【作者】 蒋向辉；

【导师】 刘选明；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 博士

【摘要】 绿原酸是金银花在生长发育过程中产生的重要次生代谢产物，近年来随着金银花研究的不断深入，绿原酸的含量、合成机制及有效地开发利用成为了生物化学、分子生物学以及代谢工程研究领域的一个热点。为了能有效地提高金银花绿原酸含量，本研究从基因克隆、基因结构与功能的生物信息学预测、基因表达等不同的方面对金银花绿原酸生物合成过程中重要的三个关键酶基因进行了相关研究，既为研究金银花绿原酸的生物合成机制奠定基础，也为基因工程在代谢工程的应用提供一定的借鉴。本论文的主要研究结果如下：（1）采用RT-PCR及RACE方法从金银花中分离到绿原酸合成紧密相关的LjHCT、LjC3H1及LjCCoAOMT1基因的全长CDS序列，LjHCT基因CDS全长1293，编码430个氨基酸，LjC3H1基因CDS全长1533，编码510个氨基酸，LjCCoAOMT1基因CDS全长744，编码267个氨基酸。通过对不同物种三个基因的亲缘关系分析，显示金银花LjHCT基因与桔梗与刺苞菜蓟HCT基因相似性最高，金银花LjC3H1基因与咖啡和红车轴草C3H1基因相似性最高，金银花LjCCoAOMT1基因与羊奶参和光皮桦的CCoAOMT1基因相似性较高，这三个基因与木本植物的相似性要高于草本植物。通过跨膜结构域的预测分析，LjC3H1蛋白有一个跨膜结构域，而其他两个蛋白没有跨膜结构域。通过亚细胞定位分析显示LjHCT蛋白可能在叶绿体或线粒体上发挥作用，LjC3H1蛋白可能在叶绿体上发挥作用，LjCCoAOMT1蛋白可能在细胞质中发挥作用。通过疏/亲水性分析结果显示，三个蛋白全为亲水性蛋白。二级结构分析显示三个蛋白都主要以α–螺旋与不规则盘绕为主。通过氨基酸保守序列分析显示LjHCT蛋白包含有转移酶的保守序列，LjC3H1蛋白包含有细胞色素P450的保守序列，LjCCoAOMT1蛋白包含有甲基转移酶的保守序列。并构建了三种蛋白的三级结构模型。（2）分析了金银花开花期绿蕾、白蕾、白花、黄花、幼叶、老叶、幼茎与茎皮中绿原酸含量，结果显示，绿原酸含量最高的部位是绿蕾，为干重的2.95%，老叶，幼叶中绿原酸含量较低，仅为0.20%和0.41%。通过荧光定量PCR的方法,对金银花不同部位相关基因的表达进行了分析，结果显示，LjHCT, LjC3H1与LjCCoAOMT1基因在金银花不同组织中广泛表达，但表达水平各有差异。LjHCT基因在白花相对表达量最高，白蕾中相对表达量最低，白花中相对表达量是白蕾中的400倍。LjC3H1基因在黄花相对表达量最高，老叶中相对表达量最低，黄花中相对表达量是老叶中的20.5倍。LjCCoAOMT1基因在绿蕾相对表达量最高，在白蕾相对表达量最低，绿蕾中相对表达量是白蕾的439.1倍，表明LjCCoAOMT1基因虽然在不同组织中都有表达，但表达差异极为显著。LjCCoAOMT1基因的表达量呈现前期减少，后期又增加的趋势。（3）利用大肠杆菌BL21对LjHCT，LjC3H1与LjCCoAOMT1基因所编码的蛋白进行了原核表达，并采用镍柱纯化的方法得到了三种可溶性重组蛋白，我们将融合蛋白用考马斯亮兰染色的方法，检测到Pcold-LjHCT融合蛋白大小约为92kDa，pCOLD-LjCCoAOMT1蛋白大小为72kDa，pCOLD-LjC3H1蛋白大小为102kDa。金银花开花期不同组织绿原酸含量与LjHCT, LjC3H1及LjCCoAOMT1基因表达量的相关关系研究表明，LjCCoAOMT1基因的表达与绿原酸含量有显著的相关性，而LjC3H1与LjHCT基因的表达与绿原酸含量的相关性没有达到显著水平。LjHCT与LjC3H1基因的表达呈一定负相关性，但没有达到显著水平，这可能与LjHCT催化p-香豆酰辅酶A变成p-香豆酰奎尼酸或p-香豆酰莽草酸的反应是一个可逆反应的有关。采用HPLC检测底物含量变化的方法鉴定了pCOLD-LjCCoAOMT1重组蛋白能催化咖啡酸甲基化生成阿魏酸。（4）构建了LjHCT，LjC3H1及LjCCoAOMT1三个基因的YFP的融合表达载体，采用原生质体转化的方法，进行了亚细胞定位研究，结果表明LjHCT与LjCCoAOMT1在细胞中各部位都有表达，其中LjHCT主要在细胞器上，LjCCoAOMT1主要在细胞质中，而LjC3H1蛋白仅在细胞器上有表达，结果与生物信息学分析结果基本一致。构建了LjCCoAOMT1基因的pCAMBIA-1301-LjCCoAOMT1植物表达载体，借助农杆菌介导的方法转化水稻，成功地获得了转基因植株，转基因水稻与野生型在株高、穗长、叶长、叶宽与千粒重方面没有明显的差异，而分蘖数和每穗粒数有显著性差异。（5）通过不同光照和赤霉素（GA3）处理金银花幼苗、转LjCCoAOMT1基因水稻和野生型水稻，进行了环境条件对LjCCoAOMT1基因表达特性影响的研究。结果显示在黑暗条件下，金银花幼苗出现黄化现象，转基因水稻比野生型水稻黄化现象更为严重，但转至蓝光下培养后，金银花幼苗转绿，转LjCCoAOMT1基因水稻比野生型水稻叶片转绿快，表明转基因水稻对光照条件的变化比野生型水稻要敏感。荧光定量PCR分析发现，金银花LjCCoAOMT1在暗处理后和蓝光处理后比在自然光下的表达量显著增加，转基因水稻OsCCoAOMT1基因在暗处理后和蓝光处理后比在自然光下的表达量同样增强，分析表明，在不同光照条件下金银花LjCCoAOMT1与水稻OsCCoAOMT1基因表达模式基本一致。不同浓度的赤霉素对金银花幼苗、转LjCCoAOMT1基因水稻和野生型水稻处理结果显示，短期喷施赤霉素，对金银花幼苗株高生长的促进效果不明显，但赤霉素能够有效地促进转LjCCoAOMT1基因水稻和野生型水稻快速生长，随着赤霉素浓度的提高，效果越好，转LjCCoAOMT1基因水稻与野生型水稻相比，前者增长速度更快，当赤霉素浓度由200mg/L增大至400mg/L时，对水稻OsCCoAOMT1基因表达的促进作用已不太明显，而对转LjCCoAOMT1基因水稻中OsCCoAOMT1基因的促进作用仍很明显。定量PCR检测分析发现，当赤霉素浓度由200mg/L增大至400mg/L，金银花幼苗LjCCoAOMT1基因表达量分别为对照（0mg/L）的1.7倍和1.8倍，而在转基因水稻中OsCCoAOMT1基因表达量分别为对照（0mg/L）的2.4倍和3.6倍，分析表明，金银花LjCCoAOMT1基因与水稻OsCCoAOMT1基因表达模式基本一致，并且在转LjCCoAOMT1基因水稻中，由于LjCCoAOMT1基因的过量表达及OsCCoAOMT1基因的协同表达，高浓度的赤霉素也能有效地发挥其生理作用。（6）本研究成功地建立了金银花组培快繁技术体系，金银花组培最合适的外植体是幼茎，适合金银花愈伤组织细胞诱导的培养基为WPM+6-BA2.0mg/L＋KT0.5mg/L＋NAA1.0mg/L培养基，适合金银花丛生芽诱导的培养基为WPM+6-BA2.0mg/L＋KT2.0mg/L＋NAA0.2mg/L，适合金银花丛生芽生根的培养基为WPM+NAA0.1mg/L＋活性炭150mg/L。以金银花幼茎为外植体，成功地建立了金银花愈伤组织细胞悬浮培养技术体系，并成功地将金银花LjCCoAOMT1基因导入悬浮细胞。在金银花悬浮细胞中，采用荧光定量PCR的方法检测LjC3H1，LjCCoAOMT1和LjHCT的相对表达量，结果显示，LjCCoAOMT1基因在转基因愈伤中过量表达，表达量是未进行遗传转化的愈伤中LjCCoAOMT1基因表达量的4.2倍，LjC3H1基因在转化LjCCoAOMT1基因的愈伤中协同表达，表达量略有增加，但LjHCT基因在转化LjCCoAOMT1基因的愈伤中表达受到抑制，表达量下降，这可能与LjHCT酶在绿原酸代谢途径中的催化作用是双重活性有关。更多还原

