title: Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.
abstract: Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silencePuroindoline a(Pina) andPuroindoline b(Pinb) hardness genes in wheat and their orthologuesSecaloindoline a(Sina) andSecaloindoline b(Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions ofPina/SinaorPinb/Sinbgenes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using thePinb-derived amiR vector for silencing ofPinborSinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using thePina-derived amiR cassette, despite large numbers of embryos attempted. Silencing ofPinbin wheat andSinbin triticale was highly efficient in the T1generation. The transcript level ofPinbin wheat was reduced up to 92% andSinbin triticale was reduced up to 98%. Moreover, intended silencing ofPinb/SinbwithPinb-derived amiR cassette was highly correlated with simultaneous silencing ofPina/Sinain the same transgenic plants. High downregulation ofPinb/Pinagenes in T1plants of wheat andSinb/Sinagenes in T1plants of triticale was associated with strong expression ofPinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, thePinb-derived amiR cassette was stably inherited in the T2generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression ofPinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.
authors.affiliation: Department of Functional Genomics, Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Blonie, Poland.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Gasparis
authors.affiliation: Department of Functional Genomics, Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Blonie, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Kała
authors.affiliation: Department of Functional Genomics, Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Blonie, Poland.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Przyborowski
authors.affiliation: Department of Genetic Engineering, Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Blonie, Poland.
authors.forename: Waclaw
authors.initials: W
authors.lastname: Orczyk
authors.affiliation: Department of Functional Genomics, Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Blonie, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Nadolska-Orczyk
keywords: Pin genes; RNAi; amiRNA; grain hardness; polyploid cereals; siRNA; triticale; wheat