Cloning And Analysis Of MiRNAs From Brassica Napus Seed

MicroRNAs (miRNAs) are a newly discovered class of non-protein-coding small RNAs with 20~25 nucleotide length. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including growth and development, stress responses, hormone signal transduction and organ biogenesis and so on.Brassica napus is regarded as one of important oil crops. Its cultivation area and gross yield make China rank the first in the world and highly link to the economy of a country and the people's life. Researching on miRNA from Brassica napus seed will promote our understanding involving regulation mechanisms of seed development, synthesis pathway of vegetable oil acid and formation of oil-body. Then our work will provide us significant theoretical reference to increase its production and improve oil quality.In our study, the two strategies---computational prediction of potential miRNAs and constructing small RNA library, were used to explore potential miRNAs in Brassica napus seed. The results are described below.1. We made use of computational prediction approach for blast search for potential miRNAs in Brassica napus. And then a total of 93 unique pre-miRNA candidates classified as 31 different miRNA families, were detected meeting a series of strict filtering criterion. Using the potential miRNAs sequences, we found 325 potential target genes for 21 miRNAs families. After analyzing function of target genes,we found that 47.7% target genes encoded transcription factors involved in plant growth and development and 37.2% target genes encoded enzyme involved in plant metabolism。2. We pooled 18-28nt RNA from Brassica napus seed of 15 DAP (Day After Pollination) and 25 DAP, and constructed two small RNA libraries. Solexa sequencing generated a total of 730974 raw sequences, including 619360 sequences from 15 DAP small RNA library and 111614 sequences from 25 DAP small RNA library.3. Processing and analyzing the raw sequences above, we get a set of 724123 reads with 412781 unique sequences ranging from 18-28nt in length, and about 83% of those unique sequences were sequenced once. The majority of the small RNAs are 20-24nt in size (approximating to 90%), and the 24nt size class is predominant.4. After analyzing all the small RNA sequences, 23134 plant miRNAs homologues with 659 unique sequences were identified, and belonged to 30 miRNA families, which included 24 predicted miRNA families above. In the meantime, 121 miRNA* sequences were identified from 16 miRNA families.5. 8 miRNAs(N1,N2,N3,N4,N5,N6,N7.1和N7.2)from 7 EST sequences were detected following a range of strict filtering criteria. We predicted target genes for 8 novel miRNAs by miRU tool on line and 15 target genes were found in Brassica napus. The potential target genes of 8 novel miRNAs in Brassica napus showed that 9 target ESTs encoded functional protein, and 6 target genes encoded unknown proteins.6. Northern blotting assay was performed to detect expression of 6 miRNAs (miR158, miR167, miR169, miR172, miR393, miR1140). The result showed that 6 miRNAs had different abundance in 6 Brassica napus tissues including root, stem, leaf, shoot apical meristem, flower and seed.