灰毡毛忍冬（Lonicera macranthoides Hand.-Mazz）中绿原酸生物合成途径及调控技术研究

发布时间：2018-05-15 11:45

  本文选题：灰毡毛忍冬 + 绿原酸　； 参考：《重庆大学》2016年博士论文

【摘要】：灰毡毛忍冬(Lonicera macranthoides Hand.-Mazz),又名山银花,是我国特有的药用植物,在西部地区大面积种植。多酚类化合物是灰毡毛忍冬重要的次生代谢产物,其中绿原酸(Chlorogenic Acids,CGA)是核心生物活性物质,因此提高绿原酸的产量是亟待解决的问题。近年来,绿原酸的含量、生物合成途径及分子机制成为研究热点。但关于忍冬属植物绿原酸生物合成的分子机理研究报道较少,目前尚未有灰毡毛忍冬中CGA生物合成途径及调控机制的系统研究。本研究以“渝蕾一号”灰毡毛忍冬为材料,基于不同组织的转录组学数据,鉴定了CGA的生物合成相关的重要功能基因以及调控因子,同时研究了不同生长环境对多酚类化合物,特别是CGA产量的影响。本论文的主要研究结果如下:(1)利用HPLC技术,对样品前处理、流动相及检测波长作了优化,建立了一种同时检测灰毡毛忍冬绿原酸、阿魏酸、香豆酸、咖啡酸、肉桂酸、芦丁和木犀草苷的方法。基于此方法,对不同花期、叶片及茎段样品中7种多酚类化合物的含量作了检测,结果发现,花中绿原酸含量最高,其次是芦丁和木犀草苷。除香豆酸外,其余6种多酚物质均在不同花期内被检出,7种物质总量在二白期最高,幼蕾期最低。对叶片和茎段而言,嫩叶中绿原酸含量最高,且高于花中绿原酸含量,其次为嫩茎,随着组织老化,绿原酸含量迅速降低,但老叶中木犀草苷含量远高于所有其他组织和发育时期。(2)利用HPLC检测了绿原酸含量,发现CGA含量在不同的组织间差异显着:幼叶(YL)幼茎(YS)成熟花(MF)。通过高通量测序获得灰毡毛忍冬转录组数据,53,533,014个高质量的reads拼接形成76,453条unigenes。共有447条unigenes被归类到与绿原酸合成相关的苯丙素生物合成途径。不同组织的RNA-seq结果表明,6831个基因在MF和YL中差异表达,691个基因在3种不同组织中均差异表达。本研究发现了CGA生物合成的两条可能途径,包含苯丙氨酸解氨酶(PAL)、肉桂酸-4-羟化酶(C4H)、4-香豆酸辅酶A连接酶(4CL)、羟基肉桂转移/羟基肉桂酰辅酶A奎尼酸转移酶(HCT/HQT)和香豆酸羟化酶(C3H)等重要酶。结合表达量与CGA含量的相关性,推测1个PAL、3个C4H和5个HCT/HQT基因为灰毡毛忍冬中绿原酸生物合成的关键基因;14个差异表达的转录因子(AP2、ERF、MYB、NAC、锌指蛋白)为潜在的绿原酸生物合成的调控因子。(3)优化了灰毡毛忍冬叶片离体再生体系,在此基础上建立了遗传转化方法;依据转录组数据库中的序列信息,克隆了Lm HQT1基因,生物信息学分析表明Lm HQT1与Ns HQT和Sl HQT高度同源;q RT-PCR结果表明,Lm HQT1在幼叶中表达量最高,显著高于其它组织,这与绿原酸含量结果一致;为了深入研究基因的功能,构建了Lm HQT1基因超表达载体并转化灰毡毛忍冬,结果发现,转基因植株中的绿原酸含量提高了约60%。(4)研究了不同光质处理对灰毡毛忍冬生长及光合作用的影响,发现红光下植株长势、生物量、茎生长及光合指标(净光合速率、胞间二氧化碳浓度、蒸腾速率和气孔导度)均达到最高,表明红光促进植株生长和光合作用;研究了培养基上的不同激素配比对植株生长和次生代谢物积累的影响,筛选了最佳生长培养基(MB+1.0mg/L BA+1.0mg/L IBA)和最佳代谢培养基(MB+2.0mg/L BA+0.2mg/L IBA);研究了不同的激素配比与光质组合对次生代谢物积累的影响,结果发现,在最佳代谢培养基上,红光最有利于酚类物质积累,白光则不利于积累;在最佳生长培养基上,白光和蓝光最有利于酚类物质的积累,绿光则不利于酚类积累。综上,该研究基于转录组学分析鉴定了灰毡毛忍冬绿原酸生物合成途径的重要功能基因和调控因子;系统建立了遗传转化技术体系,并对关键基因HQT进行了功能分析;探索了不同的培养条件对多酚物质特别是绿原酸积累的调控;为阐明灰毡毛忍冬中绿原酸生物合成的主要途径及调控机制提供了重要参考。
[Abstract]:Lonicera macranthoides Hand.-Mazz (Lonicera macranthoides) and famous mountain silver flower are the unique medicinal plants in China, which are widely cultivated in the western region. Polyphenols are important secondary metabolites of Lonicera Lonicera, and chlorogenic acid (Chlorogenic Acids, CGA) is a bioactive substance of nuclear heart. Therefore, it is urgent to improve the production of chlorogenic acid. In recent years, the content of chlorogenic acid, biosynthesis pathway and molecular mechanism have become a hot spot. But there are few reports on the molecular mechanism of chlorogenic acid biosynthesis of Lonicera genus, and there has not been a systematic study on the biosynthesis pathway and regulation mechanism of CGA in Lonicera japonica. Mao Rendong, based on the transcriptional data of different tissues, identified the important functional genes and regulatory factors related to the biosynthesis of CGA, and studied the effects of different growth environments on the yield of polyphenols, especially CGA. The main results of this paper are as follows: (1) the use of HPLC technology for sample pretreatment and flow. A method for simultaneous detection of chlorogenic acid, ferulic acid, coumaric acid, caffeic acid, cinnamic acid, rutin and luteolin was established. Based on this method, the content of 7 polyphenols in the samples of different florescence, leaves and stem segments was detected. The results showed that the content of chlorogenic acid in flowers was the highest. The second was rutin and luteolin. Except for vanill, the other 6 kinds of polyphenols were detected in different flowering stages. The total amount of the 7 substances was the highest in the two white stage and the lowest in the young bud. However, the content of luteolin in old leaves was much higher than that of all other tissues and development periods. (2) the content of chlorogenic acid was detected by HPLC, and the difference between CGA content in different tissues showed that the young leaf (YL) young stem (YS) mature flower (MF). Through high throughput sequencing, the data of the black felt winter transcriptional group were obtained, and the 53533014 high quality reads spliced into 76,45. 