银杏黄酮和木质素代谢相关基因功能分析

【作者】 程华；

【导师】 曹福亮； 程水源；

【作者基本信息】 南京林业大学 ， 森林培育， 2012， 博士

【摘要】 为了更深入了解银杏叶黄酮合成的分子机理及与苯丙氨酸代谢路径的联系，为今后利用生物技术手段提高银杏叶黄酮含量奠定基础。本文以叶用银杏家佛手为研究材料，采用同源克隆方法从银杏中分离与黄酮、木质素等积累相关的几个酶基因，它们分别是类异黄酮还原酶相似蛋白基因(GbIRL1)，二氢黄酮醇4-还原酶基因(GbDFRs)，肉桂醇脱氢酶基因(GbCAD1)和肉桂酰辅酶A还原酶基因(GbCCRs)。在得到基因序列及部分调控序列的前提下，开展相应功能研究与表达分析，主要研究结果概括如下：(1)银杏查尔酮异构酶基因(GbIRL1)的克隆、鉴定、性质及表达模式的研究。利用简并PCR技术和RACE技术从银杏叶片中克隆得到了GbIRL1的cDNA全长序列。通过信息学分析发现GbIRL1的cDNA全长为1261bp，含有一个921bp的开放式阅读框(ORF)，编码306个氨基酸序列。蛋白质同源序列分析表明，GbIRL1与IRLs家族中PCBER蛋白同源性更高；同源建模分析显示GbIRL1序列与苜蓿IRL蛋白的三维结构及活性位点高度相似。Southern blot分析表明，GbIRL1属于多基因家族；GbIRL1重组蛋白大肠杆菌表达显示，其蛋白大小与cDNA序列预测蛋白大小一致，亲和层析及Western blot分析显示，重组GbIRL1含有6×His标签，GbIRL1能在大肠杆菌中正常表达；重组GbIRL1酶活性分析表明，GbIRL1能够催化DDDC、DDC分别生成TDDC和IDDDC，具有典型的PCBER酶催化特点；RT-PCR分析显示，GbIRL1基因在银杏不同组织中都有表达，但存在较大差异，只有受伤或病虫害茎段、叶片中表达量最高，不同茎段中表达水平相对较低，胚乳和外种皮中几乎不表达。GbIRL1与银杏叶黄酮含量的年周期变化分析显示，GbIRL1基因的转录水平与黄酮含量变化间呈乘幂相关，相关系数为0.610。激素和胁迫诱导表达分析显示，虽然诱导表达模式不尽相同，但GbIRL1转录水平能被UV-B、WOU、ABA、SA、ALA和ETH诱导上调，表达模式存在差异。GbIRL1诱导表达模式与其上游的顺式调控元件较为一致，其表达特性与其他PCBER代谢、木脂素和抗毒素代谢基因表达模式类似。推测银杏GbIRL1可能主要参与银杏对病虫害防御、逆境胁迫适应方面代谢，而与黄酮代谢又有一定的联系。(2)银杏3个编码二氢黄酮醇4-还原酶基因(GbDFRs)的分离、鉴定、性质及表达模式研究。利用简并PCR技术和RACE技术从银杏叶片中分别克隆得到了GbDFR1、GbDFR2和GbDFR3的cDNA全长序列。通过信息学分析发现GbDFR1的cDNA全长为1303bp，含有一个1038bp的开放式阅读框(ORF)，编码345个氨基酸序列；GbDFR2的cDNA全长为1325bp，含有一个993bp的开放式阅读框(ORF)，编码330个氨基酸序列；GbDFR3的cDNA全长为1113bp，含有一个1002bp的开放式阅读框(ORF)，编码333个氨基酸序列。蛋白质同源序列分析表明，GbDFRs与与其他物种DFRs同源性较高，分属于3类不同DFRs；同源建模分析显示GbDFRs序列与DFRs家族蛋白的三维结构及活性位点高度相似，其中NADPH结合区域及底物结合位点保守性较高。非保守区域探针的Southern blot分析表明，GbDFRs分别属于3个不同多基因家族；GbDFRs重组蛋白大肠杆菌表达显示，其蛋白大小与cDNA序列预测蛋白大小基本一致，亲和层析及Western blot分析显示，重组GbDFRs含有6×His标签，GbDFRs能在大肠杆菌中正常表达。表达蛋白经分离纯化后酶活测定显示，GbDFR1和GbDFR3分别能够催化DHQ转化为LEU，而DFR2能够催化DHK转化为LEUC；RT-PCR分析显示，GbDFRs基因在银杏不同组织中都有表达，其中GbDFR1和在叶片中表达量最高，GbDFR2在雄蕊表达水平最高。诱导表达分析结果推测，银杏叶片中参与应对环境胁迫和伤害响应的主要是GbDFR1和GbDFR2，同时GbDFR1可能还参与花色素合成，而GbDFR3很可能在黄酮及花色素合成中功能更专一。(3)银杏肉桂醇脱氢酶基因(GbCAD1)的克隆、鉴定、性质及表达模式研究。利用RACE技术从银杏叶中克隆到GbCAD1基因的cDNA序列。得到GbCAD1的cDNA长1494bp，包含最大阅读框(ORF)为1074bp，编码一个357氨基酸多肽序列；通过软件DNAssist2.2预测编码蛋白质38.70kDa，其等电点为5.74。进化树分析结果表明银杏GbCAD1蛋白质序列与其他物种的CADs合酶同源性较高。不同组织表达分析显示，GbCAD1基因在银杏的根和茎中表达量最高，特别是在病虫害侵染部位。GbCAD1重组蛋白大肠杆菌表达显示，其蛋白大小与cDNA序列预测蛋白大小基本一致，亲和层析及Western blot分析显示，重组GbCAD1含有6×His标签，GbCAD1能在大肠杆菌中正常表达。表达蛋白经分离纯化后酶活测定显示，GbCAD1能够催化松柏醛转化为松柏醇；诱导表达分析结果显示，GbCAD1能够受到UV-B、WOU、ABA、SA和ETH显著诱导表达，而ALA对GbCAD1诱导作用不明显，GbCAD1的诱导表达模式可能受到上游苯丙氨酸代谢的影响，主要参与了木质素合成及植物逆境适应。(4)银杏肉桂酰辅酶A还原酶基因(GbCCRs)的克隆、鉴定、性质及表达模式研究。在前期研究基础上，利用简并PCR技术和RACE技术从银杏叶片中分离得到了GbCCR1和GbCCR2的cDNA全长序列。通过信息学分析发现GbCCR1的cDNA全长为1178bp，含有一个972bp的开放式阅读框(ORF)，编码323个氨基酸序列；GbCCR2的cDNA全长为1206bp，含有一个1005bp的开放式阅读框(ORF)，编码334个氨基酸序列多肽。蛋白质同源序列分析表明，GbCCRs与其他物种CCR同源性相对较高，属于同一类别CCR家族，并且属于多基因家族；同源建模分析显示GbCCRs序列与其他家族蛋白的三维结构及活性位点高度相似，CCR单亚基可以分为2个结构域，一个是由底物结合中心形成的结构域，另一个是NADPH结合区域形成的结构域，两个保守区域是酶促反应顺利进行的保证。CCRs蛋白序列进化树分析结果显示GbCCR与其他植物分化较早；Southern blot分析证实，GbCCRs属于多基因家族；GbCCR重组蛋白大肠杆菌表达显示，其蛋白大小与cDNA序列预测融合蛋白大小基本一致，亲和层析及Western blot分析显示，重组GbCCR含有6×His标签，GbCCR能在大肠杆菌中正常表达，酶活性分析显示GbCCR1的最适底物为阿魏酰-CoA，而GbCCR2的最适底物为芥子酰-CoA。RT-PCR分析显示，GbCCRs基因在银杏不同组织中都有表达，但与其他黄酮类代谢基因表达差异较大，其中GbCCR1在病虫害及老茎中表达水平较高，而GbCCR2则主要在木质素积累部位呈高水平表达。诱导表达分析显示，GbCCR1受到逆境胁迫相关激素及伤害感染的调控，可能主要参与对逆境胁迫适应及病虫害防御调节；而GbCCR2则主要受到生长相关激素调控，该基因可能主要参与组织及细胞壁中木质素的合成，与抗病虫害无明显关系。更多还原