【Abstract】 Chlorogenic acid (CGA) is an important secondary metabolite during thedevelopment and growth of Lonicera japonica Thunb. Recently there has became ahot spot in the study of content of chlorogenic acid, synthesis mechanism andeffective development and utilization in the research field of biochemistry, molecularbiology and metabolic engineering. In order to effectively improve the chlorogenicacid production of Lonicera japonica Thunb, we investigated three key enzyme genesthat are important in biosynthesis of chlorogenic acid in Lonicera japonica Thunb.using research methods and tools from genomics, proteomics and bioinformatics. Theresearch results will help us understand the biosynthesis mechanism of chlorogenicacid, and also provide a certain reference for the application of gene engineering inthe field of metabolic engineering. The major results of this study were listed asfollows:(1) By RT-PCR and RACE method LjHCT, LjC3H1and LjCCoAOMT1that areclosely related to biosynthesis of chlorogenic acid in Lonicera japonica Thunb, wereisolated. LjHCT gene coding sequence (CDS) sequence is1293bp long, coding for430amino acids; LjC3H1gene is1533bp, coding510amino acids; LjCCoAOMT1gene is744bp, coding267amino acids. The phylogenic tree analysis showed that thesequences of LjHCT, LjC3H1, LjCCoAOMT1are most closely related to HCT gene inPlatycodon grandiflorus and Cynara cardunculus Linn., C3H1gene in coffee andTrifolium pratense L., and CCoAOMT1gene in Codonopsis lanceolata and Betulaluminifera., respectively. The three genes are more homologous to woody plants thanto herbs. Based on the prediction of transmembrane domain structure LjC3H1proteinhas one transmembrane structure domain, but the other two proteins have none.Subcellular localization analysis showed that LjHCT protein may play an importantrole in the chloroplasts or mitochondria, LjC3H1protein in the chloroplasts, andLjCCoAOMT1in the cytoplasm. Hydrophobic/hydrophilic analysis showed that allthree proteins are hydrophilic. The three proteins have alpha-helix and random coil bysecondary structure analysis. Amino acid conserved sequence analysis suggested thatLjHCT, LjC3H1, and LjCCoAOMT1proteins contain transferase conservativesequence, cytochrome P450conservative sequence, methyltransferase conservativesequence, respectively. (2) This study analyzed the CGA content in white flower, yellow flower, greenbud, white bud, young shoot, old leaf, young leaf and stem bark during flowerdevelopment in Lonicera japonica Thunb., and the results showed that the CGAcontent is the highest in green bud (2.95%of the dry weight), and lowest in the youngleaves and the old leaf, only0.20%and0.41%respectively. Fluorescence quantitativePCR analyses showed that LjHCT, LjC3H1and LjCCoAOMT1genes are