3 unigenes. total of 447 unigenes were classified into the phenylpropanol biosynthesis pathway associated with the synthesis of chlorogenic acid. The RNA-seq results of different tissues showed that 6831 genes were expressed differently in MF and YL, and 691 genes were expressed differently in 3 different tissues. This study found two possible pathways, including phenylalanine, in the biosynthesis of CGA. Ammonia lyase (PAL), cinnamic acid -4- hydroxylase (C4H), 4- coumaric acid coenzyme A ligase (4CL), hydroxyl cinnamate transfer / hydroxyl cinnamyl coenzyme A quinic acid transferase (HCT/HQT) and coumaric acid hydroxylase (C3H). Combined with the correlation of expression and CGA content, 1 PAL were measured, 3 C4H and 5 genes were chlorogenic acids in Lonicera japonica. The key genes of synthesis; 14 differentially expressed transcription factors (AP2, ERF, MYB, NAC, zinc finger protein) are the regulatory factors of the potential chlorogenic acid biosynthesis. (3) the system of regeneration of Lonicera japonica Leaves in vitro was optimized. Based on this, a genetic transformation method was established. According to the sequence information in the transcriptional database, the Lm HQT1 gene was cloned and produced. Bioinformatics analysis showed that Lm HQT1 was highly homologous to Ns HQT and Sl HQT; Q RT-PCR results showed that Lm HQT1 was highest in young leaves, significantly higher than other tissues, which was in accordance with the content of chlorogenic acid. In order to study the function of genes, the Lm HQT1 gene overexpression vector was constructed and Lonicera japonica was transformed. The results were found, transgenic plant The content of chlorogenic acid in the plant increased by about 60%. (4). The effects of different light quality treatments on the growth and Photosynthesis of Lonicera japonica were studied. It was found that the plant growth, biomass, stem growth and photosynthetic indexes under red light (net photosynthetic rate, intercellular carbon dioxide concentration, transpiration rate and stomatal conductance) reached the highest, indicating that red light promoted plant growth. The effects of different hormone ratios on plant growth and secondary metabolite accumulation were studied. The optimal growth medium (MB+1.0mg/L BA+1.0mg/L IBA) and the best metabolic medium (MB+2.0mg/L BA+0.2mg/L IBA) were screened, and the effects of different hormone ratio and light quality on secondary metabolite accumulation were studied. It was found that red light was the most beneficial to the accumulation of phenolic substances on the best metabolic medium, and white light was not conducive to accumulation. On the best medium of growth, white and blue light was the most beneficial to the accumulation of phenolic substances, and green light was not conducive to the accumulation of phenols. In the sum, the biosynthesis pathway of chlorogenic acid in Lonicera japonica was identified based on the transcriptional analysis. The system of genetic transformation was established and the functional analysis of the key gene HQT was established, and the regulation of different culture conditions on the accumulation of polyphenols, especially chlorogenic acid, was explored. The main reference for clarifying the main pathway and regulation mechanism of chlorogenic acid biosynthesis in Lonicera gray felt.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.79
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本文编号：1892337