【Abstract】 The flavonoids of Ginkgo biloba have many beneficial pharmaceutical properties for humanhealth. The studies on increasing the content of flavonoids in G.biloba had been more and morepopular and important. Until now, many methods had been carried out to increase the content ofG.biloba flavonoids, and it was available method via physiological and biochemical to increaseflavonoid content. In addition, it will be an effective method by genetic engineering to increaseflavonoid content in the near future. However, the overall biosynthetic pathway of flavonoids inG.biloba is unclear at molecular genetic level, especially the Cis-element of the key gene. Inorder to deepen the research in this area, and to lay a basic research for increasing the content ofG.biloba flavonoids by using biotechnology, cloning and function analysis of several key genesGbIRL1, GbDFRs GbCAD1and GbCCRs, which involved in the accumulative process forFlavonoids, Anthocyanin and Lignin are presented in this text for the first time. Moreover, toprovide a new theory basis and practical reference for promoting the accumulation of flavonoidsof Ginkgo leaves. The main results in this research are shown as follows:(1) Molecular Cloning and Function Analysis of a Isoflavone Reductase-Like ProteinsGene from Ginkgo biloba, and Its Expression of Time course. We isolated a GbRIL gene froma cDNA library derived from the leaves cell of Ginkgo biloba L. The coding region of the gene is921bp long, and its deduced protein consists of306amino acids with a predicted molecular massof33.28kDa and a pI of6.18. The expression analysis by real-time PCR showed that GbIRL1expressed in a tissue specific manner in G.biloba. GbIRL1was also found to be up-regulated byUV, ALA, Wounding and the three phytohormone, ETH, ABA, SA, consistent with the promoterregion analysis of GbIRL1. The recombinant protein was successfully expressed in E.coli strainwith pET-28a vector. The vitro enzyme activity assay by HPLC indicated that recombinantGbIRL1protein could catalyze the formation the TDDC, IDDDC from DDDC, DDC.Correlation analysis between GbIRL1activity and flavonoid accumulation during Ginkgo leafgrowth indicated that GbIRL1might be the rate-limiting enzyme in the biosynthesis pathway offlavonoids in ginkgo leaves. Results of semi-quantitative RT-PCR analysis showed that thetranscription level of change in GbIRL1power correlated with flavonoid contents, suggestingGbIRL1gene as the specific key gene regulating lignin change in Ginkgo and effecting theaccumulation of flavonoids.