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IDENTIFICATION AND CHARACTERISATION OF MICRORNAS FROM FUSARIUM OXYSPORUM 
INFECTED ROOT TISSUES OF COTTON 
 
Ayubov M.S., Shapulatov U.M., Buriev Z.T., Shermatov Sh.E., Ubaydullayeva H.A.,
Abdurakhmonov I.Y. 
Center of Genomics and Bioinformatics, Academy of Science of Uzbekistan, Ministry of Agriculture and Water 
resource and “Uzpakhtasanoat” association 
MicroRNAs are non-coding small RNAs, containing 20-24 nucleotides. Regulatory functions of 
microRNAs were determined in plants, including differentiation of plant organs, responsibility to abiotic and 
biotic stresses and other biological processes. Experiments show that expression of miR-156, miR-160 and miR-
171 microRNAs are regularly correlated with pathogen fungi in wheat (Triticumaestivum). 
The main goal of this study was to extract and characterize small RNAs from root tissues of resistant and 
suitable cotton lines inoculated with Fusarium oxysporum sp. Vasinfectum (FOV). FOV resistant Mebane B-1 and 
suitable №11970 cotton varieties have been chosen for the experiments.
Firstly, each seeds were put into MS (Murashige and Skoog) 0.7% agar medium. Back roots grew during 
72 hours. After nearly five weeks of growth, the seedlings of each line (316 stamps, collected by Institute of 
Genetics and Plant Experimental Biology) were infected with FOV conidia suspension. Total RNA were extracted 
using Wu method. Electrophoresis was run in 15% denaturing polyacrylamide gel. Approximately 18-30 
nucleotides of the total RNA were cut from the gel, and 3’ and 5’ linkers were linked to RNAs, using miRCat
TM
cloning method (Integrated DNA Technologies, USA).
cDNA was synthesized from linked products using reverse transcription enzymes (SuperScript
TM
III, 
Invitrogen, USA). cDNAs were amplified with special primers. These PCR products were linked to each other 
using DNA ligase enzymes. Octomerized small RNAs were cloned to E.coli bacterium using TOPO TA cloning 
kit (Invitrogen, USA). Bacterial clones have been amplified with universal M13 primers. Consecutive 

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oligonucleotide sequences were analyzed on ABI Genetic Analyzer 3100xl machine (Applied Biosystems, USA). 
Before selection of small RNAs, all ABI results were reanalyzed on Sequencher 4.5 (Gene Codes, USA) computer 
program. Sequenced small RNAs were analyzed using mirBase data base collection.
In total, 4116 small RNAs, having 16-30 nucleotide sequences have been analyzed from Mebane B-1 and 
№ 11970 cotton varieties (Table 1). The mirBase collection showed that these small RNAs sequences contain 
only 4% of all sequences belong to 15 microRNA families (Table 2).
While miR2911 microRNA was found in both infected and control samples, ghr-miR3476-5p micro RNA 
was found only in FOV resistant Mebane B-1 control plants. Analysis of the ghr-miR3476-5p micro RNA showed 
that this micro RNA connected with mRNA of the “leucine-rich receptor-like” and kinase proteins. The ath-miR-
160a and ath-miR-172 were found only in Mebane B-1 control samples. The ath-miR-160a regulates the growth 
of seedlings, developing of roots and responsibility to auxin. This micro RNA is not expressed under pathogen 
stresses. This feature led them to be called as reverse correlation to pathogen stresses. The ath-miR-172 micro 
RNA is regulated by various biotic and abiotic stresses in plants at post transcription level. Homologies of ghr-
miR2949, zma-miR156 and zma-miR395 micro RNAs have found in Mebane B-1. The ghr-miR2949 micro RNA 
was found to be expressed in cotton ovules according to mirBase. Target gene of the miR395 is ATP sulfurylase 
enzyme, which catalyzes the assimilation of the inorganic sulfates. This enzyme improves the tolerance of plants 
to stresses. The zma-miR166 micro RNA was extracted from FOV non-resistant suitable № 11970 and control 
plants. This micro RNA mainly expresses in various plants under abiotic stresses.
Functions of the micro RNAs are very important for regulation of cotton resistance to biotic stresses. 
Several micro RNAs have been expressed in resistant and suitable cotton genomes after infection with fungi. 
Some micro RNAs were found from each sample, indicating them not to be important for the stress processes. 
Finding the ghr-miR2949, miR156 and zma-miR395 micro RNAs in the infected resistant cotton lines show that 
these micro RNAs may play very important role for the tolerance against biotic stresses. 
Overall, investigations of the mechanisms of FOV resistance in cotton are very important in terms of 
determination the role of the micro RNAs. Application of the gene engineering technology for this study will give 
us to get new varieties faster than traditional methods.