| The oilseed rape is one of the main oil crops in the world. The oil produced from which holds an important status in the vegetable oil and its by-product (meal) is an important source of protein in the fodder industry. The cultivated area expands gradually with the adjustment of the agricultural structure. The needs of the increase of the oil content and the improvement of the oil quality were paid more attention to by many scientists in related field. The yellow-seeded B.napus characterized with lower rate of the husk, higher oil content, more transparent oil, more protein in the meal and less polyphenol, compared with the brown- or black-one. It is considered as an excellent species. But there are only natural yellow-seeded Brassica campestris and yellow-seeded Brassica juncea, and no natural yellow-seeded B.napus. All the yellow-seeded B.napus species existed were bred by artificial selection. Some yellow-seeded B.napus was selected by Qlsson in 1960 and Lou Hou-li in 1975, respectively. From then on, many breeding scientists tried to uncover the mechanism of the formation mechanism of testa luster and related characteristics. But no breakthrough was obtained because of the heredity complexity of the yellow-seeded B.napus.The yellow-seeded rape breeding has been developed for several decades since the first yellow-seeded B. napus was bred. However, the unambiguous interpretation to the instability of yellow-seeded hereditary characteristic, which has hampered the breeding process of oilrape. Therefore, it is necessary to study the formation mechanism of the yellow luster and related characteristics systemically and deep to provide theoretic principle for the molecular breeding of yellow-seeded B.napus, which stimulates the present study.In the present study, we studied the formation mechanism of testa luster and related characteristics and the protein differential expression of testa of yellow-seeded B napus using threepairs of near iso-genetic of B.napus and another cultivar-"YU HUANG NO.1" as experimental materials. The main results are as follows:1. Study on the main color mattersThe dynamic change of the main color matter in the testa of B.napus was studied. The content of carotenoids in the testa of the yellow-seeded B.napus is not significantly defferent from that in the black-one, which indicated that the carotenoids had no significant influence to the color formation of the testa of B.napus. The content of anthocyanidin and polyphenol in the black-seeded B.napus was significantly higher than that in the yellow-one with the same change tendency throughout the seed development Based on these results and the fact that change of the testa was at the later development stage, a conclusion came to that the anthocyanidin and polyphenoll might affected the pigmentation of the testa as the precursors of the pigments. The contents of chlorophyll and melanin in the yellow-seeded B.napus were not significantly different from that in the black-seeded one during the forepart period of development, During the later period the content of chlorophyll in the yellow-seeded B.napus was significantly higher than that in the black-one, but the case was opposite for the melanin. This showed that the melanin was responsible for the difference of the pigmentation between the yellow-seed B.napus and the black-one and the polyphenol had influence to the pigmentation of the yellow testa. The difference of the content of flavonoids in the testa was not significant between the two genotypes in the later development stage but in the earlier development stage. It increased continuously in the yellow-seeded one, howerer, it decreased followed by a rise in the black-one, which suggested that it could be transited into other related substances more easily in the black-seeded than in the yellow-one. A conclusion came to from the above analysis that the flavonoids had no obvious effect on the seed color of B.napus but they affected the synthesis of the color matter.In summary, the influence of the melanin is pivotal to the pigmentation of the testa of B.napus; the content of anthocyanidin and polyphenol might cause the difference through affecting the formation of the color matter; the flavonoids might act as a kind of precursor to affect the coloration; the carotenoids had no or a little effect on the difference of the testa color.2. Study on the types of the main color matterWe identified the flavonoids (except for anthocyanidin) separated by paper chromatography with UV spectrum and colorimetry. The result showed that the flavonoids in the testa of the yellow-seeded B.napus were composed of isoflavones, isoflavone glycoside, flavonol and flavonol glycoside and that in the black-one consisted of flavones, isoflavone glycoside, flavonol and flavonol glycosides. Much more isoflavone was in the testa from the yellow-seeded rapeseed andmuch more flavone was in the black-one.We identified the melanin in the testa of B.napus using infrared and ultraviolet spectrum taking human hair mixed melanin composed of inodole type and catechol type of melanin as a reference. First, we found inodole type melanin was methanol-dissolved, in contrast and the