Arabidopsis thaliana ecotype Col-0 double-stranded RNA treatment against the chalcone synthase gene

Recent studies show that plants can accept and process exogenous double-stranded RNAs (dsRNAs) engineered to repress the transcription of various target genes. Understanding plant perception of exogenous dsRNAs may lead to the development of new biotechnological approaches to improve the valuable properties of plants for agriculture.In our work in the evening 2 plants of wild-type Arabidopsis thaliana ecotype Col-0 were treated with water, 2 plants with double-stranded RNA (dsRNA) against the chalcone synthase gene (dsCHS) and 2 plants with dsRNA against the NPT2 gene (dsNPT2), which is not present in the wild-type plant. The plants were then placed under stress conditions at +7 degrees C under constant light to induce anthocyanin biosynthesis. On day 2 after treatment, a high-molecular-weight RNA fraction and a small RNA fraction were isolated from each plant. Complementary DNA (cDNA) was obtained from high-molecular-weight RNA fractions. Small RNA fractions were sent for high-throughput sequencing using Illumina technology to Eurogen (Moscow). Incoming sample quality control was performed by horizontal electrophoresis in agarose gel. RNA was prepared for sequencing using the Qiagen Small RNA Sample Prep kit (Qiagen). The quality of the obtained libraries was checked using Fragment Analyzer. Quantitative analysis was performed by quantitative PCR. After quality control and DNA quantity estimation, the library pool was sequenced on an Illumina NovaSeq 6000 instrument (1 x 100 bp single-read sequencing). The FASTQ files were obtained using bcl2fastq v2.20 Conversion Software (Illumina). The recording format of the quality data string is Phred 33.Using the obtained cDNA, we evaluated the level of chalcone synthase (CHS) transcripts by quantitative real-time PCR. Treatment of plants with dsCHS resulted in decreased CHS gene expression levels compared to plants treated with water or dsNPT2.3' adaptor sequences: AACTGTAGGCACCATCAAT, AGATCGGAAGAGCACACGT