【作者】 石从广；

【导师】 蒋立希；

【作者基本信息】 浙江大学 ， 作物学， 2010， 博士

【摘要】 油菜是世界上重要的油料作物,含油量的提高和脂肪酸品质改良是当今油菜育种重要的目标。分子标记辅助选择育种(MMAPB)是提高作物农艺性状和种子品质性状的有效手段,现有的各种DNA分子标记的开发应用以及丰富的基因组和ESTs序列数据信息为遗传研究和分子标记辅助育种提供了极大的便利。人们对于模式植物拟南芥油脂合成相关基因功能的深入理解对于油菜品质育种具有极大的参考价值。然而据我们所知,目前几乎还没有关于油菜的油脂代谢相关基因位点的等位变异与种子脂肪酸成分积累的关系的研究报道。本文旨在研究那些偏向于在种子中表达,并且影响脂肪酸组分的关键基因位点在EMS诱导的M2群体中,以及54个地方栽培品种资源中的等位多态性,及其这些等位变异与种子品质,特别是不饱和脂肪酸含量之间的关系。研究的结果如下：1.利用甲基磺酸乙酯(EMS)溶液处理甘蓝型油菜双低品种“浙双72"种子,经田间M1代观察和M2代筛选及部分M3代验证,最终筛选到叶色、叶形、株高、茎色、花色、花瓣、雄性不育及开花期等性状发生明显变异的M2突变株；对M3种子的主要脂肪酸组分之间,以及脂肪酸组分与种子含油量进行了相关与回归分析,同时描述了各种脂肪酸组分和含油量的变异幅度与分布,提取了2558份包含突变性状的M2植株叶片的DNA。2.对基因FAD2、FAD6、FAD7和FAD8在甘蓝型油菜幼嫩种子和叶片中的转录水平差异作了分析,发现FAD2和FAD6在叶片和种子中转录水平无明显差异,而FAD7和FAD8基因更偏向于在叶片中转录。同时对6个FAD基因的12条BAC克隆序列和GeneBank的EST序列运用DNAStar5.0的MegAlign程序进行序列比对,结果以聚类分枝树的形式呈现,结果显示FAD3、FAD7和FAD8的序列相似度较高,FAD2的序列与FAD5和FAD6的序列处与不同的分枝,三者相似度较低。3.利用HRM技术分析FAD2基因的SNP变异,最终在比对区域统计出12个导致氨基酸序列变化的M2样品。在这12个样品中,、只有3个样品种子的油酸含量相对CK有显著提高,有6个油酸含量显著降低,有3个油酸含量与对照差异不明显。3个样品(代号529-3,1841-2和1823-4)种子油酸含量的升高可能与比对区域DNA序列的变异导致的FAD2酶蛋白氨基酸的突变有关。4.从拟南芥600多个与油脂合成和代谢相关基因位点中选出75个倾向于在种子中表达的基因来研究所选油菜地方种质资源的基因等位多态性,一共在上述75个基因上设计了150对引物,平均每个基因包含两对引物。在Tapitor和宁油7号两个油菜品种间进行筛选标记发现52对引物具有多态性,这些具有多态性的引物被当作ILP标记,在上述52个标记中挑出34个多态性较好的标记,这些标记被用来检测包含54个油菜地方栽培品种的油脂代谢基因的内含子的多态性。平均每对引物在覆盖的区域扩增出5.6个等位位点,分析油脂代谢相关基因的等位多态性与种子含油量和脂肪酸含量存在的相关性发现标记FAD7-ics11170(3/4)与种子中亚麻酸的积累相关,等位多态型FAD7-ics11170(3/4)-c与高亚麻酸含量相关,说明这个标记可以作为筛选高亚麻酸育种材料的有效手段。综上所述,本项研究描述了EMS诱导的M2群体和部分油菜地方品种中调控脂肪酸组成的基因位点的等位变异,以及这些等位变异与种子品质性状的关系。这些研究结论丰富了人们对于油菜种子油脂代谢分子机制的理解,并且为高不饱和脂肪酸含量分子标记辅助育种奠定了良好的基础。更多还原

