油用牡丹‘凤丹’DFR基因克隆与功能验证及木质素合成基因发掘

发布时间：2018-04-10 03:09

本文选题：凤丹　切入点：类黄酮　出处：《西北农林科技大学》2017年硕士论文

【摘要】：‘凤丹’牡丹是芍药科芍药属多年生灌木,是中国传统的观赏植物及药用植物。近年来,由其种子出油率高、油质优良等优点,而被选作主要的油用牡丹栽培品种。‘凤丹’籽油品质优良,具有很高的推广价值及发展潜力。但在生产过程中,直接用种子榨油则会出现黑色异物,因此在实际过程中需要去除种子外表坚硬的黑色种壳,对出油产量造成了一定程度的影响。色素分析表明,在油用牡丹中,控制种皮颜色的主要成分是类黄酮类物质,而种皮的主要成分则是木质素。因此,克隆并分析相关基因不仅有助于了解种壳黑色物质及木质素合成的相关机制,而且可以为下一步通过调解基因工程手段在根本上解决牡丹种壳的黑色及减少其中的木质素含量提供相应的理论基础。本实验中以‘凤丹’牡丹种子为材料,进行了类黄酮合成重要基因DFR的克隆及功能验证。结果表明:‘凤丹’二氢黄酮醇4-还原酶基因(dihydroflavonol 4-reductase,DFR)本基因含有一个长度为1092 bp的开放阅读框(ORF),共编码364个氨基酸。其中,‘凤丹’的DFR蛋白的相对分子量为40796.17 Da,理论等电点(PI)是5.76。亚细胞定位为细胞质的可能性最大;使用实时荧光定量法对PoDFR基因在‘凤丹’牡丹种子4个发育时期的表达量进行了分析,结果显示:PoDFR基因在‘凤丹’种子发育过程中表达量先上升又下降,在S2时期表达量达到最高;构建了原核表达载体,通过优化反应条件,获得PoDFR原核表达蛋白,通过与反应底物二氢槲皮素反应,证明了PoDFR蛋白能够催化合成无色花青素类物质,并且与ANS蛋白结合反应能合成花青素类物质,进一步证明了该基因的功能。分别以‘凤丹’牡丹种子种壳与种仁为材料,通过转录组测序,获得了110,151条基因序列,平均长度达到了1010 bp,基因功能注释率为50.44%。通过转录组表达水平比较,共得到了19,530条差异表达基因,从中筛选出8个木质素合成途径相关基因,其中包括咖啡酸/5-羟基阿魏酸-O-甲基转移酶(COMT)、咖啡酰辅酶-A-O-甲基转移酶(CCoAOMT)、肉桂酰CoA还原酶(CCR)、肉桂醇脱氢酶(CAD)、香豆酸-3-氢化酶(C3H)、阿魏酸-5羟基化酶(F5H)、4-香豆酸辅酶A连接酶(4CL)以及香豆酰辅酶A:莽草酸/奎宁酸香豆酰转移酶(HCT)。通过实时定量验证,进一步得到了候选基因在种壳发育过程中的表达模式,同时证明了这些基因与木质素的合成积累密切相关。综上,以上数据为进一步分离鉴定木质素合成相关基因功能,进一步阐释‘凤丹’种壳木质素合成代谢奠定了基础。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.9;Q943.2

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