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Functional characterization of microRNAs from brassica species

Student name: Ms Anuradha Bansal
Guide: Dr Anandita Singh
Year of completion: 2010
Host Organisation: The Energy and Resources Institute (TERI)

Abstract: Members of genus Brassica are important horticultural and vegetable crops. The high genetic diversity among the various species and their agricultural importance make them interesting subject for scientific studies. MicroRNAs (miRNAs) are 19–24 nt RNAs that direct the posttranscriptional silencing of transcripts of target genes with high complementarity to the miRNA. MicroRNAs are different from other classes of small RNAs (sRNAs) because they act in trans on non-self RNAs. MiR156 is a regulatory gene whose targets belong to the SPL (SQUAMOSA promoter binding protein like) class of transcription factors, which are specific to plants. The function of SPL genes with predicted miR156 target sequences largely unknown however, these SPLs are known to be upregulated during flowering. In this study, attempts have been made to clone mir156 orthologs from various species of Brassica. Bioinformatics analysis revealed the presence of two putative mir156 orthologs (mir156b and mir156e) in Brassica genome. Homology status was examined through AVID/VISTA alignment tool. Initial attempts did not amplify the targeted regions. New primers were designed and mir156b orthologs from various Brassica sp. were cloned and sequence verified. Conserved regions among Arabidopsis and Brassica were identified using DnaSP software. Spatio-temporal analysis of mir156 in Brassica juncea var. Varuna indicated the role of mir156 in phase transition from juvenile to flowering/adult stage in the apical tissues as it has been shown in Arabidopsis. Mir156 is highly expressed in inflorescence apex or flower buds to down-regulate the SPL expression and keep the tissue in juvenile stage. qRT-PCR was carried out for the expression studies and the results were analyzed with delta delta Ct method. The results were complemented with expression data of mir156 and its target SPLs in Arabidopsis.

KEYWORDS: Brassica, Arabidopsis, mir156, SPLs, qRT-PCR