【作者】 李琳玲；

【导师】 彭建营；

【作者基本信息】 河北农业大学 ， 果树学， 2010， 博士

【摘要】 为了深入研究银杏叶黄酮合成的分子机理,为今后利用生物技术手段提高银杏黄酮含量奠定基础,本文从银杏中克隆并研究了与黄酮积累相关的几个酶基因：查尔酮异构酶(GbCHI),类黄酮3’-羟化酶(F3’H),烯醇式丙酮基莽草酸3磷酸合成酶(EPSP),肉桂酰辅酶A还原酶(CCR)及查尔酮合成酶基因启动子(CHSP)序列。主要研究内容及结果如下：(1)银杏查尔酮异构酶基因(GbCHI)的克隆、性质及表达模式的研究。利用简并PCR和RACE技术从银杏叶片中克隆得到GbCHI的cDNA序列和基因组全长。通过信息学分析发现,基因组GbCHI含有两个内含子三个外显子,GbCHI cDNA全长为926bp,含有一个735bp的开放式阅读框(ORF),编码244个氨基酸序列,预测分子量为26.29kDa,等电点为7.76。蛋白质同源序列分析表明,GbCHI与TypeⅠ型CHI同源性较高。CHIs蛋白序列进化树分析结果显示GbCHI未聚合到两类中,且分化时间早于TypeⅠ和TypeⅡ型CHI。Southern blot分析表明,GbCHI属于多基因家族；GbCHI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GbCHI且含有6xHis标签,GbCHI能在大肠杆菌中正常表达；重组GbCHI酶活性分析表明,GbCHI具有TypeⅠ型CHI的酶催化特点,即催化6’-羟基查尔酮生成(2S)-黄烷酮；RT-PCR分析显示,GbCHI基因在银杏不同组织中都有表达,但存在较大差异,只有成熟叶和雄蕊中表达量最高。CHI与银杏叶黄酮含量的年周期变化分析显示,CHI基因的转录水平与CHI的酶活性呈线性相关,相关系数为0.421；酶活性与黄酮的年周期变化之间也呈线性相关,相关系数为0.373。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GbCHI转录水平能被UV-B、CCC、ABA、ALA和ETH诱导上调,而被GA抑制表达；GbCHI诱导表达模式与银杏叶黄酮的调控变化相一致,暗示GbCHI在银杏黄酮代谢过程中具有关键酶作用。(2)银杏类黄酮3’-羟化酶基因(GbF3’H)的克隆、性质及表达模式研究。利用简并PCR和RACE技术从银杏叶片中克隆得到了GbF3’H的cDNA全长序列。通过信息学分析发现GbF3’H的cDNA全长为2144 bp,含有一个1671 bp的开放式阅读框(ORF),编码556个氨基酸序列。蛋白质同源序列分析表明,GbF3’H与其他物种F3’H同源性较低,而与菊苣(Cichorium intybus)同源性最高,为56.3%。同源建模分析显示GbF3’H与P450家族蛋白的三维结构及活性位点高度相似,最终定位于微粒体膜上。F3’Hs蛋白序列进化树分析结果显示GbF3’H与其他植物分化较早。Southern blot分析表明,GbF3’H属于多基因家族。GbF3’H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GbF3’H含有6xHis标签,GbF3’H能在大肠杆菌中正常表达。RT-PCR分析显示,GbF3’H基因在银杏不同组织中都有表达,其中雄蕊表达水平最高,其次为成熟叶。激素和胁迫诱导表达分析显示,虽然诱导表达模式不相同,但GbF3’H转录水平能被UV-B、6-BA、SA、ABA和IAA诱导上调,而伤害处理对其表达量无明显改变。GbF3’H诱导表达模式与银杏ANS基因的表达模式相似,而且其上游调控序列也发现有相关的调节单元,意味着该GbF3’H基因可能参与了银杏花色素的合成代谢。(3)银杏烯醇式丙酮基莽草酸3磷酸合成酶(EPSPs)的克隆、性质及表达模式研究。利用简并PCR和RACE技术从银杏叶中克隆到EPSP合酶基因(GbEPSPs)的cDNA全长序列。信息学分析发现,GbEPSP的cDNA全长1403 bp,包含最大阅读框(ORF)为1035 bp,编码一个344氨基酸多肽序列。蛋白质同源序列分析表明,银杏EPSP合酶蛋白质序列与其他物种的EPSP合酶同源性较高,在81%-84%之间。进化树分析结果表明,在参试物种中GbEPSPs作为裸子植物与被子植物同聚为一类,但分歧时间相对更早。Southern blot分析表明,EPSPs基因有多个拷贝,属于一个小的多基因家族。不同组织表达分析显示,EPSPs基因在银杏的叶和果中表达量最高,其次为茎,根中表达水平最低。草甘膦处理能显著诱导银杏EPSPs基因表达量升高,紫外能上调银杏EPSPs基因表达,ABA则诱导GbEPSPs表达量先升后降；温度对GbEPSPs具有不同的诱导作用,其中42℃高温诱导最显著4 h达最大值,后又迅速降低。暗示银杏叶片中EPSPs基因在环境压力下表达量升高有可能与芳香族氨基酸向黄酮类物质的转化有关。(4)银杏肉桂酰辅酶A还原酶基因(GbCCR)的克隆、性质及表达模式研究。利用简并PCR和RACE技术从银杏叶片中克隆得到了GbCCR的cDNA全长序列。信息学分析发现,GbCCR的cDNA全长为1178 bp,含有一个972 bp的开放式阅读框(ORF),编码323个氨基酸序列。蛋白质同源序列分析表明,GbCCR与其他物种CCR同源性相对较高,与挪威云杉(Picea abies)同源性最高,为68.6%；同源建模分析显示GbCCR序列与其他家族蛋白的三维结构及活性位点高度相似。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分析表明,GbCCR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6xHis标签,GbCCR能在大肠杆菌中正常表达；RT-PCR分析显示,GbCCR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,其中茎和根中表达水平最高,其次为成熟叶。GA和农杆菌诱导表达分析显示,虽然二者都能诱导基因表达,但GA能够明显诱导GbCCR转录水平上调,而农杆菌侵染处理对其表达量无明显改变。GbCCR诱导表达模式显示,该基因可能主要参与组织及细胞壁中木质素的合成,而与抗病虫害无明显关系。(5)银杏查尔酮合成酶基因启动子(GbCHSP)的调控元件及功能分析。通过染色体步移方法从银杏基因组中克隆到查尔酮合成酶基因(GbCHS)翻译起始位点上游1711bp的启动子序列。生物信息学分析表明,该启动子片段中存在多个顺式作用元件,包括紫外／蓝光响应单元、植物激素响应单元、真菌诱导元件、MYB结合位点、TATA-box和CAAT-box等。亚克隆了GbCHS转录起始位点上游1402 bp序列,将其与GUS基因构建融合表达载体pBI121+CHSP,以pBI121-35s作为负对照,通过农杆菌(LBA4404)介导法分别转入烟草。结果表明,银杏CHS启动子序列能驱动GUS基因在烟草中的表达,表达具有组织差异性。其中叶片和茎中表达量较高；在诱导芽及愈伤组织中亦具有较高表达水平。结果说明,CHSP具有基本启动子功能,但表现出空间上的表达差异性更多还原

