil composition of experimental plots was deep black earth. Structure of profile is typical for northwest agro-soil region. The depth of humus horizon is 60 cm with dust-lump structure. Granulometric composition of arable layer of soil: total sand content - 61.19 %; silt content 25.08 %; clay content 11.24 %; humus content 4,8 %; salt pH 5,8 6,2. The soil was tilled to a depth of 30-32 cm and leveled before setting up the plots. Prior to sowing, fertilizer was applied at the following rates per hectare: manure at 40 tons, N at 110 tons, P at 100 kg, and K at 100 kg. Normal farming procedures were carried out in the plots including intercrop tillage and application of a leaf fungicide, Alto Super 400, at 0.5 kg/ha. Treatment and planting of sugar beets : Sugar beet seeds (Ukrainian World Cup 70) were soaked and carefully shaken for 10 min. in water solution of PGRs, Radostim, Rasostim-super, or Biogen, at the rate of 250 ml/liter, and Biolan - at the rate of 25 ml/liter. After this procedure, the seeds were dried for 2 days at room temperature. Untreated seeds were placed in water and handled the same way as the treated seeds. The treated and untreated sugar beet seeds were sown at the rate of 130,000 seeds/ha (planting date: 24 April 2010, date of occurrence of sprouts: 02 May 2010, meteorological data at planting: air temperature: 10-14C, air moisture: 80-85 %, wind speed: 5-7 m/sec, extreme weather condition: dry and hot weather on July and August). For the PGRs foliar treatment, when the sugar beet plants were at the 6-8 leaves stage (on 25 June, 2010), Biolan-extra was applied at the rate of 50 ml/250 liters of water per 1 ha. Each experiment was repeated 3 times.
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Sampling for nematodes: Soil samples were taken to assess the number of sugar beet nematodes per cm3 of soil. Soil samples were taken (1) before sowing the sugar beets seeds on 22 April, (2) after the development of the first generation of nematodes on 1 July, and (3) after harvesting of sugar beet crop on 20 September following the procedure ISO 6057:2008 outlined in the State Standard of Ukraine 6057:2008 Sugar beets. Test methods of beet nematode harmfulness. Studies carried out on an isolated plot with total area of 2 ha. Discount land - 13,5 m2, placing at randomise areas. Reversibility of the experiment - three times. Sowing was conducted 22/04/2010 with a hybrid seed Ukrainian World Cup 70. Care of crops - generally for this forest-steppe zone of Ukraine. Soil samples were taken using shovel from a depth of 10 20 cm along two diagonals or in a zigzag fashion. Ten subsamples were taken combined into one sample per plot. Each subsample contained 50 cm3 of soil and therefore each plot had a total of 500 cm3. Soil samples were placed in polyethylene bags, labelled to indicate the sampling date and plot number, and transferred to the Department of Phytopathology and Entomology, which conducted the analysis. In the laboratory soil samples were thoroughly stirred, sieved through a sieve with a diameter of 2 mm and air-dried. Beet nematode population density in the soil was determined by the number of cyst, larvae and eggs in 100 cm3 soil by the Flotation-Funnel method. A 100 cm 3 sample was placed into 1-liter cup and 750 ml of water was added. Soil was mixed with a glass rod for 2-3 min and left to settle for 5 min. The supernatant containing the nematode cysts that had floated to the surface and organic particles were processed through a sieve with pore size diameter of 0.1-0.2 mm. This procedure was repeated three times. The cysts and particles on the sieve were washed through a funnel containing a piece of filter paper. The filter paper was examined for the nematode cysts with a dissecting microscope MBS-9 (at magnification 8 X). Cysts found on the filter were transferred into water and then counted to get the total number per sub sample. The eggs and juveniles of sugar beet nematode in the soil
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were determined by counting the number of larvae and eggs per 100 cm3 soil. The effectiveness of PGRs against the sugar beet nematode in soil was determined by the percentage difference between the number of sugar beet nematodes in soil before sowing and population density after development of the first generation on July (01/07/2010). To further assess the effectiveness of the PGRs on increasing of sugar been yield and sugar content, the sugar beet was harvested and weighed from each plot to calculate the yield per hectare. In addition, the root crop sugar content was determined on the processing line "Venema" by cold digestion. The objects and methods used in the laboratory experiments: Beet and rape seeds were sprouted in Petri dishes (9.5 cm in diameter) in nematode-free aqueous medium (control) or with a suspension of nematode eggs (at the correlation of 20-50 nematode eggs/ 20 beet and rape seeds). The seeds were incubated at 23 0C and the nematode larvae hatched in 5-7 days later in average. Each experiment performed in three replicates and consisted of 4 variants: 1) seeds incubated on aqueous medium (control), 2) seeds incubated on aqueous medium with Radostim-super, 3) seeds incubated on aqueous medium with a suspension of nematode eggs, 4) seeds incubated on aqueous medium with Radostim-super and a suspension of nematode eggs. In variant 3,4 dose of nematodes for treatment was 20-50 eggs/20 beet and rape seeds. In all experiments Radostim-super was applied at the rate of 1 mg of acting substance/1ml of water (in 10-9 dilution). In the molecular-genetic experiments we determined percent of homology between cytoplasmic mRNA population and small regulatory si/miRNA of control and experimental plants using DOT-blot hybridization method [11]. Isolation of total RNA from plant cells and separation of high-purity si/miRNA preparations using our modified methods we published early [17-20]. For research on plant si/miRNA hybridization with own plant mRNA and pathogen mRNA, before receiving si/miRNA, it was intensely marked in vivo with 33P using Na2HP33O4 [17, 18]. Testing of functional (silence) activity si/miRNA, isolated from treated by
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regulator Radostim-super plants, inhibiting translation of own plant mRNA and nematode mRNA conducted in wheat germ cell-free protein synthesis system [12]. Determination of inhibition of protein synthesis in cell-free system was carried out according to index of decreasing level of incorporation [35S] methionine into proteins and was accounted on (count per min/1mg of protein). For testing its inhibitory activity in cell-free protein synthesis systems, we used unmarked si/miRNA. The dispersion statistical analysis of the data was carried out according to Student. Results and Discussion The results of soil samples revealed that the number of sugar beet nematodes before sowing averaged 4,647 eggs and larvae/100 cm3 of soil which is 23 times higher than the economic nematode threshold of 200 eggs + larvae/ 100 cm3 of soil) (Table 1). Table 1 Effect of plant growth regulators (PGRs) influence on sugar beet nematode number in soil Application rate (ml/tons) Sugar beet nematode number, eggs + larvae/ 100 cm3 of soil SE before sowing after the 1st seeds generation (April) (July) Changing nematode population density (times) **

