本文選題：蕓薹屬油菜 + 脂肪酸消減因子　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：世界上幾種主要油料作物,例如大豆、油菜、花生、向日葵以及棕櫚,油菜是排在大豆油與棕櫚油之后的第三大油料作物。在中國(guó),油菜是種植面積最大的油料作物,然而,與傳統(tǒng)高產(chǎn)油地區(qū)(德國(guó)、加拿大)相比,我們的畝產(chǎn)水平則相對(duì)較低,所以提高我國(guó)油菜單位面積產(chǎn)油量,對(duì)縮減我國(guó)與高產(chǎn)油地區(qū)之間的差距,保障我國(guó)的食用油安全,具有重要的戰(zhàn)略意義。迄今為止,已有大量的研究旨在增加油菜種子含油量,例如提高脂肪酸代謝途徑關(guān)鍵酶的活性,調(diào)控底物的競(jìng)爭(zhēng)關(guān)系,調(diào)節(jié)外源環(huán)境信號(hào)以促進(jìn)脂肪酸的合成。但是關(guān)于油菜成熟種子脂肪酸的消減,以及制約種子脂肪酸高效合成的抑制因子,至今報(bào)道很少。這里,我們開展了一系列實(shí)驗(yàn),針對(duì)以上科學(xué)問(wèn)題進(jìn)行研究:(1)成熟種子脂肪酸消減的基因型差異以及特定的轉(zhuǎn)錄組分析;(2)晝夜溫差對(duì)不同基因型油菜種子含油量的影響;(3)種子含油量抑制因子BnaC.TT2.a的等位多態(tài)性與種皮顏色、種子含油量以及組分之間的相關(guān)性。主要的研究結(jié)果如下:1.分析了 4種基因型油菜,中油511,浙油50,九二13系以及B6-3,在種子發(fā)育成熟過(guò)程中含油量的變化趨勢(shì)。發(fā)現(xiàn)中油511成熟收獲的種子,含油量相比高峰期下降了 16.8%,而其它3份基因型油菜種子含油量一直呈現(xiàn)遞增的趨勢(shì)。利用RNA-seq測(cè)序技術(shù),分析了中油511在種子成熟后期(40-DAP)以及種子發(fā)育前期(16-DAP)的全轉(zhuǎn)錄組水平的差異。結(jié)果發(fā)現(xiàn),中油511種子成熟后期顯著上調(diào)的基因有13648個(gè),顯著性下調(diào)的基因有15239個(gè)(顯著性水平:|log2(FoldChange)|1,Q0.005),對(duì)差異性基因作KEGG富集分析后發(fā)現(xiàn),在顯著性上調(diào)的基因中,與糖類碳水化合物代謝相關(guān)的基因得到顯著性富集,并且與脂肪酸消減直接相關(guān)的β-氧化途徑基因也在種子成熟后期受到顯著性上調(diào),據(jù)此,我們歸納了中油511在種子成熟后期一系列影響種子含油量的關(guān)鍵基因。2.證實(shí)了晝夜溫差環(huán)境因子對(duì)油菜種子含油量的影響,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)。結(jié)果發(fā)現(xiàn),相比于小晝夜溫差系統(tǒng),大溫差系統(tǒng)下的浙油50、九二13系種子含油量均顯著較高,浙油50的落差是13.74%(P0.05),九二13系的落差是18.9%(P0.05)。大晝夜溫差系統(tǒng)下夜晚的低溫可以抑制乙醛酸代謝途徑基因的表達(dá),并可調(diào)節(jié)植物內(nèi)源激素赤霉素的信號(hào)強(qiáng)度。處于大小晝夜溫差系統(tǒng)中的浙油50、九二13系,油滴合成基因在白天與黑夜的表達(dá)模式是相反的,進(jìn)一步分析發(fā)現(xiàn),乙醛酸代謝途徑基因的表達(dá)模式正好與之符合。對(duì)乙醛酸代謝途徑的候選基因的啟動(dòng)子序列分析發(fā)現(xiàn),大部分基因的啟動(dòng)子序列均存在著GAGA-similar的保守序列,并且,據(jù)報(bào)道該保守序列是響應(yīng)高溫調(diào)控的順式作用元件。對(duì)轉(zhuǎn)錄組數(shù)據(jù)的深入挖掘發(fā)現(xiàn),大晝夜溫差中夜晚的低溫可以調(diào)控DELLA蛋白基因的表達(dá),RT-qPCR實(shí)驗(yàn)數(shù)據(jù)也證實(shí):大晝夜溫差系統(tǒng)中夜間赤霉素信號(hào)傳導(dǎo)基因受到抑制。我們據(jù)此設(shè)計(jì)了兩年兩點(diǎn)的大田試驗(yàn),實(shí)驗(yàn)結(jié)果也佐證了赤霉素信號(hào)在脂肪酸積累過(guò)程中的負(fù)面效應(yīng),而且當(dāng)外源噴施多效唑(降解赤霉素)時(shí),可以提高油菜含油量在9-18%的水平(增油效果受種植年份以及地點(diǎn)的影響)。3.找到了脂肪酸合成抑制因素BnaC.TT2.a位點(diǎn)的等位多態(tài)變化與種皮色澤及脂肪酸含量、組分的相關(guān)性。通過(guò)收集不同生態(tài)區(qū)域的83份甘藍(lán)型油菜種質(zhì)資源,我們成功構(gòu)建了適用于關(guān)聯(lián)分析的甘藍(lán)型油菜自然群體,該群體不僅適合分析調(diào)控種子含油量的基因位點(diǎn),而且可以分析其它潛在的功能基因。分析BnaC.TT2.a堿基多態(tài)性與脂肪酸表型的關(guān)聯(lián)性時(shí),在BnaC.TT2.a上的第1個(gè)內(nèi)含子上鑒別出4個(gè)顯著性連鎖的SNPs,在第3個(gè)外顯子上的738位置鑒定出1個(gè)插入突變位點(diǎn),而且該插入位點(diǎn)導(dǎo)致TT2蛋白功能發(fā)生了改變。以上關(guān)聯(lián)結(jié)果說(shuō)明了BnaC.TT2.a在調(diào)控甘藍(lán)型油菜種皮顏色、種子亞油酸以及總脂肪酸含量等重要農(nóng)藝性狀中的作用。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S565.4

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