Identification and expression analysis of genes involved in somatic embryogenesis of centaury (Centaurium erythraea Rafn.)

Abstract

Kičica (Centaurium erythraea Rafn.) je lekovita biljka koja lako regeneriše putem somatske embriogeneze (SE) u uslovima in vitro. U cilju identifikacije gena sa ulogom u SE, sekvencirano je šest različitih tkiva kičice i de novo sastavljen transkriptom koji je funkcionalno anotiran prema NCBI nt, Swissprot i PFAM bazama podataka sa obogaćivanjem prema KOG i GO bazama. Analizom ekspresije 11 housekeeping gena u tkivima i organima različitih faza razvića utvrđeno je da su ribozomalni protein L2 (RPL2) i TATA-vezujući protein 1 (TBP1) optimalni referentni geni sa najstabilnijom ekspresijom tokom SE. U transkirptomu su identifikovani potencijalni geni markeri SE čija je ekspresija evaluirana u kolekciji tkiva i organa. Među njima se naročito izdvojila specifična ekspresija do sada nepoznatog transkripta CeNA1 u embriogenom kalusu, što ga čini potencijalnim markerom rane faze SE. Mapiranjem sekvence transkirpta na genom kičice pronađeno je sedam članova CeNA1 genske familije sa intaktnim ORF-om, čije su sekvence promotora in silico analizirane i utvrđena je egzon-intron struktura gena. Takođe, in silico je određena unutarćelijska lokalizacija i 3D struktura proteina CeNA1. Sekundarna i ciklična SE kod kičice je indukovana primenom 2,4-D i CPPU. Podloga koja sadrži 0,1 mgl-1 2,4-D i 0,25 mgl-1 CPPU omogućila je tri ciklusa SE i pokazala se optimalnom u pogledu broja primarnih somatskih embriona koji formiraju sekundarne embrione i broja i morfologije sekundarnih embriona po eksplantatu. Sekundarni embrioni sa imali visoku stopu klijavosti i konverzije u biljke i dodatno su histološki analizirani. Utvrđeno je da se ekspresija CeNA1 gena povećava u embriogenom tkivu tokom prelaska iz primarne ka prvom ciklusu sekundarne SE, a potom ostaje relativno nepromenjena. Pored toga, potvrđen je efekat genotipa na regenerativnu sposobnost embriogenog kalusa, pri čemu ekspresija odabranih gena, uključujući CeNA1, opada sa porastom diferenciranosti embriogenog tkiva.

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.