Research On The Key Genes Of Lipid Synthesis In Brassica

Plant microRNAs (miRNAs) are single-stranded RNA molecules of around 22 nucleotides (nt) in length that are associated with the RNA-induced silencing complex (RISC). They act as post-transcriptional negative regulators of gene expression mainly by guiding cleavage or attenuating the translation of target transcripts. They are processed as duplexes (miRNA/miRNA*) from non-coding miRNA transcripts (pri-miRNA) containing imperfect intramolecular stem loops by RNaseâ…¢in the nucleus. The mature guide-stranded miRNAs mediate sequence-specific, post-transcriptional repression of mRNA targets through the miRNA-induced silencing complex (miRISC). Plant miRNAs are involved in controlling diverse cellular processes including development and responses to biotic and abiotic stress, They have been confirmed to regulate a variety of processes, such as development, metabolism, signal trasduction and stress responses.In this study, we cloned and sequenced 400 small RNAs from Brassica rapa subsp. Oleifera. Analyse of the 400 small RNAs showed that nine conserved miRNA families and three potential novel miRNAs in Brassica rapa subsp. oleifera were identified. Then we compared the database of Brassica rapa subsp. oleifera and Arabidopsis. We found some potentially target of conserved miRNA. The targets of some miRNAs were predicted through computational approach and the miRNAs cleavage sites were identified by 5'RACE. We also predicted genes that can regulate lipid synthesis and are regulated by microRNAs by bioinformatic analysis. However we did not obtain any repeatable miRNA cleavage sites of predicted targets by 5'RACE.So this sites of predicted targets were not cleavage by miRNA.In this study, we found that transgenic Arabidopsis of GFP-sensor (35S-GFP-3'UTRLDP1 or 35S-GFP-3'UTRLDP1m) that the 3'UTR of LPD1 gene encoding lipoamide dehydrogenase, which is potentially targeted by miR847, or a miR847-resistant LPD13'UTR. We analyzed them by northern blot and western blot with anti-GFP antibody, the results showed that miR847 regulated GFP-sensor and LPD1 translation. This suggested that miR847 might regulated LPD1 expression in Arabidopsis.