Identification and expression analysis of genes involved in somatic embryogenesis of centaury (Centaurium erythraea Rafn.)
Identifikacija i analiza ekspresije gena uključenih u somatsku embriogenezu kičice (Centaurium erythraea Rafn.)
Abstract:
Centaurium erythraea Rafn. is a medicinal plant that easily regenerates through somatic embryogenesis (SE) in vitro. In order to identify genes involved in SE, six different centaury tissues were sequenced resulting in a assembly of a de novo transcriptome, which was functionally annotated against NCBI nt, Swissprot and PFAM databases with enrichment according to KOG and GO databases. Analysis of the expression of 11 housekeeping genes in tissues and organs at various developmental stages revealed that ribosomal protein L2 (RPL2) and TATA-binding protein 1 (TBP1) are optimal reference genes with the most stable expression during SE. In the transcriptome, potential SE marker genes were identified whose expression was evaluated in a collection of tissues and organs. Among them, the highly specific expression of the so far unknown CeNA1 transcript in the embryogenic callus stood out, which makes it a potential marker of the early stage of SE. By mapping the transcript sequence on the centaury genome, seven members of the CeNA1 gene family with an intact ORF were found, whose promoter sequences were analyzed in silico and the exon-intron structure of the gene was determined. Additionally, the intracellular localization and 3D structure of the CeNA1 protein was determined in silico. Secondary and cyclic SE in centaury were induced using 2,4-D and CPPU. A medium containing 0,1 mgl-1 2,4-D and 0,25 mgl-1 CPPU allowed three cycles of SE and proved to be optimal in terms of the number of primary somatic embryos forming secondary embryos and the number and morphology of secondary embryos per explant. Secondary embryos with a high rate of germination and conversion into plants were additionally histologically analyzed. It was observed that CeNA1 gene expression increases in embryogenic tissue during the transition from primary to the first cycle of secondary SE, and then remained relatively stable. Furthermore, the effect of genotype on the regenerative capacity of the embryogenic callus was confirmed, whereby the expression of selected genes, including CeNA1, decreased with the increase in differentiation of the embryogenic tissue.
Keywords:
Centaurium erythraea Rafn.; kičica; CeNA1; centaury; somatic embryogenesis; differentially expressed genes; secondary somatic embryogenesisSource:
University of Belgrade, Faculty of Biology, 2023, 1-184Funding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200007 (University of Belgrade, Institute for Biological Research 'Siniša Stanković') (RS-MESTD-inst-2020-200007)