Study On The Protein Differential Expression Of Yellow-and Black-seeded Oilrape (Brassica Napus)

Compared with the black-seeded oilrape (5. napus) under the same hereditary background, the yellow-seeded B. napus has some eminent characteristics such as thinner testa, a little or no pigment accumulation in the testa, higher oil content and better oil quality, higher protein content in the meal (used as fodder) and lower content of fibre and polyphenol.Tens years have witnessed the development of yellow-seeded rape breeding since the first yellow-seeded B. napus was bred but the unambiguous interpretation to the instability of yellow-seeded hereditary characteristic, which has hampered the breeding process of rapeseed and which is paid more attention to by the breeding and related field scientists in the world who studied the genetic behaviour, testa structure, pigment types in the testa, transparent testa genes and the genes encoding the enaymes involved in the pigment synthesis pathway in many aspects containing gentics, physiology and biochemistry, molecular biology and so on.Both B. napus and Arabidopsis. thaliana belong to Cruciferae and the manner of testa pigment phenotype formation in these two species may be much similar. The latest research identified more than twenty transparent testa genes in A. thaliana, the mutation of which leads to the change of the testa lustre, encode some enzymes, related regulators and pigment-transported protein. However, no research is performed on the protein differential expression (may be related to the mechanism of the pigment synthesis) between the black - and yellow -seeded B. napus up to date which stimulates the present study on the protein differential expression of black - and yellow -seeded B. napus. In present study, three pairs of near iso-genetic lines of black- and yellow-seeded B. napus were used as experimental materials and the protein differential expression at the same development stage and protein expression change during the development of the seed of black-seeded(Ll) rapeseed and yellow-ones (L2) respectively as well as the protein expression difference between the black-seeded mix (L1.L3 and L5) and yellow- ones (L2,L4 and L6) were studied. The main results are as follows:1. The common extract buffers usually used in plant proteome research including (Tris-HCl buffer, UTC buffer and acetone/TCA) were applied to extraction of the testa protein from black- and yellow-seeded B. napus for proteome research. It proved that only the acetone/TCA buffer.is the optimum one.2. The pattern of SDS-PAGE of the testa protein from LI and L2 at the same development stage indicated that the quality and quantity of protein expression is different from one to the other, but it can not offer more information about the differential protein bands except for pi. What's more, the amounts of bands displayed in the pattern of SDS-PAGE is faraway from the actual proteins amounts because of the poor resolving power. Therefore, no conclusion related to the potential protein(s)/enzyme(s)/regulator(s) involved in the pigment biosynthesis pathway can come to merely according to the SDS-PAGE pattern.3. Two-dimensional gel electrophoresis (2-D,IEF/SDS-PAGE) was applied to exploit the potential differential protein(s) between the testa proteins from LI and L2 at the same development stage (25 days postanthesis,32 days postanthesis,39 days postanthesis and 46 days postanthesis individually ) .More protein spots can be displayed in 2-D gel than that in SDS-PAGE gel. The MW of the most protein is distributed on the range of MW10-105 kD. Neither very alkaline proteins (pI>10) nor very acidic proteins (pl<4) can be observed in the 2-D gels because the IPG strips 3-10 were adopted in this study. There are many protein spots can be matched between the two gels from testa proteins of LI and L2 at the same development stages, which implies that the metabolism is similar between LI and L2 at the same development. On the other hand, there are many protein spots specific to LI or L2 and they are secund to acidic range or alkaline range, respectively.4. The testa proteins extracted by acetone/TCA from LI at different development stage were separated by IEF/SDS-PAGE individually. From the electrophoresis profile, we found that the amount of the protein spots increased with the development of the seed and reached the maximum 32 days postanthesis followed by a decline until 46 days psotanthesis but bigger than that of stage of 25 days psotanthesis .Each two 2-D gels from two close stages were analysed setting the gel from the prior stage as reference gel throughout the whole development process . Some spots can be matched between the two gels. Among the matched spots, some are up-regulated, some are down-regulated, others are equivalent. Except the matched spots , there are many spots specific to different development stage. The spots specific to the prior stage are relatively acidic,...