茶树类黄酮3′-羟基化酶功能分析及其在代谢工程中的应用

发布时间：2018-05-25 10:41

  本文选题：茶树 + 类黄酮3′-羟基化酶　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：儿茶素类物质属于类黄酮化合物,是茶的主体呈味物质和重要的保健成分。根据B环上的羟基数量,儿茶素可分为B环-3′,4′-二羟基化儿茶素和B环-3′,4′,5′-三羟基化儿茶素。类黄酮3′-羟基化酶(F3′H)负责催化柚皮素和二氢山奈酚B环3′位羟基化,生成圣草酚和二氢槲皮素,这2种化合物是合成B环-3′,4′-二羟基化儿茶素的中间体。对类黄酮3′-羟基化酶这一类黄酮生物合成中的关键酶研究,有助于通过适当措施调节茶中儿茶素类物质的合成,提高茶叶品质,同时又为类黄酮代谢工程提供基因来源。本研究首先以茶树叶片为材料,克隆了茶树类黄酮3′-羟基化酶(CsF3′H)的基因,并分析该酶的功能;进而分析了茶籽苗发育过程中、缺氮处理茶树叶片中和紫色芽叶茶树叶片中,CsF3′H的表达情况和儿茶素类物质的含量变化;最后探讨了CsF3′H在类黄酮代谢工程中的应用。主要结果如下:1.通过RECE方法克隆出一条1 706 bp编码茶树类黄酮3′-羟基化酶的基因序列,GenBank登录号为:KT180309。该基因包含1 557 bp的开放阅读框,编码518个氨基酸,分子大小为57.07 KDa,等电点为6.82。生物信息学分析发现,CsF3'H具有4个细胞色素P450酶特异保守序列(PPGPNPWP,FGAGRRISAG,E-R-R和AGTDTS)和3个类黄酮3'-羟基化酶特异保守序列(VVVAAS,GGEK和ADVRG)。CsF3'H能催化三种底物,其最适底物是柚皮素,其次是山奈酚,再次是二氢山奈酚,三种底物的Km值分别为17.08、68.06和143.64μM,Vmax值分别为0.98、0.19和0.44 pM·min-1,kcat值分别为49.09、9.86和21.88 pM·min-1·mg-1 microsome。2.在茶籽苗发育过程中,儿茶素、表没食子儿茶素没食子酸酯、没食子儿茶素没食子酸酯、B环-3′,4′-二羟基化儿茶素、B环-3′,4′,5′-三羟基化儿茶素和总儿茶素的含量呈现相同的变化趋势,即从S1到S3阶段逐渐升高,从S3到S4阶段降低;CsF3′H和其他类黄酮生物合成相关基因呈现相同的表达趋势,即从S1到S3阶段,这些基因的表达量先逐渐升高,从S3到S4阶段,它们的表达量快速下降;CsF3′H和其他类黄酮生物合成相关基因的表达量,同B环-3′,4′-二羟基化儿茶素、B环-3′,4′,5′-三羟基化儿茶素和总儿茶素累积量呈现正相关性。3.缺氮处理12天样中的B环-3′,4′-二羟基化儿茶素、B环-3′,4′,5′-三羟基化儿茶素和总儿茶素含量,分别是正常水培12天样的1.33、1.18和1.21倍。相对于正常水培样,从8天到12天,缺氮处理样中B环-3′,4′-二羟基化儿茶素、B环-3′,4′,5′-三羟基化儿茶素和总儿茶素明显增加。CsF3′H和其他10个相关基因(PAL、CHS、CHI、F3H、F3′5′H、DFR、LAR、ANS、ANR1和ANR2)在缺氮处理12天样中表达量,分别是正常水培12天样的2.61、2.59、2.03、2.83、2.98、1.61、4.66、4.31、5.33、1.66和4.02倍。从缺氮处理8天到12天,所有测试的基因表达量都呈现上调趋势。4.本试验中所用的来源于湄潭苔茶后代的紫色芽叶茶树与对照绿色芽叶茶树,在叶色上存在显著差异。相对于对照,紫芽茶树花青素含量高;总儿茶素含量低;csf3′h和其他类黄酮生物合成关键酶(pal、chs、chi、f3h、dfr、ans、anr1、anr2和f3?5?h)基因表达量上调。紫色芽叶中,各相关基因(lar除外)表达量,不仅同总儿茶素累积相关性高(r=0.84~0.99),而且与花青素累积相关性也较高(r=0.72~1.00)。绿色芽叶中,各基因(lar、csf3?h除外)表达量仅同总儿茶素累积相关性高(r=0.64~0.77)。5.为生物合成圣草酚、二氢槲皮素和槲皮素,分别以大肠杆菌和酵母作为发酵菌株进行生物合成。在大肠杆菌生物合成中,构建了4个sumo-csf3?h::atr3aa融合蛋白载体,分别转入大肠杆菌菌株top10、dh5α和bl21中表达。转化sumo-csf3?h[28-518]::atr1[49-688]3aa载体的top10菌株在25℃下发酵,转化效率最高,能将1000μm柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μm圣草酚、131.76μm二氢槲皮素和188.62μm槲皮素。sumo-csf3?h[28-518]::atr1[49-688]3aa载体中,编码csf3?h第28位氨基酸至第518位氨基酸的序列,与编码atr1第49位氨基酸至第688位氨基酸的序列由一段编码3个氨基酸的基因序列连接。以酵母菌株pyes-dest52-csf3′hwat11作为发酵菌株,分别将1000μm柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μm圣草酚、446.07μm二氢槲皮素和594.64μm槲皮素。6.为生物合成山奈酚,构建了6个sumo-f3h::fls载体,其中基因f3h和基因fls采用一段编码3个氨基酸的基因序列连接。在这6个载体转化的菌株中,sumo-csf3h::atfls3aabl21转化的终产物山奈酚产量最高,达到1579.41μm。比较共表达和融合表达的生物转化效率发现,petduet-csf3h与prsfduet-atfls共表达终产物山奈酚的转化率为37.79%;petduet-csf3h::atfls3aa融合表达的终产物转化率为35.52%;prsfduet-csf3h::atfls3aa融合表达的终产物转化率为26.64%。petduet-csf3h与prsfduet-atfls共表达生成的中间产物多,是petduet-csf3h::atfls3aa融合表达生成的中间产物的2.85倍。比较连接肽长短对融合蛋白生物转化效率的影响发现,由9个氨基酸连接肽构建的融合表达菌株sumo-csf3h::atfls9aabl21生成的终产物山奈酚达到3133.09μm,是3个氨基酸连接肽构建的融合表达菌株sumo-csf3h::atfls3aabl21的1.98倍,且sumo-csf3h::atfls9aabl21菌株生成的中间产物二氢山奈酚仅是sumo-csf3h::atfls3aabl21菌株的21.33%。酶动力学分析表明,融合蛋白csf3h::atfls9aa对底物柚皮素、二氢山奈酚的转化率大于融合蛋白csf3h::atfls3aa,连接肽的长短对csf3h::atfls融合蛋白的活性存在影响。7.为生物合成槲皮素,以5 mM柚皮素为底物,喂食pES-URA-CsF3′H和pESHIS-CsF3H::AtFLS 9AA共表达酵母菌株WAT11,在发酵36 h~48 h中,最多能生成1412.16μM圣草酚、490.25μM山奈酚、445.75μM槲皮素、66.75μM二氢槲皮素和73.50μM二氢山奈酚。
