Differential Proteomic Analysis Of Two Brassica Napus Lines Differing In The Oil Contents

Rapeseed (Brassica napus L.) is one of the most important oilseed crops in the world. Improving the oil content is the main object for breeders at present. Oil content is an important and complex metabolic trait of B. napus, Lots of studies revealed that some factors, such as seed coat, embryo, and endosperm that correlated with oil content were studied, but few reshearches were focused on the proteomic studies on seed oil content. The two-dimensional polyacrylamide gel electrophoresis (2-DE) was the main technique for proteomic analysis, which can analyze dynamic changes of proteomics of biological samples under different conditions. It is generally considered that the protein content is negatively correlated with oil content, and the size of oil bodies in B. napus was also found to have a relationship with oil content. This research established a suitable system for B. napus to analyze proteomics and follow closely to the proteomic difference between low oil content and high oil content of B.napus. The main results were shown as follows:(1) Established a suitable system for B.napus for proteomics research, found out the appropriate protein extraction method and IEF procedure. The results showed that using TCA/acetone protein extraction method, pH 4-7 IPG strip and improved IEF procedure can get good results of 2-DE gels. This system established by us can get fine results of seedling, leaf, stem and seed of B.napus, this indicate that this system is widely suitable for B. napus proteomic research in the future.(2) For TEM comparing analyze of low oil content and high oil content of B.napus, the results showed that the size of oil body in the high oil content B.napus is always normal and the array of them are inseparably close, but in the low oil content B.napus, most of oil bodies are bigger than the normal size and appear large oil body structure. Big size oil body especially large oil body goes against the space utilization and is not conducive to oil storage. In deep analyze showed that the number of protein storage vacuole in low oil content B.napus is distinct more than in high oil content B.napus, this indicate that the protein storage ability of low oil content B.napus is stronger than the high oil content B.napus, and the final protein content is higher.(3) The total protein difference in mature seeds on two B. napus cultivars, which with the oil content of 36.49% and 55.19% by using 2-DE technique, and 143 protein spots changed more than 2 times were found in total protein. 120 protein spots of total protein have been successfully identified by using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) method; peptide sequences were analyzed with MASCOT and matched against with the NCBI database. The present results revealed that these different protein spots can be classified by function to 9 classifications including: Carbohydrate Pathway protein, Cell Growth/Division protein, Defense/Desease protein, Metabolism of Amino Acids protein, Protein Biosynthesis protein, Secondary Metabolism protein, Storage Functions protein, Transporters protein and Unclear Classification protein. This indicated that the differences in the formation of fatty acids is a highly complex physiological and biochemical processes.(4) 2-DE different protein expression experiments on this two B. napus cultivars detected 50 different protein spots and successfully indentified 34 protein spots of them by MOLDI-TOF-MS, these different protein spots can be classified by function to 7 classifications including: Carbohydrate Pathway protein, Cell Growth/Division protein, Defense/Desease protein, Secondary Metabolism protein, Storage Functions protein, Transporters protein and Unclear Classification protein. Most different expression protein spots are myrosinase, myrosinase-binding protein, 12S storage protein, late embryogenesis-abundant protein, cruciferin et al. Some Cell Growth/Division protein also be detected in our experiments, these may be related with the different size of oil body and different oil content in seed embryo between different oil content B.napus.