發(fā)布時間：2018-05-15 11:45

  本文選題：灰氈毛忍冬 + 綠原酸�。� 參考：《重慶大學(xué)》2016年博士論文

【摘要】：灰氈毛忍冬(Lonicera macranthoides Hand.-Mazz),又名山銀花,是我國特有的藥用植物,在西部地區(qū)大面積種植。多酚類化合物是灰氈毛忍冬重要的次生代謝產(chǎn)物,其中綠原酸(Chlorogenic Acids,CGA)是核心生物活性物質(zhì),因此提高綠原酸的產(chǎn)量是亟待解決的問題。近年來,綠原酸的含量、生物合成途徑及分子機(jī)制成為研究熱點。但關(guān)于忍冬屬植物綠原酸生物合成的分子機(jī)理研究報道較少,目前尚未有灰氈毛忍冬中CGA生物合成途徑及調(diào)控機(jī)制的系統(tǒng)研究。本研究以“渝蕾一號”灰氈毛忍冬為材料,基于不同組織的轉(zhuǎn)錄組學(xué)數(shù)據(jù),鑒定了CGA的生物合成相關(guān)的重要功能基因以及調(diào)控因子,同時研究了不同生長環(huán)境對多酚類化合物,特別是CGA產(chǎn)量的影響。本論文的主要研究結(jié)果如下:(1)利用HPLC技術(shù),對樣品前處理、流動相及檢測波長作了優(yōu)化,建立了一種同時檢測灰氈毛忍冬綠原酸、阿魏酸、香豆酸、咖啡酸、肉桂酸、蘆丁和木犀草苷的方法�；�于此方法,對不同花期、葉片及莖段樣品中7種多酚類化合物的含量作了檢測,結(jié)果發(fā)現(xiàn),花中綠原酸含量最高,其次是蘆丁和木犀草苷。除香豆酸外,其余6種多酚物質(zhì)均在不同花期內(nèi)被檢出,7種物質(zhì)總量在二白期最高,幼蕾期最低。對葉片和莖段而言,嫩葉中綠原酸含量最高,且高于花中綠原酸含量,其次為嫩莖,隨著組織老化,綠原酸含量迅速降低,但老葉中木犀草苷含量遠(yuǎn)高于所有其他組織和發(fā)育時期。(2)利用HPLC檢測了綠原酸含量,發(fā)現(xiàn)CGA含量在不同的組織間差異顯著:幼葉(YL)幼莖(YS)成熟花(MF)。通過高通量測序獲得灰氈毛忍冬轉(zhuǎn)錄組數(shù)據(jù),53,533,014個高質(zhì)量的reads拼接形成76,453條unigenes。共有447條unigenes被歸類到與綠原酸合成相關(guān)的苯丙素生物合成途徑。不同組織的RNA-seq結(jié)果表明,6831個基因在MF和YL中差異表達(dá),691個基因在3種不同組織中均差異表達(dá)。本研究發(fā)現(xiàn)了CGA生物合成的兩條可能途徑,包含苯丙氨酸解氨酶(PAL)、肉桂酸-4-羥化酶(C4H)、4-香豆酸輔酶A連接酶(4CL)、羥基肉桂轉(zhuǎn)移/羥基肉桂酰輔酶A奎尼酸轉(zhuǎn)移酶(HCT/HQT)和香豆酸羥化酶(C3H)等重要酶。結(jié)合表達(dá)量與CGA含量的相關(guān)性,推測1個PAL、3個C4H和5個HCT/HQT基因為灰氈毛忍冬中綠原酸生物合成的關(guān)鍵基因;14個差異表達(dá)的轉(zhuǎn)錄因子(AP2、ERF、MYB、NAC、鋅指蛋白)為潛在的綠原酸生物合成的調(diào)控因子。(3)優(yōu)化了灰氈毛忍冬葉片離體再生體系,在此基礎(chǔ)上建立了遺傳轉(zhuǎn)化方法;依據(jù)轉(zhuǎn)錄組數(shù)據(jù)庫中的序列信息,克隆了Lm HQT1基因,生物信息學(xué)分析表明Lm HQT1與Ns HQT和Sl HQT高度同源;q RT-PCR結(jié)果表明,Lm HQT1在幼葉中表達(dá)量最高,顯著高于其它組織,這與綠原酸含量結(jié)果一致;為了深入研究基因的功能,構(gòu)建了Lm HQT1基因超表達(dá)載體并轉(zhuǎn)化灰氈毛忍冬,結(jié)果發(fā)現(xiàn),轉(zhuǎn)基因植株中的綠原酸含量提高了約60%。(4)研究了不同光質(zhì)處理對灰氈毛忍冬生長及光合作用的影響,發(fā)現(xiàn)紅光下植株長勢、生物量、莖生長及光合指標(biāo)(凈光合速率、胞間二氧化碳濃度、蒸騰速率和氣孔導(dǎo)度)均達(dá)到最高,表明紅光促進(jìn)植株生長和光合作用;研究了培養(yǎng)基上的不同激素配比對植株生長和次生代謝物積累的影響,篩選了最佳生長培養(yǎng)基(MB+1.0mg/L BA+1.0mg/L IBA)和最佳代謝培養(yǎng)基(MB+2.0mg/L BA+0.2mg/L IBA);研究了不同的激素配比與光質(zhì)組合對次生代謝物積累的影響,結(jié)果發(fā)現(xiàn),在最佳代謝培養(yǎng)基上,紅光最有利于酚類物質(zhì)積累,白光則不利于積累;在最佳生長培養(yǎng)基上,白光和藍(lán)光最有利于酚類物質(zhì)的積累,綠光則不利于酚類積累。綜上,該研究基于轉(zhuǎn)錄組學(xué)分析鑒定了灰氈毛忍冬綠原酸生物合成途徑的重要功能基因和調(diào)控因子;系統(tǒng)建立了遺傳轉(zhuǎn)化技術(shù)體系,并對關(guān)鍵基因HQT進(jìn)行了功能分析;探索了不同的培養(yǎng)條件對多酚物質(zhì)特別是綠原酸積累的調(diào)控;為闡明灰氈毛忍冬中綠原酸生物合成的主要途徑及調(diào)控機(jī)制提供了重要參考。
[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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S567.79
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本文編號：1892337