山核桃油脂合成基因家族分析及候选基因功能验证

发布时间：2018-05-21 20:17

本文选题：山核桃 + 油脂合成　；参考：《浙江农林大学》2017年硕士论文

【摘要】：山核桃(Carya cathayensis Sarg.)是我国特有的特色优质干果,具有极高的营养价值,富含优质脂肪酸和多种微量元素,是高油酸木本油料作物。在成油方面,通过对山核桃果实发育阶段粗脂肪含量和脂肪酸组分的测定,结合山核桃果实发育阶段的转录组数据,分析油脂的来源、合成及降解,构建了山核桃成油网络,揭示了山核桃油脂积累过程中的基因表达规律。本研究运用实验和生物信息学的研究手段对山核桃油脂合成过程中的脂肪酸去饱和酶基因家族做了全面分析,以期揭示山核桃高不饱和脂肪酸油脂组分机理,此外,通过转基因手段验证油脂合成候选基因功能,以期从分子水平解释山核桃高油机制,筛选出提高含油率和特定脂肪酸含量的关键基因,为选育获得高油和高品质的山核桃品种提供理论依据。主要结果如下:1、山核桃油主要由棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)以及亚麻酸(C18:3)五种组分组成,成熟阶段的山核桃不饱和脂肪酸含量为92.45%,山核桃是高油酸植物,成熟阶段的油酸占总不饱和脂肪酸的比例可达85.57%,此外亚油酸和亚麻酸含量也较为丰富,ω-6型脂肪酸/ω-3型脂肪酸比值为1.69:1,是多不饱和脂肪酸含量的最佳比值,有很高的营养价值。2、扩增得到山核桃SAD家族成员3个,分别是:CcSAD-1;CcSAD-2,CcSAD-3,都含有典型的Ferritin-like superfamily功能结构域,含有1个保守基序(DLLNKYLY),同时,定量结果表明SAD基因表达在山核桃果实成熟过程中存在差异,随着CcSADs基因表达上升,山核桃油中油酸含量增加。3、扩增得到山核桃FAD家族成员9个,分别是:CcFAD2;CcFAD3,3个拷贝,记为CcFAD3-1、CcFAD3-2、CcFAD3-3;CcFAD6;CcFAD7;CcFAD8,3个拷贝,记为CcFAD8-1、CcFAD8-2、CcFAD8-3,都含有典型的Membrance-FADS-like super family功能结构域,含有2个保守的组氨酸基序(Hisbox),同时,定量结果表明FAD基因表达在山核桃果实成熟过程中存在差异,随着CcFADs基因表达上升,山核桃油中不饱和脂肪酸比例增加。4、构建3个油脂合成候选基因转基因体系:35Spro-Uni11、35Spro-Uni72、35SproUni58,35Spro-Uni11转基因拟南芥表型较野生型叶型小、叶片数多、抽薹率高、侧枝多,但种子油含量未明显增加;35Spro-Uni72和35Spro-Uni58转基因拟南芥T1代表型较野生型拟南芥侧枝多,株型更饱满,但是从T2代转基因拟南芥中未再检测到相应基因,猜测原因是拟南芥的自我保护机制,切除了异源基因。
[Abstract]:Carya cathayensis Sarg.) It is a unique high quality dried fruit with high nutritional value, rich in high quality fatty acids and trace elements, and is a high oleic acid woody oil crop. In the aspect of oil formation, the crude fat content and fatty acid composition of pecan fruit were determined, and the source, synthesis and degradation of oil were analyzed according to the transcriptional data of pecan fruit development stage, and the oil forming network of pecan was constructed. The rule of gene expression during the accumulation of pecan oil was revealed. In this study, the fatty acid desaturase gene family in the synthesis of pecan oil was analyzed by means of experiments and bioinformatics, in order to reveal the mechanism of the fatty acid desaturase gene in pecan oil synthesis. In order to explain the mechanism of high oil content in pecans at molecular level and to screen out the key genes to increase oil content and specific fatty acid content, the function of candidate genes for oil synthesis was verified by transgenic methods. To provide theoretical basis for breeding high oil and high quality pecan varieties. The main results are as follows: the pecan oil is composed mainly of five components: palmitic acid C16: 0, stearic acid C18: 0, oleic acid C18: 1, linoleic acid C18: 2) and linolenic acid (C18: 3). The unsaturated fatty acid content of pecan is 92.455.The pecan is a high oleic acid plant. The ratio of oleic acid to total unsaturated fatty acid in mature stage is 85.57, and the content of linoleic acid and linolenic acid is also relatively rich. The ratio of 蠅 -6 fatty acid to 蠅 -3 fatty acid is 1.69: 1, which is the best ratio of polyunsaturated fatty acid content. There are three members of the pecan SAD family, namely: CcSAD-1, CcSAD-2CcSAD-3, which contain a typical functional domain of Ferritin-like superfamily, a conserved motif DLLNKYYYY, and a conserved motif DLNKYLYYY. The quantitative results showed that there were differences in the expression of SAD gene during fruit ripening. With the increase of CcSADs gene expression, oleic acid content in pecan oil increased by .3.Nine members of the FAD family of pecan were obtained by amplification, which were respectively: 1 / CcFAD2CcFAD3, 3 copies. CcFAD3-1, CcFAD3-2, CcFAD3-3, CcFAD6, CcFAD7, CcFAD8, and CcFAD8-2, CcFAD8-3, all contained typical Membrance-FADS-like super family functional domains and two conserved histidine sequence Hisboxbox. The quantitative results showed that the expression of FAD gene was different in the ripening process of pecan fruit. With the increase of CcFADs gene expression, the proportion of unsaturated fatty acids in pecan oil increased .4.The transgenic Arabidopsis thaliana transgenic with three candidate genes for oil synthesis was constructed, the phenotype of Arabidopsis thaliana was smaller than that of wild type leaves, the number of leaves was more, the bolting rate was higher, and the lateral branches were more than those of wild type Arabidopsis thaliana. However, the seed oil content was not significantly increased in transgenic Arabidopsis thaliana and 35Spro-Uni58 transgenic Arabidopsis thaliana, the T1 representative type was more than that of wild type Arabidopsis thaliana, but the corresponding gene was not detected from T2 generation transgenic Arabidopsis thaliana. The hypothetical reason is the self-protection mechanism of Arabidopsis thaliana and the excision of allogenic genes.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S664.1

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