發(fā)布時(shí)間：2018-05-25 10:41

  本文選題：茶樹 + 類黃酮3′-羥基化酶　； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：兒茶素類物質(zhì)屬于類黃酮化合物,是茶的主體呈味物質(zhì)和重要的保健成分。根據(jù)B環(huán)上的羥基數(shù)量,兒茶素可分為B環(huán)-3′,4′-二羥基化兒茶素和B環(huán)-3′,4′,5′-三羥基化兒茶素。類黃酮3′-羥基化酶(F3′H)負(fù)責(zé)催化柚皮素和二氫山奈酚B環(huán)3′位羥基化,生成圣草酚和二氫槲皮素,這2種化合物是合成B環(huán)-3′,4′-二羥基化兒茶素的中間體。對類黃酮3′-羥基化酶這一類黃酮生物合成中的關(guān)鍵酶研究,有助于通過適當(dāng)措施調(diào)節(jié)茶中兒茶素類物質(zhì)的合成,提高茶葉品質(zhì),同時(shí)又為類黃酮代謝工程提供基因來源。本研究首先以茶樹葉片為材料,克隆了茶樹類黃酮3′-羥基化酶(CsF3′H)的基因,并分析該酶的功能;進(jìn)而分析了茶籽苗發(fā)育過程中、缺氮處理茶樹葉片中和紫色芽葉茶樹葉片中,CsF3′H的表達(dá)情況和兒茶素類物質(zhì)的含量變化;最后探討了CsF3′H在類黃酮代謝工程中的應(yīng)用。主要結(jié)果如下:1.通過RECE方法克隆出一條1 706 bp編碼茶樹類黃酮3′-羥基化酶的基因序列,GenBank登錄號為:KT180309。該基因包含1 557 bp的開放閱讀框,編碼518個(gè)氨基酸,分子大小為57.07 KDa,等電點(diǎn)為6.82。生物信息學(xué)分析發(fā)現(xiàn),CsF3'H具有4個(gè)細(xì)胞色素P450酶特異保守序列(PPGPNPWP,FGAGRRISAG,E-R-R和AGTDTS)和3個(gè)類黃酮3'-羥基化酶特異保守序列(VVVAAS,GGEK和ADVRG)。CsF3'H能催化三種底物,其最適底物是柚皮素,其次是山奈酚,再次是二氫山奈酚,三種底物的Km值分別為17.08、68.06和143.64μM,Vmax值分別為0.98、0.19和0.44 pM·min-1,kcat值分別為49.09、9.86和21.88 pM·min-1·mg-1 microsome。2.在茶籽苗發(fā)育過程中,兒茶素、表沒食子兒茶素沒食子酸酯、沒食子兒茶素沒食子酸酯、B環(huán)-3′,4′-二羥基化兒茶素、B環(huán)-3′,4′,5′-三羥基化兒茶素和總兒茶素的含量呈現(xiàn)相同的變化趨勢,即從S1到S3階段逐漸升高,從S3到S4階段降低;CsF3′H和其他類黃酮生物合成相關(guān)基因呈現(xiàn)相同的表達(dá)趨勢,即從S1到S3階段,這些基因的表達(dá)量先逐漸升高,從S3到S4階段,它們的表達(dá)量快速下降;CsF3′H和其他類黃酮生物合成相關(guān)基因的表達(dá)量,同B環(huán)-3′,4′-二羥基化兒茶素、B環(huán)-3′,4′,5′-三羥基化兒茶素和總兒茶素累積量呈現(xiàn)正相關(guān)性。3.缺氮處理12天樣中的B環(huán)-3′,4′-二羥基化兒茶素、B環(huán)-3′,4′,5′-三羥基化兒茶素和總兒茶素含量,分別是正常水培12天樣的1.33、1.18和1.21倍。相對于正常水培樣,從8天到12天,缺氮處理樣中B環(huán)-3′,4′-二羥基化兒茶素、B環(huán)-3′,4′,5′-三羥基化兒茶素和總兒茶素明顯增加。CsF3′H和其他10個(gè)相關(guān)基因(PAL、CHS、CHI、F3H、F3′5′H、DFR、LAR、ANS、ANR1和ANR2)在缺氮處理12天樣中表達(dá)量,分別是正常水培12天樣的2.61、2.59、2.03、2.83、2.98、1.61、4.66、4.31、5.33、1.66和4.02倍。從缺氮處理8天到12天,所有測試的基因表達(dá)量都呈現(xiàn)上調(diào)趨勢。4.本試驗(yàn)中所用的來源于湄潭苔茶后代的紫色芽葉茶樹與對照綠色芽葉茶樹,在葉色上存在顯著差異。相對于對照,紫芽茶樹花青素含量高;總兒茶素含量低;csf3′h和其他類黃酮生物合成關(guān)鍵酶(pal、chs、chi、f3h、dfr、ans、anr1、anr2和f3?5?h)基因表達(dá)量上調(diào)。