【作者】 许锋；

【导师】 程述汉； 程水源； 束怀瑞；

【作者基本信息】 山东农业大学 ， 果树学， 2008， 博士

【摘要】 银杏叶类黄酮具有重要的药用价值,提高其含量已成为银杏研究的热点与难题。研究表明,采用生理生化途径调控银杏叶类黄酮含量是一种有效的方法;利用植物基因工程技术提高银杏叶类黄酮含量也具有良好的前景。然而,银杏类黄酮生物合成途径在分子水平上的报道还较少。为深入研究银杏类黄酮的合成途径,并为今后利用基因工程技术提高银杏类黄酮含量奠定基础,本文首次克隆并研究了银杏类黄酮合成途径中的两个重要的基因,它们分别是苯丙氨酸解氨酶基因(GbPAL)和花色素合成酶基因(GbANS)。针对生理生化途径,本文研究了5-氨基乙酰丙酸(ALA)处理对银杏叶片类黄酮含量影响,以期为提高银杏叶类黄酮含量提供理论依据。其主要研究内容与结果如下:(1)银杏苯丙氨酸解氨酶基因的克隆、性质以及表达模式研究。利用RACE技术从银杏中克隆获得一个苯丙氨酸解氨酶基因GbPAL的cDNA和基因组全长(GenBank Accession No. EU071050)。GbPAL基因基因组序列与cDNA序列一样,无内含子。GbPAL基因cDNA全长为2886 bp,含有一个2172 bp的开放阅读框(ORF),编码724个氨基酸。蛋白质同源比对分析表明,GbPAL与其它植物的PAL蛋白高度同源,且包含苯丙氨酸解氨酶所必需的保守氨基酸位点和保守模式。用同源建模的方法得到GbPAL蛋白的三维模型,这个模型与欧芹PAL(PcPAL)的三维结构高度相似。PAL基因的系统进化树分析结果表明,GbPAL与裸子植物的PAL具有更近的亲缘关系。Southern blot结果表明,GbPAL基因属于一个多基因家族。实时定量PCR(real-time PCR)分析表明,GbPAL基因在银杏不同器官中组成型表达,但在茎和叶中的表达相对较高。Real-time PCR分析结果显示,GbPAL基因的表达受UV-B、机械损伤、水杨酸和低温的诱导,暗示GbPAL基因参与了银杏对逆境胁迫的抗性。线性回归分析结果表明银杏叶类黄酮含量与GbPAL基因表达量之间存在显著正相关关系,表明GbPAL基因可能是银杏叶类黄酮合成途径中的关键基因之一。(2)银杏花色素合成酶基因的克隆、功能分析及响应非生物胁迫的表达模式。利用RACE技术首次从裸子植物银杏中克隆得到一个花色素合成酶基因GbANS的cDNA全长(EU600206)和基因组序列(EU600205)。GbANS基因全长cDNA为1441 bp,含有一个1062 bp的ORF,编码354个氨基酸。蛋白序列多重比对结果表明,GbANS蛋白与其它植物的ANS蛋白具有很高的相似性,GbANS存在结合亚铁离子和酮戊二酸的保守位点。GbANS基因含有三个外显子和两个内含子。通过基因组步移的方法获得了GbANS基因的5’侧翼序列,该区域含有包括TATA盒在内的启动子必需元件,序列预测表明,这个区域还含有光、冷、水杨酸、乙烯和脱落酸应答等顺式作用元件。同源建模产生的GbANS三维结构与功能已知的、拟南芥ANS(AtANS)的三维结构一样,具有2-ODD酶特征性的扭曲果冻状结构。类黄酮特异2-ODD酶进化树分析表明,GbANS与其它植物的ANS具有共同的祖先。Southern blot结果显示,GbANS基因属于一个多基因家族。Real-time PCR分析表明,GbANS基因的表达水平在不同器官以及器官不同时期均有差异性,在成熟果中的表达量最高,胚珠和雄蕊,茎和根中未检测到有表达,花青苷在各器官中的含量显示了与GbANS基因器官与发育表达图谱良好的一致性。real-time PCR分析结果显示GbANS基因的表达水平受UV-B、脱落酸、低温、水杨酸、乙烯以及蔗糖上调,这一结果与GbANS基因启动子序列预测分析的结果相吻合,表明GbANS基因可能参与银杏对上述逆境胁迫下的抗性。原核表达的重组GbANS蛋白的分子量大小和通过生物信息学预测的分子量大小相同。Western blot试验结果表明,重组GbANS蛋白的N末端含有6×His标签,说明GbANS基因编码的GbANS蛋白可以在大肠杆菌中正确的表达。利用HPLC分析纯化的重组GbANS蛋白体外酶活性,结果表明重组GbANS蛋白具有两种功能,即(1)催化无色花青素生成花青素,(2)催化二氢槲皮素生成槲皮素。(3) ALA对银杏类黄酮含量的影响。以3年生盆栽银杏实生苗为试材,研究了叶面处理浓度分别为10、100 mg/L的ALA对银杏叶光合作用、叶绿素含量、可溶性糖含量、类黄酮含量、总多酚含量、花青苷、黄酮合成相关的酶(PAL、CHS和CHI)活性的影响。结果表明,在处理后的第4天,相对对照而言,ALA处理的银杏叶的光合速率显著提高,ALA的这种促进作用一直稳定维持到处理后的第16天。ALA处理能显著提高银杏叶的叶绿体和可溶性糖含量,且随着处理时间的延长增幅加大,但对于叶绿体a/叶绿体b的比值无显著性差异。总酚、类黄酮以及花青苷含量在ALA的处理下迅速增加,其增加速率显著高于对照。ALA处理下PAL、CHS和CHI的活性变化与类黄酮含量的变化趋势相似,表明ALA可显著诱导PAL、CHS和CHI的活性。上述处理效果均具有浓度效应,以100mg/L ALA的效果较佳。更多还原

