沙棘果肉和种子油脂合成积累及转录表达差异研究

发布时间：2018-01-15 04:05

本文关键词：沙棘果肉和种子油脂合成积累及转录表达差异研究　出处：《东北林业大学》2016年博士论文　论文类型：学位论文

【摘要】：木本油料的开发利用是缓解当前食用油供应不足且耕地逐渐减少的有效途径。沙棘(Hippophae rhamnoides)为胡颓子科(Elaeagnaceae)沙棘属植物,多年生小乔木或灌木,适应性强,具有抗旱、抗盐碱、抗寒特性,能适应-40～40℃的生长环境。沙棘果实中含有多种生物功能活性成分,如脂肪酸、维生素、植物甾醇、类胡萝卜素、黄酮类化合物和有机酸等。果肉油和种子油是沙棘中最有价值的成分,果肉油中高积累自然界中稀有的棕榈油酸(omega-7脂肪酸),而种子油中高富集亚油酸(omega-6脂肪酸)和亚麻酸(omega-3脂肪酸),omega脂肪酸在维持人体正常生理功能方面具有重要作用。但与大田油料作物相比,沙棘果肉和种子含油量相对较低,制约了沙棘油的高效开发和利用。目前影响沙棘油脂合成的关键基因及途径尚未清晰,特别是沙棘果肉中高积累棕榈油酸的机制未见报道。研究沙棘果肉和种子中油脂和脂肪酸组分的动态变化规律,以及发育过程中相关功能基因的转录表达模式,为通过现代组学技术探索沙棘油脂合成积累机理研究奠定基础,也为进一步提高沙棘油脂含量提供技术支撑。主要研究结果如下：1.通过51份沙棘种质的成熟果肉和种子含油量分析和ISSR分子标记亲缘关系鉴定,发现蒙古沙棘亚种的干果肉含油量(27.2±0.9%)显著高于中蒙沙棘杂交种的干果肉含油量(12.0±1.2%)(P0.01)；首次识别到4个与沙棘高果肉含油量呈正相关,2个与高种子含油量呈正相关的ISSR标记；筛选获得1对近缘高低果肉油种质(品系TF2-36和56)和1对近缘高低种子油种质(品系XE3和SJl),果肉组样品含油量相差30.58%(干重),DICE系数为0.752；种子组样品含油量相差4.98%,DICE系数为0.756。2.通过8个时期的高、低果肉油和种子油样品的含油量和脂肪酸组分分析发现,果肉油脂积累模式为先上升后下降,种子油脂积累模式为先下降后上升再微量下降；品系TF2-36的干果肉含油量一直显著高于品系56,大多数时期品系XE3的种子含油量显著高于品系SJ1,品系XE3的干果肉含油量也显著高于其种子含油量。果肉中高积累棕榈酸(18～36%)和棕榈油酸(1～41%),且均与干果肉含油量呈正相关,亚油酸和亚麻酸相对含量呈下降趋势；种子中高积累亚油酸(26～47%)和亚麻酸(20～37%),亚油酸与种子含油量呈正相关,棕榈酸和棕榈油酸呈明显下降趋势；果肉中的单不饱和/饱和脂肪酸、多不饱和/饱和脂肪酸,以及种子中的单不饱和/饱和脂肪酸、亚油酸/亚麻酸比值均符合健康食用油的国际标准。3.转录组de novo测序解决了无参考基因组的沙棘等木本油料功能基因研究的困境,首次基于Illumina平台构建沙棘根、茎、叶、果肉和种子转录组,组装获得62166条unigene,N50为1780 bp,总注释率78.87%。KEGG pathway分析发现碳水化合物代谢途径获注释unigene最多(2798条),注释到14条与脂类代谢相关途径,包括甘油酯代谢、甘油磷脂代谢、脂肪酸生物合成、脂肪酸延伸、不饱和脂肪酸生物合成等途径,共1794条umgenes。通过5种组织部位表达谱的两两比较获得566～945个与脂类代谢相关的差异表达基因,DGAT、GPD1、KAS Ⅱ和FAD7等基因可能与沙棘果肉和种子油脂和脂肪酸生物合成有关。4.利用qRT-PCR分析验证了12个与沙棘油脂和脂肪酸合成相关基因的表达特性,研究发现果肉中KAS Ⅱ基因的持续低表达可能限制了C16脂肪酸向C18脂肪酸转化,而Δ9D基因的上调表达则促进了棕榈油酸的合成积累,同时高果肉油品系TF2-36中GPD1、DGAT1和DGAT2基因的显著上调表达,可能加速了3-磷酸甘油醛(G3P)与高积累的C16脂肪酸组装合成甘油三酯(TAG)；KAS Ⅱ、FAD2、FAD3、FAD7、FAD8、GPD1和DGAT2基因在种子油脂快速积累期间有明显的表达量高峰,这可能与种子高积累C18：2和C18：3且促其参与TAG合成有关。KAS Ⅱ、Δa9D、FAD2、FAD3、FAD7和FAD8是影响果肉和种子脂肪酸组分差异的关键基因,而GPDl、DGAT1和DGAT2是影响沙棘TAG合成积累的关键基因。5.为了深层次挖掘影响沙棘油脂合成的关键基因,对4个生育期的近缘高低果肉和种子油样品进行RNA-Seq表达谱分析,筛选获得了11个与油脂和脂肪酸合成相关的差异表达基因(LPIN、plcC、adhE、ACSL、PLD12、CER10、GPD1、GPAT、FATA、FAD2和KCS)(log2fold change1, Q value0.05),经qRT-PCR验证获得了与RNA-Seq分析相似的基因表达模式。LPIN, plcC和adhE基因参与果肉油脂合成且有显著上调表达过程；KCS和CER10基因是定位于沙棘内质网的显著差异表达基因,ACSL、 FATA和CER10基因的下调表达,限制了果肉中C18脂肪酸合成及其与甘油酯的结合。GPD1、PLD1_2和GPAT基因参与种子油脂合成且有显著上调表达过程。
[Abstract]:The development and utilization of woody oil is the effective way to alleviate the current shortage of edible oil and cultivated land decreased gradually. Sea buckthorn (Hippophae rhamnoides) (Elaeagnaceae) as the Elaeagnaceae Hippophae plants, perennial small trees or shrubs, with strong adaptability, drought resistance, cold resistance, alkali resistance, can adapt to the growth of -40 to 40 DEG C containing environment. A variety of biological active components of sea buckthorn fruit, such as fatty acids, vitamins, plant sterols, carotenoids, flavonoids and organic acids. Pulp oil and seed oil is the most valuable component in seabuckthorn, palmitoleic acid rare nature high accumulation in pulp oil (omega-7 