【Abstract】 Brassica oilseeds are important sources of edible oil in many parts of the world. Molecular Marker-Assisted Plant Breeding (MMAPB) is highly efficient in improving crop agronomic and seed quality traits. The availability of various kinds of DNA molecular markers and the sequence data of genomes and ESTs facilitate genetic research and MMAPB programs. Knowledge about the genes implicated in lipid biosynthesis acquired from the model plant Arabidopsis is useful in understanding the formation of seed oil in Brassicas.Nevertheless, little is known about allelic variations of the genes regulating lipid biosynthesis and their association with a certain kind of fatty acid accumulation in Brassica oilseeds. The current study was envisaged to identify the DNA polymorphisms of functional genes that are preferably expressed in seeds and are putatively important to the fatty acid composition among EMS-induced M2 individual plants as well as a collection of 54 local Brassica oilseed cultivars. The process of our research and the results are as followed:1. The ethyl methan sulfonate (EMS) was applied to treat the pure seeds of the ’double low’cultivar "Zhe-Shuang 72" (Brassca napus L.). The mutants with abnormal appearance of leaf color, branch number, plant height, stem or flower color, floral fertility and life cycle were observed in M1 population, screened in the M2 population and partly verified in the M3 population. In general, the mutants accounts for 8.46% of the total population in the M2 generation. Correlation analysis among the fatty acids and between the fatty acids and total oil content has been conducted in the M3 seeds. The variation degrees of the fatty acid components and oil content of the M3 seeds are described. A DNA pool from 2558 individual M2 plants were constructed for the analysis of allelic variations of the major genetic loci regulating grain quality traits. Materials with particular mutated traits are available for breeding projects targeting genetic improvement of oilseed rape.2. The comparison of the transcription level of the FATTY ACID DESATURATION genes as FAD2, FAD6, FAD7 and FAD8 between leaf and seed tissues revealed that FAD2 and FAD8 transcribed with similar level in seeds and leaves, whereas FAD7 and FAD8 preferably expressed in leaves to seeds. Alignment among sequences of the FAD genes indicated a higher similarity among FAD3, FAD7 and FAD8 genes. On the other hand, the sequences of FAD2, FAD5 and FAD6 were more different from each other and also diverged significantly from the group including the FAD3, FAD7 and FAD8 genes. Three primer pairs covering the coding region of FAD2 were used for High Resolution Melting (HRM) PCR and SNP detection.3. A total of 12 SNPs that gave rise to non-synonymous amino acid changes were identified on the basis of one-thousand M2 individual plants. Six of the corresponding mutants were characterized with lower oleic acid content, and three of the mutants demonstrated higher oleic acid content, whereas the other three mutants was not significantly different from the control in terms of oleic acid content.4. Polymorphic markers at the loci putative for the seed oil formation between two geographically different genotypes:the Chinese cultivar Ningyou-7 and the European cultivar Tapidor were systemically screened. These primer pairs (150) were designed based on 75 Brassica genes that were Arabidopsis orthologues implicated in the oil formation. A total of 52 out of the 150 primer pairs associated with 47 of the 75 genes showed polymorphisms between the two genotypes. The type of polymorphisms that could be detected on capillary electrophoresis images and their respective visual futures are described. Further,34 polymorphic markers were selected to scan allelic variations and rich DNA polymorphisms among the 54 Brassica oilseed cultivars were found. On the average, each primer pair resulted in 5.6 alleles at the region that was covered. The correlation between the alleles and seed quality traits revealed that the alleles of BnFAD7 were related to the variation of linolenic acid (C18:3) contents among the cultivars. The allele FAD7-ics11170 (3/4)-b that was significantly correlated with high linolenic acid content can be used as an efficient marker for the selection of breeding materials with high linolenic acid content.Taken together, we described the allelic variation of some loci that regulate seed fatty acid composition among an EMS-induced M2 population as well as a collection of Brassica oilseeds germplasm. Our results could be an enriching addition to the body of work that attempts to understand the accumulation of fatty acids in Brassica oilseeds. The start-up work to screen for alleles correlating with high content unsaturated fatty acids laid a solid foundation for MMAPB project towards the improvement of the seed quality of Brassica crops.更多还原