本文選題：鳳丹　切入點：類黃酮　出處：《西北農(nóng)林科技大學》2017年碩士論文

【摘要】：‘鳳丹’牡丹是芍藥科芍藥屬多年生灌木,是中國傳統(tǒng)的觀賞植物及藥用植物。近年來,由其種子出油率高、油質(zhì)優(yōu)良等優(yōu)點,而被選作主要的油用牡丹栽培品種�！P丹’籽油品質(zhì)優(yōu)良,具有很高的推廣價值及發(fā)展?jié)摿�。但在生產(chǎn)過程中,直接用種子榨油則會出現(xiàn)黑色異物,因此在實際過程中需要去除種子外表堅硬的黑色種殼,對出油產(chǎn)量造成了一定程度的影響。色素分析表明,在油用牡丹中,控制種皮顏色的主要成分是類黃酮類物質(zhì),而種皮的主要成分則是木質(zhì)素。因此,克隆并分析相關(guān)基因不僅有助于了解種殼黑色物質(zhì)及木質(zhì)素合成的相關(guān)機制,而且可以為下一步通過調(diào)解基因工程手段在根本上解決牡丹種殼的黑色及減少其中的木質(zhì)素含量提供相應(yīng)的理論基礎(chǔ)。本實驗中以‘鳳丹’牡丹種子為材料,進行了類黃酮合成重要基因DFR的克隆及功能驗證。結(jié)果表明:‘鳳丹’二氫黃酮醇4-還原酶基因(dihydroflavonol 4-reductase,DFR)本基因含有一個長度為1092 bp的開放閱讀框(ORF),共編碼364個氨基酸。其中,‘鳳丹’的DFR蛋白的相對分子量為40796.17 Da,理論等電點(PI)是5.76。亞細胞定位為細胞質(zhì)的可能性最大;使用實時熒光定量法對PoDFR基因在‘鳳丹’牡丹種子4個發(fā)育時期的表達量進行了分析,結(jié)果顯示:PoDFR基因在‘鳳丹’種子發(fā)育過程中表達量先上升又下降,在S2時期表達量達到最高;構(gòu)建了原核表達載體,通過優(yōu)化反應(yīng)條件,獲得PoDFR原核表達蛋白,通過與反應(yīng)底物二氫槲皮素反應(yīng),證明了PoDFR蛋白能夠催化合成無色花青素類物質(zhì),并且與ANS蛋白結(jié)合反應(yīng)能合成花青素類物質(zhì),進一步證明了該基因的功能。分別以‘鳳丹’牡丹種子種殼與種仁為材料,通過轉(zhuǎn)錄組測序,獲得了110,151條基因序列,平均長度達到了1010 bp,基因功能注釋率為50.44%。通過轉(zhuǎn)錄組表達水平比較,共得到了19,530條差異表達基因,從中篩選出8個木質(zhì)素合成途徑相關(guān)基因,其中包括咖啡酸/5-羥基阿魏酸-O-甲基轉(zhuǎn)移酶(COMT)、咖啡酰輔酶-A-O-甲基轉(zhuǎn)移酶(CCoAOMT)、肉桂酰CoA還原酶(CCR)、肉桂醇脫氫酶(CAD)、香豆酸-3-氫化酶(C3H)、阿魏酸-5羥基化酶(F5H)、4-香豆酸輔酶A連接酶(4CL)以及香豆酰輔酶A:莽草酸/奎寧酸香豆酰轉(zhuǎn)移酶(HCT)。通過實時定量驗證,進一步得到了候選基因在種殼發(fā)育過程中的表達模式,同時證明了這些基因與木質(zhì)素的合成積累密切相關(guān)。綜上,以上數(shù)據(jù)為進一步分離鑒定木質(zhì)素合成相關(guān)基因功能,進一步闡釋‘鳳丹’種殼木質(zhì)素合成代謝奠定了基礎(chǔ)。
[Abstract]:'Fengdan' is the peony peony families so peony perennial shrub is Chinese traditional ornamental plants and medicinal plants. In recent years, the seeds of high oil yield, good quality, and was chosen as the main oil with peony cultivars. 'Fengdan' seed quality, has the value of popularization and development the potential is very high. But in the production process, the direct use of oil seeds will appear black bodies, so in the actual process of the need to remove the black seed shell looks hard, it has a certain influence on the oil yield. Pigment analysis showed that in the peony oil, the main component is the control of seed coat color flavonoids, while the main component of seed coat is lignin. Therefore, cloning and analysis of genes related to not only help to understand the mechanism of shell black substance and lignin synthesis, but also for the next step through the mediation of genetic engineering The process means to solve the Black Peony shell fundamentally reduce the wood and provide a theoretical basis. The content of 'Fengdan' peony seeds as materials in this experiment, the cloning and characterization of flavonoid biosynthesis gene DFR. The results show that the 'Fengdan' two flavanonols 4- reductase gene (dihydroflavonol 4-reductase, DFR) of this gene contains a length of 1092 BP open reading frame (ORF), encoding 364 amino acids. Among them, "the relative molecular weight of Fengdan 'DFR protein is 40796.17 Da, isoelectric point (PI) is the subcellular localization of 5.76. cytoplasm is most likely to use; real time fluorescent quantitative assay the expression of PoDFR gene in' Fengdan 'peony seed 4 developmental stages were analyzed, the results showed that PoDFR gene expression in the process of development of Fengdan' seeds increased first and decreased during the S2 table Reached the highest; prokaryotic expression vector was constructed, by optimizing the reaction conditions, obtain the prokaryotic expression of PoDFR protein by reaction with substrate two hydrogen quercetin, proved that PoDFR protein can catalyze the synthesis of anthocyanin and colorless, the binding reaction can synthesize anthocyanin and ANS protein, indicating that the gene function respectively. To 'Fengdan' peony seed shell and seed as material, through transcriptome sequencing, obtained 110151 gene sequences, the average length of 1010 BP, gene functional annotation was 50.44%. by transcriptome expression level, a total of 19530 differentially expressed genes, screened 8 genes related to wood pigment biosynthesis from, including caffeic acid ferulic acid /5- hydroxy -O- methyl transferase (COMT), caffeoyl COA -A-O- methyltransferase (CCoAOMT), cinnamoyl CoA reductase (CCR), cinnamyl alcohol Dehydrogenase (CAD), p-coumaric acid -3- hydrogenase (C3H), -5 ferulic acid hydroxylase (F5H), 4- coumaric acid coenzyme A ligase (4CL) and p-coumaric acyl coenzyme A: shikimic acid / quinic acid p-coumaric acyl transferase (HCT). Through real-time quantitative validation, further expression model of candidate genes in the shell in the process of development, and that is closely related to the accumulation of these genes and the synthesis of lignin. Therefore, the above data for further separation and identification of lignin synthesis related gene function, further explanation of 'Fengdan' shell lignin synthesis metabolism laid the foundation.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.9;Q943.2

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