INVESTIGADORES
RUIZ Oscar Adolfo
congresos y reuniones científicas
Título:
EDITING OF KEY STRUCTURAL GENES FOR PROANTHOCYANIDIN BIOSYNTHESIS IN LOTUS CORNICULATUS
Autor/es:
DAMIANI F.; MONTENOVO E.; BELIA A.; ESCARAY F.J.; RUIZ O.A.; PAOLOCCI F
Lugar:
VERONA
Reunión:
Congreso; 62.mo SIGA Annual Congress ; 2018
Institución organizadora:
SIGA-Italian Society of Agricultural Sciences-
Resumen:
Proanthocyanidins (PAs),also known as condensed tannins, are a class of polymers of flavan-3-ol units,represented by the sole epicatechin (EC) in the model Arabidoppsis or by bothEC and catechin (C)units in may crop species. The concentration, structure anddegree of polymerization of PAs highly influence forage digestibility inruminants and, consequently, their productivity and environmental footprint.Among forage legumes, Lotus corniculatus is regarded as a model species fordissecting 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) andanthocyanidin reductase (ANR), enzymes deputed to the synthesis of C and ECunits, respectively, have been previously isolated by our group and theirexpression profiles investigated in a number of PA mutants obtained via i) theoverexpression of MYB and bHLH activator or repressor genes; ii) interspecifichybridization between L.corniculatus and PA depleted Lotus tenuis.Nevertheless, the contribution of the two metabolic branches to the overall PAaccumulation and the in vivo role of LAR in producing C units remain openquestions. To address these points we embarked on the production of a loss offunction LAR and ANR mutants by means of CRISPR/Cas9 genome editing tool. Tothis end, a diploid L. corniculatus line was stably transformed withAgrobacterium rhizogenes binary vectors harbouring an editing cassette wherethe CAS9 nuclease was driven by the Petroselinum crispum UBI4-2 promoter andthe single guide RNA(sgRNA) by the Arabidopsis U6-26 promoter. For both genestwo sgRNAs were designed and employed. Several transgenic plants for each ofthe four transforming events were obtained and the sequencing of the targetlocus was performed in a few of them. Indeed, the occurrence of editing,although with a difference extent, was observed in all lines analysed. Theydisplayed deletions of a few nucleotides, ranging from 6 to 15, upstream theprotospacer adiacent motif (PAM). Phenotypic, metabolic and gene expressionanalyses in primary transformants and their progeny will help us to elucidatethe contribution of and interplay between the ANR and LAR branches in buildingthe PA trait in forage legumes.