AUTHOR=Gasparis Sebastian , Kała Maciej , Przyborowski Mateusz , Orczyk Waclaw , Nadolska-Orczyk Anna 

TITLE=Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations

JOURNAL=Frontiers in Plant Science

VOLUME=7

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2016.02017

DOI=10.3389/fpls.2016.02017

ISSN=1664-462X

ABSTRACT=<p>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 silence <italic>Puroindoline a</italic> (<italic>Pina</italic>) and <italic>Puroindoline b</italic> (<italic>Pinb</italic>) hardness genes in wheat and their orthologues <italic>Secaloindoline a</italic> (<italic>Sina</italic>) and <italic>Secaloindoline b</italic> (<italic>Sinb</italic>) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of <italic>Pina</italic>/<italic>Sina</italic> or <italic>Pinb</italic>/<italic>Sinb</italic> genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the <italic>Pinb</italic>-derived amiR vector for silencing of <italic>Pinb</italic> or <italic>Sinb</italic>, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the <italic>Pina</italic>-derived amiR cassette, despite large numbers of embryos attempted. Silencing of <italic>Pinb</italic> in wheat and <italic>Sinb</italic> in triticale was highly efficient in the T<sub>1</sub> generation. The transcript level of <italic>Pinb</italic> in wheat was reduced up to 92% and <italic>Sinb</italic> in triticale was reduced up to 98%. Moreover, intended silencing of <italic>Pinb</italic>/<italic>Sinb</italic> with <italic>Pinb</italic>-derived amiR cassette was highly correlated with simultaneous silencing of <italic>Pina</italic>/<italic>Sina</italic> in the same transgenic plants. High downregulation of <italic>Pinb</italic>/<italic>Pina</italic> genes in T<sub>1</sub> plants of wheat and <italic>Sinb</italic>/<italic>Sina</italic> genes in T<sub>1</sub> plants of triticale was associated with strong expression of <italic>Pinb</italic>-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, the <italic>Pinb</italic>-derived amiR cassette was stably inherited in the T<sub>2</sub> generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of <italic>Pinb</italic>-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.</p>