fatty acid), and seed oil high enrichment of linoleic acid (omega-6 fatty acid) and linolenic acid (omega-3 fatty acid), omega fatty acid plays an important role in maintaining the normal physiological function of human body. But compared with field crops, sea buckthorn pulp And the seed oil content is relatively low, restricts the efficient development and utilization of sea buckthorn oil. The effect of Hippophae oil key gene synthesis pathway and has not been clear, especially high palmitoleic acid accumulation in sea buckthorn pulp mechanism has not been reported. Study on dynamic changes of sea buckthorn pulp and seed oils and fatty acid composition, expression patterns of transcription related genes during development and function, through the modern group lay the foundation on research synthesis and accumulation mechanism of Hippophae oil technology, also provided technical support for the further improvement of sea buckthorn oil content. The main results are as follows: 1. through 51 analysis of seabuckthorn seed mature pulp and seed oil content and ISSR molecular marker genetic relationship identification the oil content of dried meat, found the Mongolia subspecies (27.2 + 0.9%) was significantly higher than that of dried meat of the oil content of seabuckthorn Mongolia hybrids (12 + 1.2%) (P0.01); For the first time to identify 4 positive and high oil content of seabuckthorn fruit flesh, 2 ISSR markers were positively associated with high seed oil content; 1 to close level of pulp oil germplasm screening (lines TF2-36 and 56) and 1 of the relatives of low seed oil germplasm (strains XE3 and SJl), flesh group the sample content is 30.58% (dry weight), DICE coefficient is 0.752; the oil content of seed samples was 4.98%, DICE coefficient is 0.756.2. through 8 periods of high oil content and fatty acid composition of low pulp oil and seed oil sample analysis, the pulp oil accumulation pattern of decline after rising first, seed oil the accumulation mode is first decreased and then increased again trace decline; dried meat oil content line TF2-36 was significantly higher than that of strain 56, seed oil content during the most of strain XE3 was significantly higher than that of strain SJ1, dried meat oil content line XE3 is significantly higher than that of the seed oil content in pulp. In the high accumulation of palmitic acid (18 ~ 36%) and palmitic acid (1 ~ 41%), and all with the dried meat of oil content was positively correlated with linoleic acid and linolenic acid relative content decreased; linoleic acid high accumulation in seeds (26 ~ 47%) and linolenic acid (20 ~ 37%), linoleic acid and positively the seed oil content, palmitic acid and palmitic acid decreased significantly; the flesh of the mono unsaturated / saturated fatty acids, polyunsaturated / saturated fatty acid, and the seeds of mono unsaturated / saturated fatty acid, linoleic acid and linolenic acid / health edible oil ratios are