(2) Molecular cloning and function analysis of three Dihydroflavonol4-Reductasegenes from G.biloba, and its expression in abiotic stress responses. Dihydroflavonol4-Reductase(GbDFRs), is a member of the SDR superfamily, which catalyzes monooxygenasereactions dependent upon NADPH. In the flavonoid pathway DFRs hydroxylates the3’-positionof the B ring of naringenin and dihydrokaempferol to generate eriodictyol and dihydroquercetin,respectively which are important intermediates for biosynthesis of anthocyanins andproanthocyanidins, major coloration substances of flowers and seed coat. The full-length cDNA sequences of DFR1gene (designated as GbDFR1) were isolated from G.biloba for the first time.The full-length cDNA of GbDFR1contained a1038bp open reading frame (ORF) encoding a345-amino-acid protein; The full-length cDNA of GbDFR2contained a993bp open readingframe (ORF) encoding a330-amino-acid protein; The full-length cDNA of GbDFR3contained a1002bp open reading frame (ORF) encoding a333-amino-acid protein. The deduced GbDFRsprotein showed high identities to other plant DFRs, which belong to three different DFRs family.The secondary structure of GbDFRs is mainly composed of a NADPH binding site domain and asubstrate binding domain. Southern blot analysis show that GbDFRs belong to three differentmulti-gene family respectively. Phylogenetic tree analysis revealed that GbDFRs shared the sameancestor with other DFRs. The expression recombinant protein of three GbDFRs in E.colishowed that the size of their protein and cDNA sequences predict protein size is basically thesame. Affinity chromatography and western blot analysis indicated that recombinant GbDFRscontaining6×His tag. The recombinant protein were purified and activity nanalysis, GbDFR1and GbDFR3respectively catalyzed DHQ converted to LEU, while the DFR2could catalyzeDHK convers to LEUC. The expression analysis by RT-PCR showed that GbDFRs expressed ina tissue manner in G.biloba, which were in good agreement with the pattern of ANSaccumulation in G.biloba. the expression profile suggesting that GbDFR1and GbDFR2mainlyinvolved in responding to environmental stress and damage response, at the same time GbDFR1also be involved in anthocyanin synthesis, while GbDFR3is likely more specific functions in thesynthesis of flavonoids and anthocyanin.(3) Cloning and expression analysis of Cinnamyl Alcohol Dehydrogenase Gene FromGinkgo biloba L. RNA was isolated respectively from Ginkgo biloba leaves by CTAB method;the RACE technology was used for cloning the full-length cDNA of GbCAD1gene from Ginkgofor the first time. Ginkgo Cinnamyl Alcohol Dehydrogenase gene is a total length of1494bp, itscDNA largest reading frame (ORF) is1074bp, encoding a357amino acid peptide sequence.DNAssist2.2software predicted that it coded a protein of38.70KD, and its isoelectric point was5.74. Phylogenetic tree analysis showed that the homology of GbCAD1protein sequences washigher with the CADs synthase of other species. RT-PCR analysis showed that GbCAD1have ahigher expression level in stems and roots, especially in the parts of the pest and disease infection.Southern blot analysis showed that GbCAD1belong to a multi-gene