[Abstract]:Catechins belong to the flavonoids and are the main flavors and important health components of tea. According to the number of hydroxyl groups on the B ring, catechin can be divided into B ring -3 ', 4' - two hydroxy catechin and B ring -3 ', 4', 5 '- three hydroxylated catechin. The flavonoid 3' - hydroxylase (F3 'H) is responsible for the catalysis of naringin and two hydropomelenol B The hydroxylation of the ring 3 'is hydroxylation to produce St. alacetol and two hydroxy quercetin. These 2 compounds are intermediates in the synthesis of B ring -3', 4 '- two hydroxy catechin. The study of the key enzymes in the flavonoid biosynthesis of flavonoids 3' - hydroxylase helps to adjust the synthesis of catechin in tea and improve the quality of tea by appropriate measures. In this study, the genes of tea tree flavonoids 3 '- hydroxylase (CsF3' H) were cloned and the function of the enzyme was analyzed. The expression of CsF3 'H and the expression of CsF3' H in tea leaves and purple bud leaf tea leaves during the development of tea seedlings were analyzed. The changes in the content of the tea substance; finally, the application of CsF3 'H in the metabolic engineering of flavonoids was discussed. The main results were as follows: 1. the sequence of 1706 BP encoded tea flavonoids 3' - hydroxylase was cloned by RECE method. The GenBank login number was: KT180309. this gene contains open reading frame containing 1557 BP, and 518 amino acids are encoded. The molecular size is 57.07 KDa, and the isoelectric point is 6.82. bioinformatics analysis. It is found that CsF3'H has 4 specific conservative sequences of cytochrome P450 enzyme (PPGPNPWP, FGAGRRISAG, E-R-R and AGTDTS) and 3 specific conservative sequences of the flavonoid 3'- hydroxylase (VVVAAS, GGEK and ADVRG) can catalyze three substrates. The most suitable substrate is naringin, followed by mountain The Km value of the three substrates was two hydrogen and 143.64 mu M respectively, the values of Vmax were 0.98,0.19 and 0.44 pM min-1 respectively, and kcat values were 49.09,9.86 and 21.88 pM. Min-1 mg-1 respectively in the development of tea seedlings, catechins, epigallocatechin gallate and gallate gallate. The content of B ring -3 ', 4' - two hydroxylated catechin, B ring -3 ', 4', 5 '- three hydroxylated catechin and catechin showed the same trend of variation, that is, from S1 to S3 stage, gradually decreasing from S3 to S4 phase, CsF3' H and other genes related to flavonoid biosynthesis are present in the same expression trend, from S1 to S3 stage, these genes The expression level was increased first, from S3 to S4. The expression of CsF3 'H and other flavonoid biosynthesis related genes were the same as B ring -3', 4 '- two hydroxylated catechin, B ring -3', 4 ', 5' - three hydroxylated catechin and total catechin accumulation of B ring -3 ', 4 in the 12 day sample of.3. nitrogen deficiency treatment. '- two hydroxy catechin, B ring -3', 4 ', 5' - three hydroxy catechin and catechin, respectively, 1.33,1.18 and 1.21 times of normal hydroponic culture 12 days respectively. Compared with normal hydroponic samples, from 8 days to 12 days, B ring -3 ', 4' - two hydroxy catechin, B ring -3 ', 4', 5 '- three hydroxy catechin and catechin are obvious .CsF3 'H and 10 other related genes (PAL, CHS, CHI, F3H, F3' 5 'H, DFR, LAR, ANS, ANR1, and ANR1) were expressed in the 12 days of nitrogen deficiency treatment, respectively, and 4.02 times as much as 12 days in normal hydroponic culture. From the nitrogen deficiency to 12 days from 8 days to 12 days, the expression of all the tests was up to up. There are significant differences in leaf color between the purple bud leaf tea tree and the control green bud leaf tea tree from the offspring of Meitan moss tea. Compared with the control, the content of anthocyanins in the purple bud tea tree is high; the content of the total catechin is low; csf3 'H and other key enzymes of the flavonoid biosynthesis (pal, CHS, Chi, F3H, DFR, ans, anr1, anr2 and F3? 