Overexpression Of BnOLE Produces Larger Seeds And Organs In Brassica Napus

Rapeseed(Brassica napus) is one of the three major oil rape varieties, and the most widely planted oil crops in our country. Rapeseed oil accounted for about half of Chinese total consumption of vegetable oil. As China’s oil crop production can not be self-sufficient, we need to import large amounts of vegetable edible oil from Canada and Australia every year. Since seed size is an important agronomic trait and one of the main factors affecting the yield of rapeseed. It is important to find the genes affecting the seed sizeand and to explore the possible mechanism which may play an important role in crop breeding and agricultural production.We cloned a BnOLE gene encoding OLEOSIN protein from the cDNA library of Brassica napus. Bioinformatics analysis showed that BnOLE had oleosin superfamily domain. Protein motif analysis revealed that BnOLE has three different sizes of motifs, which are mainly composed of basic and conserved amino acids. By phylogenetic analysis, we found that genetic relationship between BnOLE and AtOLE5 of Arabidopsis is closest. Then we use the Arabidopsis database to analyse the AtOLE5(the homologous gene of BnOLE). We found that AtOLE5 expressed highest in the Ripe Seed during analyzing the expression of AtOLE5 in different tissues in the microarray database of Arabidopsis thaliana.We constructed an overexpression vector of BnOLE, and transform them into wild type rapeseed through In Planta Agrobacterium-mediated transformation. After screening by Basta, PCR identification and expression level analysis, we obtained several independent BnOLE overexpression transgenic lines. Among them, 3 highest expression lines were selected for further study. Observation and anlysis of the statistical data on rape seed, cotyledon, leaf, flowers, siliques, embryo, hypocotyl and cotyledon of the seed, as well as cells and oil body in cells demonstrated that overexpression of BnOLE increased the size of seed through promoting the number of cells increase.We predicted the genes which directly interacted with AtOLE5(BnOLE homologous genes in Arabidopsis thaliana) in the Arabidopsis genome database which contains 9264 genes and 40008 relationship between those genes by the Cytoscape software. The results showed that there were only two genes interacting with AtOLE. One of them is the encoding gene of transduction protein W40 upstream of AtOLE5 and the other one is the encoding gene of Ring-type E3 ubiquitin-protein transferase downstream of AtOLE5. AmiGO 2 analysis showed that E3 ubiquitin protein transferase had anaphasepromoting complex activity, ubiquitin ligase activity and so on. GW2 in rice and DA2 in Arabidopsis(they belong to homologous gene) encode an E3 ubiquitin ligase involved in proteasomal degradation pathway to regulater the seed size. We speculate that there may be a relationship between BnOLE and BnDA2, and the seed size was regulated by BnDA2 in the proteasome degradation pathway. Analysis of expression of BnDA2 in BnOLE overexpression plants and wild type plants by qRT-PCR showed that BnDA2 was severely inhibited in BnOLE overexpression plants. Consequently, we speculate that overexpression of BnOLE inhibit the expression of BnDA2 and remove the inhibition of BnDA2 on its downstream substrate, then promote cell proliferation, and ultimately lead to increase of seed size, which are consistent with our phenotypic analysis.