【作者】 蒋金金；

【导师】 王幼平；

【作者基本信息】 扬州大学 ， 植物学， 2013， 博士

【摘要】 黄籽甘蓝型油菜与褐籽甘蓝型油菜相比具有很多优点,如种皮薄、含油量高、高蛋白、纤维素和木质素含量低、油质清澈等,已经成为近年来油菜育种改良的重要目标之一。本研究利用前人从甘蓝型油菜-白芥体细胞杂种后代筛选获得的黄籽株系,通过NIRS、FTIR、NMR等方法比较该黄籽材料与褐籽甘蓝型油菜(扬油6号)的品质差异,借助LC-ESI-MSn比较分析黄、褐籽种子成熟过程中类黄酮等色素成分含量的差异；通过比较蛋白质组学(2-DE)研究了黄、褐籽材料种子成熟过程的蛋白质差异,利用iTRAQ定量比较分析黄、褐籽材料成熟种子油体蛋白的差异；通过数字基因表达谱(DGE)比较黄、褐籽材料种子成熟过程中基因表达的差异,并通过qRT-PCR进行了验证、以及与蛋白质组差异进行了关联分析,探讨与甘蓝型油菜黄籽性状形成的相关基因。主要研究结果如下：黄、褐籽甘蓝型油菜的品质分析NIRS分析表明黄籽甘蓝型油菜的含油量、蛋白质含量、硫代葡萄糖苷和芥酸较褐籽甘蓝型油菜扬油6号有所改善,黄籽株系的NDF、ADF、 ADL含量比扬油6号低；ATR-FTIR分析表明白芥与扬油6号脂类物质、蛋白质酰胺键等化学基团的振动峰相比差异明显,黄籽材料间也存在差异,黄籽株系的这些振动峰与白芥或扬油6号相似,但是黄、褐籽材料种皮的碳水化合物没有明显差异；NMR分析鉴定出油菜种皮中代表不同化学基团的谱峰,发现白芥与扬油6号在代表木质素相关基团的谱峰上有明显差异,扬油6号NMR谱图中代表纤维素和木质素的峰明显比白芥和黄籽株系强,这与黄籽材料的木质素和纤维素含量比褐籽材料低的结果是一致的。黄、褐籽甘蓝型油菜类黄酮等的HPLC分析通过LC-ESI-MSn我们成功鉴定了油菜种皮中的主要多酚化合物(芥子碱、芥子酸和1,2-芥子酰基蔗糖)和16种类黄酮物质,包括表儿茶素及其单体聚合物、槲皮素、山奈酚、异鼠李素-二己糖苷、山奈酚-芥子酰-三己糖苷、异鼠李素-芥子酰-三己糖苷、异鼠李素-己糖硫酸、异鼠李素-3-O-糖苷。大部分黄酮类物质随着种子成熟过程不断积累,部分类黄酮在种子成熟后期含量减少,黄籽株系的类黄酮含量比褐籽低。此外,不溶性原花青素、可溶性酚酸的含量在黄、褐籽材料中也有明显差异。黄、褐籽甘蓝型油菜种子成熟过程中比较蛋白质组分析通过蛋白质组学方法比较了黄、褐籽甘蓝型油菜种子成熟过程蛋白质组学的变化,以期寻找与黄籽性状相关的蛋白质表达特征。利用双向电泳(2-DE)对种子总蛋白进行分离,MALDI-TOF-MS质谱分析鉴定了27个差异蛋白点,成功鉴定出8个蛋白,包括蛋白激酶、烯醇化酶、磷酸异构酶、双加氧酶等。基于差异蛋白的推测氨基酸序列设计引物进行验证,表明蛋白点H3-5为远缘杂交后代的新出现的蛋白,H5-2来自于亲本白芥,且该黄籽材料与现有其他来源的黄籽不同。黄、褐籽甘蓝型油菜成熟种子油体蛋白的比较通过分离纯化褐籽扬油6号(Bn)、白芥(Sa)、黄籽株系(W82)的油体,并将提取的油体蛋白进行水解后,经iTRAQ标记肽段,经串联质谱对肽段进行鉴别和定量。我们成功鉴定3091个特异多肽片段,通过同源比对分析鉴定到1511种蛋白,其中包括常见的油体蛋白：15个油质蛋白(oleosin)、5个钙结合油质蛋白(steroleosin)、3个固醇油质蛋白(caleosin)。此外,我们检测出p-葡糖苷酶、α-葡糖苷酶、黑芥子酶、黑芥子酶结合蛋白、胚胎特异蛋白(ATS)、热激蛋白(HSP)和抗氧化蛋白等,以及cruciferin和napin等种子贮藏蛋白；一些质体膜蛋白、膜转移酶、膜受体、ATP合成酶以及ATP结合蛋白也成功检测到,这些蛋白可能源自蛋白体、线粒体等的污染。通过对蛋白的定量比较分析,我们发现白芥和W82之间有270个差异蛋白,其中92个蛋白在W82中上调、178个蛋白表达量下调：白芥与扬油6号相比有167个蛋白上调、86个蛋白下调；W82与扬油6号相比27个蛋白上调、37个蛋白下调。对差异表达蛋白进行聚类分析,我们发现85个蛋白在3个比较组合(Bn vs W82, Sa vs W82, Bn vs Sa)中均有差异,其中包括油质蛋白S1-2、S4-1和S4-3。黄、褐籽甘蓝型油菜种子成熟过程的数字基因表达谱分析利用DGE技术比较黄籽后代(W82)与褐籽亲本种子成熟过程中RNA表达水平的差异,通过基因表达注释及差异表达基因的分析,筛选与黄籽性状相关的差异表达基因。我们发现黄、褐籽不同发育时期类黄酮生物合成途径的基因表达差异很大,脂肪酸生物合成、代谢途径的基因表达也有差异。通过对参与类黄酮生物合成途径的差异基因进行qRT-PCR验证,发现这些基因的表达水平随着种子的成熟也呈下降趋势,这与HPLC的分析结果是一致的。新型黄籽甘蓝型油莱遗传规律分析将黄籽株系与褐籽扬油6号杂交,对杂种F1、F2代种皮色泽进行统计；同时将杂种F1分别与黄籽株系与褐籽扬油6号进行回交,推断该黄籽性状由2对独立遗传基因控制,当这2对基因都隐性纯合时,种皮表现为黄色。更多还原

【Abstract】 Yellow-seeded Brassica napus has several admirable advantages over its black-seeded phenotype, including thinner seed coat, higher oil and protein content, lower lignin and fiber content, and purer oil quality. Development of yellow-seeded cultivars is becoming an optimum pathway to improve rapeseed quality and quantity. The objective of this study was to conduct a detailed comparison of yellow-seeded progenies from B. napus-Sinapis alba hybrids and black seeds of B. napus. With the assistant of NIRS, FTIR and NMR analysis, we compared the quality differences between yellow-and black-seeded materials. LC-ESI-MSn analysis of flavonoid extracted from yellow-and black-seeded B. napus revealed the different accumulation and content of these chemicals. Comparative proteomics of seed