Treatments

Seeds incubated on 4420125 5384176 water medium Seeds treated by PGRs: Radostim 250 3671112 248796* Radostim113134* super 250 4375134 Biogen 250 4625142 207463* Biolan 25 4336116 3367107 * - availability of differences between groups, p<0.05, n=3 **- (-) decreasing; (+) - increasing

(+) 1.2

(-)1.5 (-) 3.9 (-) 2.2 (-)1.3

Results showed the effectiveness of PGRs applied for treatment of sugar beet seeds and spraying in vegetation period against beet nematode varied and ranged from 22.4 to 74.2% (Table 1). High effectiveness in reducing the number of beet nematode was shown with the application of Radostim-super. The number of nematode in the soil showed a decrease of 74.2%. Seed treatment by Biogen provided somewhat lower anti-nematode action with a reduction of the pest population density by 55.2%. Application of Biolan and Radostim provided decline of beet nematode numbers in soil by 22.4% and 32.2%. On the contrary, in control experiments (seeds treated by water) the beet nematode numbers in soil increased by 22 %. In addition to the reduction in nematode numbers, the application of PGRs increased the sugar beet yield and sugar content which were significantly higher than in the control by 1.7-6.5 and 0.6-1.4 tons/ha, respectively (Table 2). The highest indexes of sugar beet yield and sugar content were obtained after treatment of sugar beet seeds by Biogen and Radostim-super, 40.3 and 40.0 t/ha and 6.1 and 6.2 t/ha respectively. Treatment of sugar beet seeds by Radostim and Biolan provided increase of root crop yield by 2.7 and 1.7 t/ha, respectively compared to control sugar beet seeds treated with water. Table 2 Plant growth regulators influence on sugar been yield and sugar content at the end of September Yield data* Experiment variants Norm, ml/ton sugar beet yield, tons/ha SE 33.61.2 36.31.6 Sugar content, % SE 14.20.02 15.20.03* sugar yield, tons/ha SE

Control (seed treated with water) Seeds treated by PGRs: Radostim

250

4.80.01 5.50.02*
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Radostim-super Biogen Biolan

250 250 25

40.01.8* 40.11.7* 35.31.1

15.60.04* 15.30.02* 15.20.01*

6.20.03* 6.10.03* 5.40.02*

* - availability of differences from control, p<0.05, n=3 Our molecular-genetic experiments were based on the assumption that in organisms infected with different types of pathogens or parasites is induced synthesis of si/miRNA pools specific to own plant cell mRNA (which exhibited increased expression level at specialized feeding cells in infected roots and involves plant developmental processes) [7, 16] or to pathogen mRNA [8, 9, 14] or parasite mRNA [1, 23] with high degree of homology. We further assumed that the PGRs stimulated synthesis of si/miRNA which improved plant immunity through the specified mechanism of si/miRNA action. We need to be sure that our assumptions are correct so that a new generation of PGRs with the properties of selective activation of synthesis of si/miRNA specific to own plant cell mRNA or to mRNA of pathogen or parasite (with highly degree of homology) can be created. Table 3 provides the data on the level of si/miRNA synthesis in control plants, plants treated with Radostim-super, plants incubated with nematodes or plants infected with nematodes treated with Radostim-super. Our results show that Radostim-super dramatically increased cell si/miRNA synthesis, and on the contrary plant infection with nematodes reduced sharply synthesis of this class of RNA. Radostim-super compensated somewhat si/miRNA synthesis but this level, though higher than in the control, did not reach the level of synthesis induced by the plant growth regulator without nematodes.