catechol type one was non-methanol-dissolved. The spectrum character of the methanol-dissolved melanin and non-methanol-dissolved. melanin from B.napus (yellow- and black-seeded) was similar to that of inodole type and catechol type from the human hair melanin , respectively. It indicated that the melanins from the testa of B.napus were composed of two types, namely inodole type and catechol type. What's more, the content of methanol-dissolved melanin was much less than that of non-methanol-dissolved one, suggesting the catechol type melanin was predominant in the testa of B.napus.3.Study on the relationship between the synthesis of the testa pigment and the corresponding precursorsThe author studied the dynamic change of nine secondary metabolites and analyzed the relationship between the nine secondary metabolites and the biosynthesis of die pigments in the testa from B.napus taking LI and L2 as experimental materials. The precursors of melanin in the black-seeded testa might be constituted of the precursors of anthocyanidin, favlones, condensed tannins, tannins, caffeic acid, ferulic acid and sinapic acid, which were less contained because they might be transited into other substances in the yellow-one. This might lead to higher melanin content in the black-one than that in the yellow-one. The caffeic acid was changed into some substance or the precursors of the pigments, which affected the synthesis of the pigments. The p-coumaric acid, ferulic acid and sinapic acid affected the content of polyphenols, which influenced melanin formation leading to the difference of the testa color. In the yellow-seeded rapeseed testa, the precursors might be transited into colored anthocyanidin and condensed tannins but melanin in the later stage of the seed development. The cinnamic acid transformed faster in the black-seeded rapeseed than in the yellow-seeded one, indicating that the poor transmission of cinnamic acid into p-coumaric acid might be the clues of the formation of the lighter color of the yellow-seeded rapeseed.4.Study on the mechanism of the formation of the testa thicknessStudies on the dynamic change of the content of husk, fibre, lignin and sugars were performed. The husk content in the black-seed rapeseed was higher than that in the yellow-one, and was significantly higher especially after the maturation of rapeseed. The difference of content of fibrebetween the two genotypes was not obvious indicating that the fibre might not contribute to the difference .The difference of lignin content was significant between the two genotypes (the yellow-seeded B.napus and the black-one) and it surpassed the difference of the husk content, which suggested that the lignin was responsible for not only the difference of husk content but also the variation of the testa luster. The content of the total sugars was significantly different between the two genotypes, indicating that the husk content was related to the content of the total carbohydrate.5.Study on the relationship between the synthesis of lignin and the corresponding precursors.We studied the dynamic change of the secondary metabolites such as cinnamic acid, p-coumaric acid, ferulic acid, caffeic acid and sinapic acid and analyzed the relationship between them and the synthesis of lignin. The above secondary metabolites had significant influence to the synthesis of lignin. Much more sinapic acid was in the testa of the black-seeded B.napus than that in the yellow-one, which indicated that sinapic acid might be one main factor contributing to the difference of testa color of the two genotypes. The poor conversion of cinnamic acid into p-coumaric acid in the testa from the yellow seed might be another factor responsible for the variation of the content of lignin. In the testa of black-seeded rapeseed, most of p-coumaric acid, ferulic acid might be the precursors of melanin and the rest was changed into lignin of the precursors of lignin. The caffeic acid was related to the lignin or the precursors of the lignin. The process of transition of p-coumaric acid into other substances in the yellow-seeded testa might be hampered.The precursors of lignin in the testa from the yellow-seeded rapeseed consisted of caffeic acid, sinapic acid and ferulic acid, which indicated that the lignins were G-type and S-type; the precursors of lignin in the testa from the black-one were composed of p-coumaric acid, ferulic acid, caffeic acid and sinapic acid, which suggested that the lignis were G-type, H-type and S-type.6.Study on the dynamic change of some enzymes related to the characteristics of the testa from B.napus.The dynamic change of the activity of PAL, PPO, POD and TRO were measured throughout the seed development process. The results showed that the activity of the fore three kinds of enzymes (PAL, PPO, POD) had more important influence to the characteristics of the testa than that of TRO. The activity of PAL showed different change tendency between the two genotypes, which reached the climax more early in the black-seeded genotype and was significantly higher than that of the yellow-one, which suggested that the massive precursors began to be synthesized at the prior development