widelyexpressed in different tissues of Lonicera japonica Thunb., but the expression levelsare different from each other. The expression of LjHCT gene is the highest in whiteflower, but lowest in white bud, and the relative expression of LjHCT gene in whiteflowers is400times higher than in white bud. The expression of LjC3H1genes is thehighest in yellow flower, but lowest in the old leaf, and the relative expression ofLjC3H1in yellow flower is20.5times higher than in the old leaf. The expression ofLjCCoAOMT1genes is the highest in green bud, but lowest in the white bud, and therelative expression of LjCCoAOMT1in green bud is439.1times higher in white bud,which showed that LjCCoAOMT1gene was expressed in different tissues, but therelative expression of the three different proteins differ greatly. The relativeexpression of LjCCoAOMT1gene reduces at earlier stage, and increase at later stage.(3) We expressed the coding proteins of LjHCT, LjC3H1and LjCCoAOMT1genes in E. coli BL21. By the Ni-chelating affinity chromatography methods, thethree kinds of soluble recombinant proteins of pCOLD-LjHCT,pCOLD-LjCCoAOMT1and pCOLD-LjC3H1were obtained. Using the coomassiebrilliant blue dyeing method, we found that the sizes of three fusion proteins(pCOLD-LjHCT, pCOLD-LjCCoAOMT1, pCOLD-LjC3H1) are92kDa,72kDa, and102kDa, respectively. The correlations between CGA content in different tissues andrelative gene expression of LjHCT, LjC3H1and LjCCoAOMT1were analyzed, and theresults showed that the chlorogenic acid content is highly correlated withLjCCoAOMT1gene expression, but this high correlation with chlorogenic acidcontent is not found in the other two genes. There was negative correlation betweenLjHCT and LjC3H1gene expression, but the correlation was not significant, whichmay be due to the reversible reaction that LjHCT catalyzes chlorogenic acid reactingto form caffeic acid. Using HPLC testing the change in substrate content, weidentified activity of the LjCCoAOMT1recombinant protein, and the results showedLjCCoAOMT1can catalyze caffeic acid to form ferulic acid.(4) We constructed three YFP fusion expression vectors, which are: YFP-LjHCT,YFP-LjC3H1and YFP-LjCCoAOMT1, using protoplast transformation method. The subcellular localization assays showed that LjHCT protein was located in differentorganelles，LjCCoAOMT1protein mainly in the cytomembrane, and LjC3H1proteinonly in the organelles, which are generally the same as the prediction frombioinformatics analysis. We constructed the pCAMBIA-1301::LjCCoAOMT1plantexpression vector, and transformed the vector in rice using the agrobacteriummediated transformation method. Compared to the wild type rice, the successfullytransgenic plants have no significant difference in plant height, spike length, leaflength, leaf width and grain weight, but does have significant difference in tillernumber and grain number per panicle.