Table 3 Impact of Radostim-super plant growth regulator on incorporation of Na2HP33O4 in si/miRNA in cells of 5-day beet sprouts incubated and nonincubated with nematode Count per min/mg of si/miRNA SE 1. Control (sprouts of plants incubated on aqueous medium) 2680 98.0 2. Sprouts got on aqueous medium with Radostim-super 4309 121* 3. Sprouts got on aqueous medium with nematode larvae 1970 83* 4. Sprouts got on aqueous medium with Radostim-super and 3760 112* nematode larvae Note: five-day sprouts of the plants were incubated with Na2HP33O4 during one hour in Petri dishes. Regulatory si/miRNA being anti-sense, complementary to mRNA were separated by our procedure of obtaining highly purified native preparations of si/miRNA. Aliquots of radioactive si/miRNA were placed on nitrocellulose substrate with subsequent counting of radioactivity. * - availability of differences from control, p<0.05, n=3 Table 4 shows the data revealing the essence of the results listed in the Table 3. As it can be seen, this is connected to the fact that nematode infection reduces synthesis of plant si/miRNA, which increases significantly at plants non-infected by nematodes and treated with Radostim-super, but this increased level is suppressed by larvae. At the same time, synthesis of protective anti-nematode si/miRNA (targeted to own plant mRNA, synthesized in specialized feeding cells in infected roots and involves plant developmental processes, as well as nematode mRNA with high degree of homology) is increased in plants, infected by nematodes and treated with Radostim-super. No. Experiment variants

Table 4
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Increase of anti-nematode properties of si/miRNA of 5-day sugar beet sprouts under impact of Radostim-super and sugar beet nematode juveniles Level of homology Experiment of hybridization variants mRNA with 33 si/miRNA (count per min/20g SE of mRNA) Hybrids of mRNA 8724 146 33 1 with si/miRNA (100%) of control plants Hybrids of mRNA 2 from control plants with 33 6850 224 si/miRNA (83%)** from plants treated with Radostimsuper Hybrids of mRNA 3 from control 5583 164 33 plants with (64%)** si/miRNA from plants incubated with nematode larvae Hybrids of mRNA 4 from control 6358 182 33 plants with (73%)** si/miRNA from plants incubated with Radostimsuper and nematode larvae Indicator of inhibition of protein synthesis in cell-free system (count per min/1mg of protein)* mRNA from plants mRNA from 33 + si/miRNA larvae from plants + 33 si/miRNA from plants 100 % 10 %

82 %

15 %

46 %

36 %

65 %

58 %

Note: free-cell system of protein synthesis from wheat sprouts was used; S 35 methionine amino acid was taken as a marked predecessor. * the same experiment variants as in the investigations on hybridization were used in the cell-free protein synthesis system. ** - availability of differences from control, p<0.05, n=3
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Similar results were obtained in our experiments and with the rape plants infecting by nematode larvae. Data presented at Table 5 allows to see that conformity with a law in the changes of si/miRNA synthesis at rape as it is observed at beet plants: the considerable increasing level of si/miRNA synthesis under action of growth regulator Radostim-super; decreasing level of own cellular si/miRNA synthesis because plants are infected by nematode larvae; decreasing of nematode infection under growth regulator action due to increasing level of synthesis si/miRNA with antinematode activity. Table 5 Impact of Radostim-super plant growth regulator on incorporation of Na2HP33O4 in si/miRNA in cells of 5-day rape sprouts incubated and nonincubated with nematode No. 1 2 3 4 Experiment variants Count per min/mg of si/miRNA SE 4760 146 6420 208* 2910 117* 5380 185*

Control (sprouts of plants incubated on aqueous medium) Sprouts got on aqueous medium with Radostim-super Sprouts got on aqueous medium with nematode larvae Sprouts got on aqueous medium with Radostim-super and nematode larvae Note: five-day sprouts of the plants were incubated with Na 2HP33O4 during one hour in Petri dishes. Regulatory si/miRNA being anti-sense, complementary to mRNA were separated by our procedure of obtaining highly purified native preparations of si/miRNA. Aliquots of radioactive si/miRNA were placed on nitrocellulose substrate with subsequent counting of radioactivity. * - availability of differences from control, p<0.05, n=3 Conclusions Thus, the results of molecular-genetic experiments indicate considerable increase in plant cells of si/miRNA synthesis (i. e. plant immunity), that leads to

inhibition of reproduction nematode larvae, formation of cyst in sugar beet cells and according to decreasing accumulation of nematode in soil (the cycle or exclusive circle of nematode reproduction are destroyed), therefore plant productivity - crop capacity per unit of sown area increases.

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