5?) In the purple bud leaves, the expression of each related gene (except lar) was not only associated with the accumulation of total catechin (r=0.84~0.99), but also with the accumulation of anthocyanins (r=0.72~1.00). In green buds, the expression of each gene (except LAR, csf3? H) was only associated with the accumulation of total catechin (r=0.64~0.77).5. as biosynthesis in green bud leaves. In the biosynthesis of Escherichia coli and yeast, 4 sumo-csf3? H:: atr3aa fusion protein vectors were constructed, which were transferred into Escherichia coli TOP10, DH5 alpha and BL21 respectively. Sumo-csf3 h[28-518]: Top: atr1[49-688]3aa carrier top. The 10 strain was fermented at 25 C, and the conversion efficiency was the highest. It could convert 1000 u m naringin, two HMH and kaempferol to 287.93 mu m, 131.76 m two and 188.62 mu m quercetin.Sumo-csf3? H[28-518]: atr1[49-688]3aa vector, encoding csf3? H twenty-eighth amino acids to 518th amino acids, and coded ATR1 The sequence of forty-ninth amino acids to 688th amino acids is connected by a sequence of genes encoding 3 amino acids. The yeast strain pyes-dest52-csf3 'hwat11 is used as a fermentative strain, and 1000 mu m naringin, two hydro acetaminophen and kaempferol, respectively, can be converted to 734.32 mu m, 446.07, two hydrogen quercetin and 594.64 u m quercetin.6. For biosynthesis of kaempferol, 6 sumo-f3h:: FLS vectors were constructed, in which gene F3H and gene FLS were connected by a sequence of genes encoding 3 amino acids. Among the strains transformed by the 6 vectors, the highest yield of the final product of sumo-csf3h:: atfls3aabl21 transformation was to reach 1579.41 mu m. and to compare the co expression and fusion expression of the biotransformation. It was found that the conversion rate of the final product of petduet-csf3h and prsfduet-atfls was 37.79%, and the conversion rate of the final product of petduet-csf3h:: atfls3aa fusion expression was 35.52%, and the conversion rate of the final product of prsfduet-csf3h:: atfls3aa fusion expression was more of the intermediate products produced by the co expression of 26.64%.petduet-csf3h and prsfduet-atfls, which was petduet. -csf3h:: atfls3aa fusion expressed 2.85 times the production of the intermediate product. Comparing the effect of the length of the connective peptide on the bioconversion efficiency of the fusion protein, the fusion expression strain, sumo-csf3h:, constructed by 9 amino acid connective peptides, was found to be 3133.09 mu m of the final product of atfls9aabl21, and a fusion protein constructed by 3 amino acid connective peptides. Strain sumo-csf3h:: 1.98 times of atfls3aabl21, and sumo-csf3h:: the intermediate product of sumo-csf3h:: atfls9aabl21 was only sumo-csf3h:: 21.33%. enzyme kinetic analysis of atfls3aabl21 strain showed that the fusion protein csf3h:: atfls9aa to substrate naringin, two of the conversion of HMC was greater than that of the fusion protein csf3h:: atfls3aa, connective peptide. The activity of csf3h:: atfls fusion protein affects.7. as the biosynthesis of quercetin, with 5 mM naringenin as the substrate, feeding pES-URA-CsF3 'H and pESHIS-CsF3H:: AtFLS 9AA co expression yeast strain WAT11. In the 36 h~48 h, the maximum can be generated by 1412.16 mu, 490.25 mu, 445.75 Mu quercetin and 66.75 Mu two hydrogen quercetin. And 73.50 micron M two HMH.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S571.1
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