紫色芽葉中,各相關(guān)基因(lar除外)表達(dá)量,不僅同總兒茶素累積相關(guān)性高(r=0.84~0.99),而且與花青素累積相關(guān)性也較高(r=0.72~1.00)。綠色芽葉中,各基因(lar、csf3?h除外)表達(dá)量僅同總兒茶素累積相關(guān)性高(r=0.64~0.77)。5.為生物合成圣草酚、二氫槲皮素和槲皮素,分別以大腸桿菌和酵母作為發(fā)酵菌株進(jìn)行生物合成。在大腸桿菌生物合成中,構(gòu)建了4個(gè)sumo-csf3?h::atr3aa融合蛋白載體,分別轉(zhuǎn)入大腸桿菌菌株top10、dh5α和bl21中表達(dá)。轉(zhuǎn)化sumo-csf3?h[28-518]::atr1[49-688]3aa載體的top10菌株在25℃下發(fā)酵,轉(zhuǎn)化效率最高,能將1000μm柚皮素、二氫山奈酚和山奈酚,分別轉(zhuǎn)化生成287.93μm圣草酚、131.76μm二氫槲皮素和188.62μm槲皮素。sumo-csf3?h[28-518]::atr1[49-688]3aa載體中,編碼csf3?h第28位氨基酸至第518位氨基酸的序列,與編碼atr1第49位氨基酸至第688位氨基酸的序列由一段編碼3個(gè)氨基酸的基因序列連接。以酵母菌株pyes-dest52-csf3′hwat11作為發(fā)酵菌株,分別將1000μm柚皮素、二氫山奈酚和山奈酚,最多能轉(zhuǎn)化生成734.32μm圣草酚、446.07μm二氫槲皮素和594.64μm槲皮素。6.為生物合成山奈酚,構(gòu)建了6個(gè)sumo-f3h::fls載體,其中基因f3h和基因fls采用一段編碼3個(gè)氨基酸的基因序列連接。在這6個(gè)載體轉(zhuǎn)化的菌株中,sumo-csf3h::atfls3aabl21轉(zhuǎn)化的終產(chǎn)物山奈酚產(chǎn)量最高,達(dá)到1579.41μm。比較共表達(dá)和融合表達(dá)的生物轉(zhuǎn)化效率發(fā)現(xiàn),petduet-csf3h與prsfduet-atfls共表達(dá)終產(chǎn)物山奈酚的轉(zhuǎn)化率為37.79%;petduet-csf3h::atfls3aa融合表達(dá)的終產(chǎn)物轉(zhuǎn)化率為35.52%;prsfduet-csf3h::atfls3aa融合表達(dá)的終產(chǎn)物轉(zhuǎn)化率為26.64%。petduet-csf3h與prsfduet-atfls共表達(dá)生成的中間產(chǎn)物多,是petduet-csf3h::atfls3aa融合表達(dá)生成的中間產(chǎn)物的2.85倍。比較連接肽長短對融合蛋白生物轉(zhuǎn)化效率的影響發(fā)現(xiàn),由9個(gè)氨基酸連接肽構(gòu)建的融合表達(dá)菌株sumo-csf3h::atfls9aabl21生成的終產(chǎn)物山奈酚達(dá)到3133.09μm,是3個(gè)氨基酸連接肽構(gòu)建的融合表達(dá)菌株sumo-csf3h::atfls3aabl21的1.98倍,且sumo-csf3h::atfls9aabl21菌株生成的中間產(chǎn)物二氫山奈酚僅是sumo-csf3h::atfls3aabl21菌株的21.33%。酶動(dòng)力學(xué)分析表明,融合蛋白csf3h::atfls9aa對底物柚皮素、二氫山奈酚的轉(zhuǎn)化率大于融合蛋白csf3h::atfls3aa,連接肽的長短對csf3h::atfls融合蛋白的活性存在影響。7.為生物合成槲皮素,以5 mM柚皮素為底物,喂食pES-URA-CsF3′H和pESHIS-CsF3H::AtFLS 9AA共表達(dá)酵母菌株WAT11,在發(fā)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二氫槲皮素和73.50μM二氫山奈酚。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S571.1
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本文編號：1933136