filling between yellow-and black-seeded materials was taken out using two-dimentional electrophoresis (2-DE). Oil body proteins extracted from yellow and black seed B. napus, S. alba were quantitatively compared using iTRAQ and Q-TOF MS/MS. DGE analysis was also applied to discriminate transcriptome difference between yellow-and black-seeded B. napus, and the differentially expressed genes related to flavonoid biosynthesized pathway were verified using qRT-PCR. The research would aid in dissecting the genes related to the formation of yellow-seeded B. napus. The main results were shown as followings:Quality analysis of yellow-and black-seeded B. napus NIRS analysis revealed that yellow seed lines used in this research possess higher oil and protein content, low glucosinolate and erucic acid than black-seeded Yangyou6. The content of NDF, ADF and ADL in yellow seeds was lower than Yangyou6. ATR-FTIR analysis showed many differences in lipid feature, protein feature were observed between yellow-and black-seeded B. napus, and the FTIR spectra was also different among yellow seed lines. Variation of carbohydrate features in yellow and black seed materials was not obvious. By comparing the NMR spectra of yellow and black seed materials, we found the difference between S. alba and Yangyou6were most obvious at peaks representing for lignin. The resonance peaks standing for lignin and fiber were stronger in Yangyou6than S. alba and yellow seed lines, which are consistent with the perspective that yellow seed material possess lower fiber and lignin than black B. napus.HPLC analysis of flavonoids in yellow-and black-seeded B. napus We applied LC-ESI-MSn to identify phenylpropanoid and flavonoid in rapeseeds. The most abundant phenolic compounds (sinapine and sinapic acid) and1,2-disinapoylglucose,16different flavonoids were identified and quantified, including (-)-epicatechin and its five monocharged oligomers, quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin-sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids was lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties.Proteomics analysis of seed filling between yellow-and black-seeded B. napus Comparative proteomics of seed filling between yellow and black seed were taken out to find proteins related to yellow seed character. By using two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS analysis, eight out of the27discriminate proteins were identified by MASCOT comparison, including protein kinase, enolase, triosephosphate isomerase and dioxygenase. PCR primers contrived for the putative genes were applied for further identification of progenies and both parent, indicating H3-5might be the novel protein of intergeneric hybrid, H5-2derived from S.alba. Applying these specific primers, this study demonstrates that the new yellow seed germplasm is different from the existing yellow seed materials.Comparison of oil body