甘蓝型油菜脂肪酸合成抑制与消减因素的研究

发布时间：2018-06-28 11:42

本文选题：芸薹属油菜 + 脂肪酸消减因子　；参考：《浙江大学》2016年博士论文

【摘要】：世界上几种主要油料作物,例如大豆、油菜、花生、向日葵以及棕榈,油菜是排在大豆油与棕榈油之后的第三大油料作物。在中国,油菜是种植面积最大的油料作物,然而,与传统高产油地区(德国、加拿大)相比,我们的亩产水平则相对较低,所以提高我国油菜单位面积产油量,对缩减我国与高产油地区之间的差距,保障我国的食用油安全,具有重要的战略意义。迄今为止,已有大量的研究旨在增加油菜种子含油量,例如提高脂肪酸代谢途径关键酶的活性,调控底物的竞争关系,调节外源环境信号以促进脂肪酸的合成。但是关于油菜成熟种子脂肪酸的消减,以及制约种子脂肪酸高效合成的抑制因子,至今报道很少。这里,我们开展了一系列实验,针对以上科学问题进行研究:(1)成熟种子脂肪酸消减的基因型差异以及特定的转录组分析;(2)昼夜温差对不同基因型油菜种子含油量的影响;(3)种子含油量抑制因子BnaC.TT2.a的等位多态性与种皮颜色、种子含油量以及组分之间的相关性。主要的研究结果如下:1.分析了 4种基因型油菜,中油511,浙油50,九二13系以及B6-3,在种子发育成熟过程中含油量的变化趋势。发现中油511成熟收获的种子,含油量相比高峰期下降了 16.8%,而其它3份基因型油菜种子含油量一直呈现递增的趋势。利用RNA-seq测序技术,分析了中油511在种子成熟后期(40-DAP)以及种子发育前期(16-DAP)的全转录组水平的差异。结果发现,中油511种子成熟后期显著上调的基因有13648个,显著性下调的基因有15239个(显著性水平:|log2(FoldChange)|1,Q0.005),对差异性基因作KEGG富集分析后发现,在显著性上调的基因中,与糖类碳水化合物代谢相关的基因得到显著性富集,并且与脂肪酸消减直接相关的β-氧化途径基因也在种子成熟后期受到显著性上调,据此,我们归纳了中油511在种子成熟后期一系列影响种子含油量的关键基因。2.证实了昼夜温差环境因子对油菜种子含油量的影响,分析高含油量基因型(浙油50)以及低含油量基因型(九二13系)。结果发现,相比于小昼夜温差系统,大温差系统下的浙油50、九二13系种子含油量均显著较高,浙油50的落差是13.74%(P0.05),九二13系的落差是18.9%(P0.05)。大昼夜温差系统下夜晚的低温可以抑制乙醛酸代谢途径基因的表达,并可调节植物内源激素赤霉素的信号强度。处于大小昼夜温差系统中的浙油50、九二13系,油滴合成基因在白天与黑夜的表达模式是相反的,进一步分析发现,乙醛酸代谢途径基因的表达模式正好与之符合。对乙醛酸代谢途径的候选基因的启动子序列分析发现,大部分基因的启动子序列均存在着GAGA-similar的保守序列,并且,据报道该保守序列是响应高温调控的顺式作用元件。对转录组数据的深入挖掘发现,大昼夜温差中夜晚的低温可以调控DELLA蛋白基因的表达,RT-qPCR实验数据也证实:大昼夜温差系统中夜间赤霉素信号传导基因受到抑制。我们据此设计了两年两点的大田试验,实验结果也佐证了赤霉素信号在脂肪酸积累过程中的负面效应,而且当外源喷施多效唑(降解赤霉素)时,可以提高油菜含油量在9-18%的水平(增油效果受种植年份以及地点的影响)。3.找到了脂肪酸合成抑制因素BnaC.TT2.a位点的等位多态变化与种皮色泽及脂肪酸含量、组分的相关性。通过收集不同生态区域的83份甘蓝型油菜种质资源,我们成功构建了适用于关联分析的甘蓝型油菜自然群体,该群体不仅适合分析调控种子含油量的基因位点,而且可以分析其它潜在的功能基因。分析BnaC.TT2.a碱基多态性与脂肪酸表型的关联性时,在BnaC.TT2.a上的第1个内含子上鉴别出4个显著性连锁的SNPs,在第3个外显子上的738位置鉴定出1个插入突变位点,而且该插入位点导致TT2蛋白功能发生了改变。以上关联结果说明了BnaC.TT2.a在调控甘蓝型油菜种皮颜色、种子亚油酸以及总脂肪酸含量等重要农艺性状中的作用。
[Abstract]:Several major oil crops in the world, such as soya beans, rape, peanuts, sunflowers, and palm, and palm, and palm, are the third major oil crops in the soybean oil and palm oil. In China, rape is the largest oil-bearing crop, however, compared with the traditional high oil region (Germany, Canada), our yield is relatively low. In order to improve the oil yield per unit area of rapeseed in China, it is of great strategic significance to reduce the gap between China and high yield oil areas and ensure the safety of edible oil in China. So far, a large number of studies have been conducted to increase the oil content of rape seeds, for example, to improve the activity of key enzymes of fatty acid metabolites and to regulate the competitive relationship of substrates. Regulation of exogenous environmental signals to promote the synthesis of fatty acids. But there are few reports on the decline of fatty acids in rapeseed mature seeds and inhibition factors that restrict the efficient synthesis of fatty acids. Here, we have carried out a series of experiments to study the above scientific problems: (1) the genotypic difference of fatty acid reduction in mature seeds And specific transcriptional analysis; (2) the effect of day and night temperature difference on oil content in the seeds of different genotypes; (3) the correlation between the allelic polymorphism of the seed oil content inhibitor BnaC.TT2.a and the seed coat color, the seed oil content and the components. The main results are as follows: 1. analysis of 4 types of rapeseed, 511 of oil, 50, nine of Zhejiang oil. Two 13 lines and B6-3, the change trend of oil content in the process of seed maturation. It was found that the seed of 511 mature harvest, oil content decreased by 16.8% compared to the peak period, and the oil content of the other 3 rapeseed seeds has been increasing gradually. Using RNA-seq sequencing technology, the medium oil 511 in the late seed maturity (40-DAP) was analyzed. The difference in the total transcriptional level of 16-DAP was found. The results showed that 