【Abstract】 The flavonoids of Ginkgo biloba have many beneficial pharmaceutical properties for human health. The studies on increasing the content of flavonoids in G. biloba had been more and more popular and important. Until now, many methods had been carried out to increase the content of G. biloba flavonoids, and it was available method via physiological and biochemical to increase flavonoid content. In addition, it will be an effective method by genetic engineering to increase flavonoid content in the near future. However, the overall biosynthetic pathway of flavonoids in G. biloba is unclear at molecular genetic level. In order to deepen the research in this area, and to lay a groundwork for increasing the content of ginkgo flavonoids by using bioengineering, cloning and characterization of two key genes GbPAL and GbANS, which encoding phenylalanine ammonia-lyase and anthocyanidin synthase, involved in the biosynthetic pathway of flavonoids, are presented in this text for the first time. Moreover, to provide a new theory basis and practical reference for promoting the accumulation of flavonoids of ginkgo leaves, the effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins, in a low level (10 and 100 mg/l) on photosynthesis, flavonoids accumulation in ginkgo leaves were also first investigated in this research. The main contents and results in the present study are as following:(1) Molecular cloning, characterization and expression of phenylalanine mmonia-lyase gene from G. biloba.A full-length cDNA and genomic DNA of phenylalanine ammonia-lyase gene, which catalyzes the first step in the flavonoid biosynthetic pathway, were isolated from G. biloba for the first time (designated as GbPAL, GenBank Accession No. EU071050). The cDNA and genomic sequences of GbPAL were the same; in other words, this gene is intronless. The coding region of the gene was 2172 bp long, and its deduced protein consists of 724 amino acids with a predicted molecular mass of 79.1 kDa and a pI of 5.96. The deduced GbPAL protein showed high identities to other plant PALs. Southern hybridization analysis indicated that GbPAL belonged to a small multi-gene family. Tissue expression analysis by real-time PCR revealed that GbPAL constitutively expressed in all the tested tissues, especially highly in leaf and stem. GbPAL was also observed to be induced by a variety of stresses including UV-B, wounding, cold and salicylic acid. Temporal expression profiling analyses showed that the transcription levels of GbPAL were significantly correlated with flavonoid accumulation, suggesting that GbPAL might play a regulatory role in flavonoid biosynthesis in leaves of G. biloba at the transcriptional level.(2) Molecular cloning and function analysis of anthocyanidin synthase gene from G. biloba, and its expression in abiotic stress responses.Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA (EU600206) and genomic DNA (EU600205) sequences of ANS gene (designated as GbANS) were isolated from G. biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA sequence analysis showed that GbANS gene had three exons and two introns. The 5′flanking region of GbANS was isolated by genome walking method, and some main cis-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids ligating ferrous iron (H-X-D) and residues participating in 2-oxoglutarate binding (R-X-S) were found in GbANS at the similar positions like other ANSs. 3D structure modeling showed that GbANS had a jellyroll motif in the enzyme core consisted ofβ-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including ANSs. Phylogenetic tree analysis revealed that GbANS shared the same ancestor with other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba, which were in good agreement with the pattern of anthocyanins accumulation in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, namely UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.(3) Effect of 5-aminolevulinic acid on flavonoids accumulation in the leaves of G. biloba. The effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins, in the low levels (10 and 100 mg/l) on photosynthesis, flavonoids accumulation and the activities of three enzymes involved in flavonoids biosynthesis in Ginkgo biloba leaves were investigated. The results showed that photosynthetic rates of leaves that treated with both concentrations of ALA, increased significantly at day 4 compared with that of control and remained so for 16 days. ALA at concentrations of 10 and 100 mg/l significantly increased the contents of chlorophyll and soluble sugar (P<0.05) at day 4 and these increments kept increase tendency until day 16, while the ratio of Chl a/b remained constant after ALA treatment. ALA-treatment enhanced the contents of total polyphenols, flavonoids and anthocyanins, as well as PAL, CHS and CHI activities from day 4 to 16. The effects on those determined targets mentioned above were all concentration-dependent, and 100mg/L ALA had greater effect than 10 mg/L ALA. These results suggestted that foliar treatment with the low concentration of ALA might provide a useful means of improving pharmacological properties in G. biloba leaves.更多还原