consistent with the international standard.3. transcription de group novo sequencing for the study of gene reference genome of sea buckthorn and woody oil function dilemma, first build the Illumina platform based on Seabuckthorn root, stem, leaf, fruit and seed transcriptome assembly, 62166 UniGene, N50 1780 BP, the total notes rate of 78.87%.KEGG pathway analysis showed that the hydration of carbon Complex metabolic pathways by UniGene (2798) notes up to 14 notes, related pathways and lipid metabolism, including triglyceride metabolism, glycerophospholipid metabolism, fatty acid biosynthesis, fatty acid, unsaturated fatty acid biosynthesis pathway, a total of 1794 umgenes. expression compared to 22 from 566 to 945 and lipid metabolism genes differentially expressed by 5 kinds of tissue DGAT, GPD1, KAS II and FAD7 gene may be associated with sea buckthorn pulp and seed oils and fatty acid biosynthesis of.4. using qRT-PCR to analyze and verify the expression characteristics of 12 and Hippophae oil and fatty acid synthesis related genes, the study found that low expression of KAS II in the flesh continued genes may limit C16 fatty acids to C18 fatty acids, while 9D up-regulated gene promoted the synthesis and accumulation of palmitic acid, while high pulp oil line TF2-36 GPD1, DGAT1 and DG Up regulate the expression of AT2 gene, may accelerate the glyceraldehyde 3- phosphate (G3P) and C16 fatty acid synthesis and triglyceride accumulation assembly (TAG); KAS II, FAD2, FAD3, FAD7, FAD8, GPD1 and DGAT2 genes during rapid accumulation in seed oil has obvious peak expression, which may be related to the high seed the accumulation of C18:2 and C18:3 and promote their participation in the synthesis of TAG.KAS II, Delta a9D, FAD2, FAD3, FAD7 and FAD8 are key genes, pulp and seed fatty acid composition and the difference of GPDl, DGAT1 and DGAT2 are the key genes influencing sea buckthorn TAG biosynthesis and accumulation of.5. in order to dig deep sea buckthorn oil synthesis key the 4 gene, growth period related level of pulp and seed oil samples were RNA-Seq expression analysis of 11 genes were obtained with lipid and fatty acid synthesis differences related to the expression of LPIN, plcC, adhE (screening, ACSL, PLD12, CER10, GPD1, GPAT, FATA, FAD2 and K CS (log2fold) change1, Q, value0.05) were obtained and verified by qRT-PCR RNA-Seq analysis of similar gene expression patterns of.LPIN, plcC and adhE genes involved in lipid synthesis and pulp increased expression of KCS and CER10; gene expression gene, significant differences in positioning Seabuckthorn endoplasmic reticulum of ACSL, down regulate the expression of FATA and CER10 gene the limit of C18 fatty acid synthesis in pulp and glycerol combined with.GPD1, PLD1_2 and GPAT genes involved in seed oil synthesis and were significantly up-regulated during.

【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S793.6

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