13648 genes were significantly up-regulated in the late maturity of the seed oil 511, and 15239 significantly down regulated genes (significant levels: |log2 (FoldChange) |1, Q0.005). After KEGG enrichment and analysis of the differential genes, the genes were significantly up-regulated and sugar was significantly increased. The genes related to carbohydrate metabolism were significantly enriched, and the beta oxidation pathway genes directly related to fatty acid reduction were also significantly up-regulated in the late stage of seed maturity. Accordingly, we concluded that a series of key genes affecting seed oil content in the late stage of seed maturity by.2. confirmed the day and night temperature difference environment. The effect of the seed oil content of rape seed was analyzed, and the oil content genotype (Zhejiang oil 50) and the low oil content genotype (92 13 lines) were analyzed. Compared with the small day and night temperature difference system, the oil content in the 50 and 92 13 lines of Zhejiang oil under the large temperature difference system was significantly higher, the drop of Zhejiang oil 50 was 13.74% (P0.05), and the drop of 92 13 system was 18.9% (P0.05). The low temperature at night at night temperature difference system can inhibit the expression of glyoxylic acid metabolism pathway gene and regulate the signal intensity of the plant endogenous hormone gibberellin. In the 50 and 92 13 lines of Zhejiang oil in the day and night temperature difference system, the oil drop synthetic gene is opposite to the expression pattern of day and night. Further analysis and analysis of glyoxylic acid The expression pattern of the metabolic pathway gene coincides with it. Analysis of the promoter sequence of the candidate genes for glyoxylic acid pathway shows that most of the genes have a conservative sequence of GAGA-similar, and it is reported that the conservative sequence is a cis acting element in response to high temperature regulation. It was found that the low temperature at night in the large day and night temperature difference could regulate the expression of DELLA protein gene. The RT-qPCR experimental data also confirmed that the nocturnal gibberellin signal transduction gene was suppressed in the large day night temperature difference system. We designed a two two two point field experiment accordingly. The results also supported the results of the gibberellin signal in the accumulation of fatty acids. Negative effects, and when exogenous spray of Paclobutrazol (degrading gibberellin), the oil content of rape can be increased at the level of 9-18% (the effect of planting year and location)..3. found the allelic polymorphism of the BnaC.TT2.a locus of fatty acid synthesis, the correlation of the seed color and fatty acid content, and the correlation of the components. 83 Brassica napus germplasm resources in different ecological regions, we successfully constructed the natural population of Brassica napus which is suitable for correlation analysis. This group is not only suitable for analyzing gene loci regulating seed oil content, but also analyzing other potential functional genes. The relationship between BnaC.TT2.a base polymorphism and fatty acid phenotype is analyzed. In the first introns on the BnaC.TT2.a, 4 distinct linkage SNPs were identified, and 1 insertion mutation sites were identified in 738 exons of third exons, and the insertion site resulted in a change in the function of TT2 protein. The above correlation results showed that BnaC.TT2.a was used to regulate the color of the seed coat of Brassica napus, seed linoleic acid and The role of total fatty acid content and other important agronomic traits.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

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