本文關(guān)鍵詞：沙棘果肉和種子油脂合成積累及轉(zhuǎn)錄表達(dá)差異研究　出處：《東北林業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 沙棘 油脂 轉(zhuǎn)錄組測序 差異表達(dá)基因 生物合成途徑

【摘要】：木本油料的開發(fā)利用是緩解當(dāng)前食用油供應(yīng)不足且耕地逐漸減少的有效途徑。沙棘(Hippophae rhamnoides)為胡頹子科(Elaeagnaceae)沙棘屬植物,多年生小喬木或灌木,適應(yīng)性強(qiáng),具有抗旱、抗鹽堿、抗寒特性,能適應(yīng)-40～40℃的生長環(huán)境。沙棘果實(shí)中含有多種生物功能活性成分,如脂肪酸、維生素、植物甾醇、類胡蘿卜素、黃酮類化合物和有機(jī)酸等。果肉油和種子油是沙棘中最有價(jià)值的成分,果肉油中高積累自然界中稀有的棕櫚油酸(omega-7脂肪酸),而種子油中高富集亞油酸(omega-6脂肪酸)和亞麻酸(omega-3脂肪酸),omega脂肪酸在維持人體正常生理功能方面具有重要作用。但與大田油料作物相比,沙棘果肉和種子含油量相對較低,制約了沙棘油的高效開發(fā)和利用。目前影響沙棘油脂合成的關(guān)鍵基因及途徑尚未清晰,特別是沙棘果肉中高積累棕櫚油酸的機(jī)制未見報(bào)道。研究沙棘果肉和種子中油脂和脂肪酸組分的動態(tài)變化規(guī)律,以及發(fā)育過程中相關(guān)功能基因的轉(zhuǎn)錄表達(dá)模式,為通過現(xiàn)代組學(xué)技術(shù)探索沙棘油脂合成積累機(jī)理研究奠定基礎(chǔ),也為進(jìn)一步提高沙棘油脂含量提供技術(shù)支撐。主要研究結(jié)果如下：1.通過51份沙棘種質(zhì)的成熟果肉和種子含油量分析和ISSR分子標(biāo)記親緣關(guān)系鑒定,發(fā)現(xiàn)蒙古沙棘亞種的干果肉含油量(27.2±0.9%)顯著高于中蒙沙棘雜交種的干果肉含油量(12.0±1.2%)(P0.01)；首次識別到4個(gè)與沙棘高果肉含油量呈正相關(guān),2個(gè)與高種子含油量呈正相關(guān)的ISSR標(biāo)記；篩選獲得1對近緣高低果肉油種質(zhì)(品系TF2-36和56)和1對近緣高低種子油種質(zhì)(品系XE3和SJl),果肉組樣品含油量相差30.58%(干重),DICE系數(shù)為0.752；種子組樣品含油量相差4.98%,DICE系數(shù)為0.756。2.通過8個(gè)時(shí)期的高、低果肉油和種子油樣品的含油量和脂肪酸組分分析發(fā)現(xiàn),果肉油脂積累模式為先上升后下降,種子油脂積累模式為先下降后上升再微量下降；品系TF2-36的干果肉含油量一直顯著高于品系56,大多數(shù)時(shí)期品系XE3的種子含油量顯著高于品系SJ1,品系XE3的干果肉含油量也顯著高于其種子含油量。果肉中高積累棕櫚酸(18～36%)和棕櫚油酸(1～41%),且均與干果肉含油量呈正相關(guān),亞油酸和亞麻酸相對含量呈下降趨勢；種子中高積累亞油酸(26～47%)和亞麻酸(20～37%),亞油酸與種子含油量呈正相關(guān),棕櫚酸和棕櫚油酸呈明顯下降趨勢；果肉中的單不飽和/飽和脂肪酸、多不飽和/飽和脂肪酸,以及種子中的單不飽和/飽和脂肪酸、亞油酸/亞麻酸比值均符合健康食用油的國際標(biāo)準(zhǔn)。3.轉(zhuǎn)錄組de novo測序解決了無參考基因組的沙棘等木本油料功能基因研究的困境,首次基于Illumina平臺構(gòu)建沙棘根、莖、葉、果肉和種子轉(zhuǎn)錄組,組裝獲得62166條unigene,N50為1780 bp,總注釋率78.87%。KEGG pathway分析發(fā)現(xiàn)碳水化合物代謝途徑獲注釋unigene最多(2798條),注釋到14條與脂類代謝相關(guān)途徑,包括甘油酯代謝、甘油磷脂代謝、脂肪酸生物合成、脂肪酸延伸、不飽和脂肪酸生物合成等途徑,共1794條umgenes。通過5種組織部位表達(dá)譜的兩兩比較獲得566～945個(gè)與脂類代謝相關(guān)的差異表達(dá)基因,DGAT、GPD1、KAS Ⅱ和FAD7等基因可能與沙棘果肉和種子油脂和脂肪酸生物合成有關(guān)。4.利用qRT-PCR分析驗(yàn)證了12個(gè)與沙棘油脂和脂肪酸合成相關(guān)基因的表達(dá)特性,研究發(fā)現(xiàn)果肉中KAS Ⅱ基因的持續(xù)低表達(dá)可能限制了C16脂肪酸向C18脂肪酸轉(zhuǎn)化,而Δ9D基因的上調(diào)表達(dá)則促進(jìn)了棕櫚油酸的合成積累,同時(shí)高果肉油品系TF2-36中GPD1、DGAT1和DGAT2基因的顯著上調(diào)表達(dá),可能加速了3-磷酸甘油醛(G3P)與高積累的C16脂肪酸組裝合成甘油三酯(TAG)；KAS Ⅱ、FAD2、FAD3、FAD7、FAD8、GPD1和DGAT2基因在種子油脂快速積累期間有明顯的表達(dá)量高峰,這可能與種子高積累C18：2和C18：3且促其參與TAG合成有關(guān)。KAS Ⅱ、Δa9D、FAD2、FAD3、FAD7和FAD8是影響果肉和種子脂肪酸組分差異的關(guān)鍵基因,而GPDl、DGAT1和DGAT2是影響沙棘TAG合成積累的關(guān)鍵基因。5.為了深層次挖掘影響沙棘油脂合成的關(guān)鍵基因,對4個(gè)生育期的近緣高低果肉和種子油樣品進(jìn)行RNA-Seq表達(dá)譜分析,篩選獲得了11個(gè)與油脂和脂肪酸合成相關(guān)的差異表達(dá)基因(LPIN、plcC、adhE、ACSL、PLD12、CER10、GPD1、GPAT、FATA、FAD2和KCS)(log2fold change1, Q value0.05),經(jīng)qRT-PCR驗(yàn)證獲得了與RNA-Seq分析相似的基因表達(dá)模式。LPIN, plcC和adhE基因參與果肉油脂合成且有顯著上調(diào)表達(dá)過程；KCS和CER10基因是定位于沙棘內(nèi)質(zhì)網(wǎng)的顯著差異表達(dá)基因,ACSL、 FATA和CER10基因的下調(diào)表達(dá),限制了果肉中C18脂肪酸合成及其與甘油酯的結(jié)合。GPD1、PLD1_2和GPAT基因參與種子油脂合成且有顯著上調(diào)表達(dá)過程。
[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.

【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S793.6

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