Regulation Of Growth And Seed Development Of Brassica Napus By MIR394

microRNAs (miRNAs) are a class of non-coding,20-24 nt, single-stranded and endogenous small RNA, which play important roles in plant development, signal transduction and responses to environmental stressors by inhibiting expression or translation of their target genes. F-box containing proteins are a integral part of ubiquitin-proteasome degradation pathway involved in plant growth and development and abiotic stress responses. miRNA394 (miR394) is a conserved miRNA. Its target gene LCR (LEAF CURLING RESPONSIVENESS LCR) is a member of the F-box protein family. Rapeseed (Brassica napus) is the world's third largest oil economic crop. In this paper, we cloned miR394 and its target gene BnLCR from Brassica napus using homology cloning approach. CDS of LCR is 1404 bp, encoding 467 amino acids, which has 88% similarity to Arabidopsis LCR. The DNA sequence of LCR is 2013 bp in length and contains a 629 bp intronTo characterize BnmiR394, we generated transgenic Brassica napus plants 35S::BnMIR394a,35S::BnMIR394b,35S::antiBnLCR,35S::BnLCR and miR394-resistant Bnm6LCR (35S::Bnm6LCR) respectively using Agrobacterium-mediated genetic transformation system. Analysis of transcripts in the genetic plants showed that expression of BnmiR394 precursor (pre-BnmiR394) and BnmiR394 was increased, while the expression of BnLCR was decreased in 35S::BnMIR394a and 35S::BnMIR394b plants, indicating that the BnMIR394a and BnMIR394b were successfully transferred to Brassica napus and could down-regulate the expression of its target gene LCR. In 35S::BnLCR and 35S::Bnm6LCR plants, expression of BnLCR were increased and more transcripts were observed in 35S::Bnm6LCR plants, suggesting that Bnm6LCR is resistant to the cutting of BnmiR394. AntiBnLCR was also successfully transferred to Brassica napus.Functional analysis showed that down-regulation of BnLCR in 35S::BnMIR394a, 35S::BnMIR394b and 35S::antiBnLCR plants resulted in enlarged seed size; the seed mass was increased by 32.5 to 50.1% compared with untranformants; it was also found that the transgenic plants usually have increased seed protein, glucosinolate content, but decreased oil content. In addition to the above properties, the fatty acid composition changed in 35S::BnMIR394b. Linoleic acid and erucic acid contents were increased. In 35S::BnLCR and 35S::Bnm6LCR plants, however, smaller seed size was observed, with mass weight decreased by 10.5-26.7% compared with untransformants. There were higher oil and lower protein and glucosinolate contents, with litte change in fatty acid composition in LCR over-expression plants. With regard to the growth and development,35S::BnMIR394a, 35S::BnMIR394b and 35S::antiBnLCR showed taller, stronger, higher branch position, more and larger leaves and flowering postponed. On the contrary,35S::BnLCR and 35S:: Bnm6LCR plants were smaller and shorter, with lower branch position, less leaves and earlier flowering. These results indicate that BnmiR394 and its target BnLCR are involved in regulating the growth and development processes and oil formation of Brassica napus.