Presentazione, 2018, ENG

EDITING OF KEY STRUCTURAL GENES FOR PROANTHOCYANIDIN BIOSYNTHESIS IN LOTUS CORNICULATUS

DAMIANI F.*, MONTENOVO E.*, BELIA A.*, ESCARAY F.J.**, RUIZ O.A.**, PAOLOCCI F.*

*) Institute of Biosciences and Bioresources CNR, sect. Perugia, Via Madonna Alta 130, 06128 Perugia (Italy) **) IIB-INTECH (UNSAM-CONICET), Chascomús (Argentina)

Proanthocyanidins (PAs), also known as condensed tannins, are a class of polymers of flavan3-ol units, represented by the sole epicatechin (EC) in the model Arabidoppsis or by both EC and catechin (C)units in may crop species. The concentration, structure and degree of polymerization of PAs highly influence forage digestibility in ruminants and, consequently, their productivity and environmental footprint. Among forage legumes, Lotus corniculatus is regarded as a model species for dissecting the genetic control of PAs, since it accumulates both EC and C- based PAs in edible organs and is amenable for genetic transformation. L. corniculatus genes coding for leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), enzymes deputed to the synthesis of C and EC units, respectively, have been previously isolated by our group and their expression profiles investigated in a number of PA mutants obtained via i) the overexpression of MYB and bHLH activator or repressor genes; ii) interspecific hybridization between L.corniculatus and PA depleted Lotus tenuis. Nevertheless, the contribution of the two metabolic branches to the overall PA accumulation and the in vivo role of LAR in producing C units remain open questions. To address these points we embarked on the production of a loss of function LAR and ANR mutants by means of CRISPR/Cas9 genome editing tool. To this end, a diploid L. corniculatus line was stably transformed with Agrobacterium rhizogenes binary vectors harbouring an editing cassette where the CAS9 nuclease was driven by the Petroselinum crispum UBI4-2 promoter and the single guide RNA(sgRNA) by the Arabidopsis U6-26 promoter. For both genes two sgRNAs were designed and employed. Several transgenic plants for each of the four transforming events were obtained and the sequencing of the target locus was performed in a few of them. Indeed, the occurrence of editing, although with a difference extent, was observed in all lines analysed. They displayed deletions of a few nucleotides, ranging from 6 to 15, upstream the protospacer adiacent motif (PAM). Phenotypic, metabolic and gene expression analyses in primary transformants and their progeny will help us to elucidate the contribution of and interplay between the ANR and LAR branches in building the PA trait in forage legumes.

LXII SIGA Annual Congress, Verona, Italy - 25/28 September, 2018, 25/28 September, 2018

Keywords

proanthocyanidins, CRISPR/Cas9, genome editing, gene expression

CNR authors

Damiani Francesco, Paolocci Francesco

CNR institutes

IBBR – Istituto di Bioscienze e Biorisorse

ID: 440081

Year: 2018

Type: Presentazione

Creation: 2020-12-23 12:30:09.000

Last update: 2020-12-23 12:31:07.000

External links

OAI-PMH: Dublin Core

OAI-PMH: Mods

OAI-PMH: RDF

External IDs

CNR OAI-PMH: oai:it.cnr:prodotti:440081