proteins in yellow-and black-seeded B. napus Oil body from Yangyou6,S. alba, yellow seed line were purified for protein extraction. The oil body protein were digested and labeled with iTRAQ reagents, and peptides were analyzed and quantified by Q-TOF MS/MS mass spectrometer. Finally, we successfully identified3091unique peptides representing for1511proteins, including fifteen oleosin proteins, five steroleosin and three caleosin. Besides the main OB proteins, beta-glucosidase and alpha-glucosidase, myrosinase, myrosinase-binding protein, myrosinase-associated protein, embryo specific protein (ATS), heat shock protein (HSP) and peroxiredoxin were also identified. Obvious contamination of storage proteins, including cruciferin and napin were found in protein storage vesicles. Besides, mitochondrial import inner membrane translocase, mitochondrial import receptor, secretory carrier membrane protein, mitochondrial ATP synthase and ATP binding protein were also included as contamination from mitochondria. Among all the identified proteins,270proteins displayed significant differences (p<0.05) between W82and S. alba, of which92proteins were up-regulated and178proteins were down-regulated in W82.167proteins were up-regulated and86proteins were down-regulated in S. alba compared with Yangyou6.27proteins were up-regulated and37proteins were down-regulated in W82compared with Yangyou6. Clustering analysis of differentially expressed proteins between each comparison (Bn vs W82, Sa vs W82, Bn vs Sa) was carried out,85identified proteins were found to have commonly changed in each comparison, of which only three oleosins were included (oleosin S1-2, oleosin S4-1and oleosin S4-3).Digital gene expression (DGE) analysis on seed filling of yellow-and black-seeded B. napus We compared gene expression difference of yellow seed line (W82) and Yangyou6using DGE analysis. By GO annotation and pathway analysis of differentially expressed genes (DEG), we selected DEGs that might be related to yellow seed character. Obvious gene expression difference was found in the flavonoid biosynthesis pathway, fatty acid biosynthesis, fatty acid metabolism. Transcriptome changes of genes participating in the flavonoid pathway were discovered by qRT-PCR analysis. Consistent with flavonoid changes identified by HPLC analysis, the expression for most genes in the flavonoid biosynthetic pathway was also downregulated.Genetic analysis of the new yellow-seeded B. napus In this research, the yellow seed line was hybridized with Yangyou6to obtain Fi and F2, the seed coat color of this two generation was analyzed. F1generation was also backcrossed with the yellow seed parent and Yangyou6, respectively. By calculation of the seed coat color segregation, we deduced that the yellow seed character was controlled by two pairs of independent genes. The yellow seed phenotype was presented only when both pairs of genes were recessive.更多还原