in the black-seeded rapeseed. However, much less precursors began to be synthesizedat the later stage. All of these implied that the PAL was the crucial enzyme contributing to the color difference between the two genotypes. The activity value of PPO and POD reached the maximum 41 days postanthesis (the time was pivotal to the testa coloration ) in the two genotypes, but it was higher in the black-seeded oilrape testa than that in the yellow-one, which indicated PPO and POD played important roles in the coloration of the testa. The activity of TRO was significantly higher in the black-seeded than that in the yellow-one, but the change model was similar between them, implying that TRO had a little influence to the testa coloration.7.Study on the effect of the metabolism regulation on the testa pigment and related substances.The pods and the whole plants were treated with p-Hydroxybenzoic acid (a PAL inhibitor), polyvinylpyridoxal (a PPO inhibitor), urea (a protein synthesis promoter) or chloramphenicol (CM, a plastid protein synthesis inhibitor), Vc (reducant) and the different light quality during the seed development.The application of inhibitors of PPO and PAL and the reducant decreased the content of testa pigments as well as the content of husk and lignin.; the application of the protein synthesis regulators had biggish influence on the content of the pigments and the thickness of the testa; the PAL activity was sensitive to red light but not blue light; the PAL activity was high under the treatment of red light. The results suggested that the luster and the thickness of the testa were related to the shikimate pathway and related to the activity of PPO and PAL as well as the metabolism process of protein. Besides this, we found that the inhibition of the activity of single enzyme and regulation of a single metabolism process could not change the color of the testa, suggesting that the formation of the characteristics of the testa was affected by many metabolism processes which could self-regulate.8. Study on the relationship of seed color with protein and lipid.The content of the protein in the testa rose gradually to the maximum with the seed development followed by a decline, but the difference of the content did not exist between the two types rapeseed until the maturation of the seed. The variance analysis results showed that the content of the protein varied with the species, implying that the heredity background affected the content of protein remarkably. ;The activity of GS was not significantly different between the two genotypes. But it was significantly different among species or different development stages, which meant the content of protein had no obvious effect on the GS activity.We analyzed the relationship of the synthesis of protein and the activity of GS and PAL. Theinhibition of PAL using the inhibitor and the promotion of GS by treating with blue-light affected the synthesis of protein. The GS activity affected the synthesis of protein indirectly and PAL was the primary factor that caused the difference of the content of protein.The oil content in the testa from the different genotypes was similar, a conclusion from which came to that the genotype had no or little influence to the testa oil content. On the contrary, the oil content of the whole seed in one type was obviously different from that from the other, what's more, the seed of the yellow-seeded rapeseed had much more oil than the black-one. In addition, the difference of the oil of the whole seed among the different species was significant. In summary, the oil content of the seed (the embryo, accurately ) varied with the species.9.Study on the protein differential expression between the yellow- and the black-seeded B.napusTwo-dimensional gel electrophoresis (2-DJEF/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 ).The MW of the most protein was distributed on the range of MW10-105 kD and no high MW protein was detected . There were many protein spots could be matched between the two gels from testa proteins of LI and L2 at the same development stage, which implied that the metabolism was similar between LI and L2 at the same development. On the other hand, there were many protein spots specific to LI or L2 and they were secund to acidic range or alkaline range , respectively.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. Some spots could be matched between the two gels from two closer development stages. Among the matched spots, some were up-regulated, some were down-regulated, others were equivalent. Except the matched spots, there were many spots specific to different development stage. The spots specific to the prior stage were relatively acidic, however, spots specific to the latter stage were relatively alkaline. The same phenomenon could be found in analysis ofL2.Three pairs of near iso-genetic lines of black- and yellow-seeded B. napus were sorted into two groups including black-seeded lines (LI, L3 and L5) and yellow-seeded lines (L2, L4 and L6). After the analysis of the 2-D gels of testa proteins from the two groups 40 days postanthesis, PMF of two... |
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