family. Phylogenetic treeanalysis revealed that GbCAD1shared the same ancestor with other DFRs. The expressionrecombinant protein of GbCAD1in E.coli showed that the size of their protein and cDNAsequences predict protein size is basically the same. Western blot analysis indicated thatrecombinant GbCAD1containing6×His tag. The purified protein of GbCAD1could to catalyzethe transformation of coniferyl aldehyde to coniferyl alcohol. Inducible expression analysis ofGbCAD1suggest that the treatment of UV-B, WOU, ABA, SA and ETH could improve theexpression level of mRNA sinificantly, ALA have a little effect on the level. The expressionprofile of GbCAD1may be effecte by upstream of phenylalanine metabolism, mainly involved in lignin synthesis and plant stress adaptation.(4) Molecular cloning and function analysis of Cinnamoyl-CoA Reductase gene fromG.biloba, and its expression in abiotic stress responses. Cinnamoyl-CoA Reductase (CCR, EC1.2.1.44) catalyses the first step of the lignin pathway. Two full-length cDNA sequences of CCRgene (designated as GbCCR2) were isolated from G.biloba according to preliminary work. Thefull-length cDNA of GbCCR1contained a972-bp open reading frame (ORF) encoding a323-amino-acid protein; GbCCR2contains a full-length cDNA of1206bp, which contains a1005bp open reading frame encoding a334amino acid sequences of peptides. The two deducedGbCCR protein showed high identities to other plant CCRs. They both contain a commonsignature which is thought to be involved in the catalytic site of CCRs. Phylogenetic tree analysisrevealed that GbCCRs shared the same ancestor with other CCRs. Southern blot analysisindicated that GbCCRs belonged to a multi-gene family. Homology modeling analysis showedthat GbCCRs sequence is highly similar to the other family of protein structure. GbCCRs singlesubunit can be divided into two domains, one is the center of the substrate binding site, and theother is NADPH domain binding region, two conserved regions of the enzymatic reaction toensure the reaction. Recombinant protein of GbCCRs were consistent with the cDNA sequencespredicted fusion protein size. Western blot analysis and affinity chromatography showed that therecombinant protein contain6×His tag. Enzyme activity analysis showed that ferulic acyl-CoA isthe optimum substrate for GbCCR1, mustard acyl-CoA for GbCCR2. The expression analysis byRT-PCR showed that GbCCRs expressed in a tissue-specific manner in G.biloba, GbCCR1havea higher levels of expression in the old or pest injected stem, while GbCCR2showed a higherlevels in the parts tissue of lignin accumulation. Induced expression analysis indicated thatGbCCR1maily regulated by stress-related hormones and injury infection, which maybe mainlyinvolved in stress adaptation and pest defense. GbCCR2mainly involved in the organization andcell wall lignin synthesis, and regulated by growth hormone, while have no obvious proportionalrelationship with anti-pest, diseases infection.更多还原