(5) We investigated the impact of environmental conditions on LjCCoAOMT1gene expression (light and gibberellic acid (GA3)) in Lonicera japonica Thunb.seedling, transgenic LjCCoAOMT1rice and wild type rice. The result showed that inthe dark conditions, Lonicera japonica Thunb. turned yellow, and transgenic riceturned yellow more seriously, but after being transferred to blue light environments,Lonicera japonica Thunb. turned green, and the LjCCoAOMT1transgenic rice turnedgreen more quickly than the wild type rice, which suggests that transgenic rice wasmore sensitive to light conditions than the wild type rice. Through the fluorescencequantitative PCR analysis, we found in that Lonicera japonica Thunb. the relativeexpression of LjCCoAOMT1increases under dark or blue light conditions, thetransgenic rice OsCCoAOMT1expression also increase more quickly than in naturallight. The result showed that gene expression patterns of Lonicera japonica Thunb.LjCCoAOMT1and rice OsCCoAOMT1are basically identical. Lonicera japonicaThunb., LjCCoAOMT1transgenic rice and wild type rice were treated with differentconcentration of GA3. We found that promotion effect of small dose of GA3onLonicera japonica Thunb. growth is not obvious, but GA3can effectively promoteboth transgenic LjCCoAOMT1rice and wild type rice growing, and the promotingeffect becomes greater with increasing concentration. However, the LjCCoAOMT1transgenic rice grew faster. The OsCCoAOMT1expression quantity increased alongwith the increasing of GA3in transgenic LjCCoAOMT1rice and wild type rice, butmore significantly in transgenic LjCCoAOMT1rice. When GA3concentrationincreased from200mg/L to400mg/L, OsCCoAOMT1gene expression in wild typerice did not increase obviously, but in transgenic LjCCoAOMT1rice the differencewas very obvious, which indicated that under high concentration the physiologicalactivity of GA3was weakened, and in LjCCoAOMT1transgenic rice, the GA3canstill perform their normal physiological function under high concentration. (6) This study successfully set up the rapid reproduction technology of Lonicerajaponica Thunb. The most appropriate explant is young stem, the most suitablemedium for callus induction is WPM+6-BA2.0mg/L+KT0.5mg/L+NAA1.0mg/L medium, the most suitable medium for cluster bud induction is WPM+6-BA2.0mg/L+KT2.0mg/L+NAA0.2mg/L medium, and the most suitable medium forcluster bud rooting induction is WPM+NAA0.1mg/L+activated carbon150mg/Lmedium. Using the young stem for explants, we successfully set up the callus cellsuspension culture technology system of Lonicera japonica Thunb., and successfullytransferred LjCCoAOMT1gene to suspended cells. In suspension cells of Lonicerajaponica Thunb., we used the fluorescent quantitative PCR method to detect therelative expression quantity of LjC3H1, LjCCoAOMT1and LjHCT. the result showedthat LjCCoAOMT1gene expression was strengthened in transgenic callus, the expressquantity being4.2times for wild type callus. The expression of LjC3H1gene in thetransformation LjCCoAOMT1callus strengthened too, but LjHCT gene in thetransformation LjCCoAOMT1callus was restrained, which may result from the factthat LjHCT enzyme is double active in the chlorogenic acid metabolic pathway.更多还原