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Design of artificial miRNA for redundant silencing of Brassica SHP1 and SHP2: transient assay-based validation of transcript cleavage from polyploid Brassicas

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Abstract

Asynchronous pod-shatter results in significant yield losses in canola. SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) regulate pod shatter in Brassicaceae. This study describes the potential of an artificial RNA (amiRNA)-based approach for redundant silencing of SHP1/SHP2 homologs in Brassicas for introducing shatter resistance. After surveying the natural variation in Brassica SHP homologs, amiR-bnashp1 and amiR-bnashp2 were designed and processed from miR319a precursor in N. benthamiana. Analysis of thermodynamic interaction between amiRNAs and SHP1/SHP2 homologs predicted high binding energy for amiR-bnashp2 [total free energy of binding (TFEB): −23.54 kcal/mol] and not for amiR-bnashp1 (TFEB: 6.01 kcal/mol). As predicted, agro-co-infiltration assay validated amiR-bnashp2-mediated cleavage of SHP1 and SHP2 homologs from B. napus, B. juncea and A. thaliana, while amiR-bnashp1 failed to direct cleavage of these homologs. In conclusion, for trait manipulation in polyploid genomes deployment of amiRNAs is suggested to silence redundant genes. Furthermore, an a priori knowledge on transcriptome potential of genome concerned is a prerequisite for predicting efficiency and specificity of target selection by amiRNA(s).

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Acknowledgments

This work was supported by grants (BT/PR10071/AGR/36/31/2007 and BT/PR628/AGR/36/674/2011) received from Department of Biotechnology, Govt. of India. The authors thank Dr. Sandip Das for offering valuable suggestions. Plasmid pRS300 was received as a kind gift from Prof. Detlef Weigel. Financial assistance as SRF to Priyanka Dhakate from Council of Scientific and Industrial Research and to S.M. Shivaraj from Department of Biotechnology, Govt. of India is gratefully acknowledged. Infrastructural support from TERI and TERI University are duly acknowledged.

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  1. Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi, 110070, India

    Priyanka Dhakate, S. M. Shivaraj & Anandita Singh

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  1. Priyanka Dhakate
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  2. S. M. Shivaraj
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  3. Anandita Singh
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Correspondence to Anandita Singh.

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Communicated by E. Schleiff.

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Dhakate, P., Shivaraj, S.M. & Singh, A. Design of artificial miRNA for redundant silencing of Brassica SHP1 and SHP2: transient assay-based validation of transcript cleavage from polyploid Brassicas. Acta Physiol Plant 36, 2125–2135 (2014). https://doi.org/10.1007/s11738-014-1589-6

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  • DOI: https://doi.org/10.1007/s11738-014-1589-6

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