Clonging, Construction And Transformation Of MiR394a/b/c And Its Target F-Box From Brassica Napus

microRNAs (miRNAs) are a new class of endogenous non-coding small RNAs (-22nt). They play important roles in development, hormone response, signal transduction, plant disease and stressful response. miRNAs base-pair their target genes to repress their translation or induce their degradation. We previously constructed a library of small RNAs from B. napus under sulfate deficiency. Sequencing the library resulted in identification of 9 known miRNAs including miR156, miR159, miR167a, miR167b, miR167c, miR168, miR169m, miR393 and miR395. Additionally,6 novel miRNAs were detected. To get insights into the regulatory role of these miRNAs, we performed experiments focusing on trasncription analysis of the miRNAs under sulfate deprivation. Expressions of these miRNAs were shown to be differentially regulated by sulfate starvation. We also identified miR394 as one of the newly found miRNAs. Its target F-box is an important component of SCF complex and plays important roles in hormone response, lateral root formation, and light signalling. In this study I cloned cDNA sequence of a putative F-box from Brassica napus and analyzed how it was regulated by miR394. It has an open reading frame (ORF) of 1404 bp, encodes a 467-amino acid protein. The complementary vector of F-box gene was also constructed. Finally, the overexpression vector pCAMBIA1304-pre-miR394a/b/c was constructed and transgenic seeds were obtained.