【Abstract】 In order to study biosynthetic mechanism of flavonoids in G.biloba and develop biotechnology of increasing the content of the flavonoids, several key genes such as GbCHI, GbF3’H, GbCCR, GbEPSPs and GbCHSp which are involved in the flavonoids accumulation process have been cloned and studied in this paper. The main results are as follows:(1) Molecular cloning, characterization and expression models of chalcone isomerase gene from Ginkgo biloba. A full-length cDNA and genomic DNA of chalcone isomerase gene were isolated from Ginkgo biloba L. by using PCR and RACE technologies. Compared with the cDNA sequence, the genomic sequence contain two intron and three extron. The coding region of the gene is 735 bp long, and its deduced protein consists of 244 amino acids with a predicted molecular mass of 26.29 kDa and a pI of 7.76. Protein sequence analysis reveals that GbCHI had closer relationship with TypeⅠCHIs. Phylogenetic tree analysis revealed that GbCHI does not belong to TypeⅠor typeⅡgroup. Southern blot analysis indicated that GbF3’H belonged to a multi-gene family. The results of prokaryotic expression showed that the product of recombinant GbCHI protein was in accordance with the anticipation. The vitro enzyme activity assay by HPLC indicated that recombinant GbCHI protein could catalyze the formation the (2S) naringenin from 6’-hydroxychalcone. The expression analysis by RT-PCR showed that GbCHI constitutively expressed in all the tested tissues, and had tissue specific manner in G.biloba. GbCHI was also found to be up-regulated by UV, ALA, ETH, ABA, CCC, and be down-regulated by GA. Correlation analysis between CHI activity and flavonoid accumulation during gingkgo leaf growth indicated that GbCHI might be the rate-limiting enzyme in the biosynthesis pathway of flavonoids in ginkgo leaves. Results of quantitative RT-PCR analysis showed that CHI activity correlated with the transcription level of change in CHI gene, suggesting CHI gene as the specific key gene regulating flavonoid accumulation in ginkgo.(2) Molecular cloning, characterization and expression models of Flavonoid 3’-hydroxylase gene from G.biloba. Flavonoid 3’-hydroxylase (GbF3’H), is a member of the P450 superfamily, which catalyzes monooxygenase reactions dependent upon NADPH and O2. In the flavonoid pathway F3’H hydroxylates the 3’-position of the B ring of naringenin and dihydrokaempferol to generate eriodictyol and dihydroquercetin, respectively which are important intermediates for biosynthesis of anthocyanins and proanthocyanidins, major coloration substances of flowers and seed coat. The full-length cDNA sequences of F3’H gene (designated as GbF3’H) were isolated from G.biloba for the first time. The full-length cDNA of GbF3’H contains a 1671 bp open reading frame (ORF) encoding a 556 amino acid protein. The 5’flanking region of GbF3’H 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 GbF3’H protein showed low identities to other plant F3’Hs, but had closer relationship with Cichorium intybus and shares 56.3% homology.3D structure modeling showed that GbF3’H bears all conserved motifs featured and have high similarity with P450s. Phylogenetic tree analysis revealed that the divergence time of GbF3’H from other F3’Hs is earlier. Southern blot analysis indicated that GbF3’H belonged to a multi-gene family. The expression analysis by RT-PCR showed that GbF3’H expressed in a tissue-specific manner in G.biloba, with the highest level in stamen and next in mature leaves. GbF3’H was also found to be up-regulated by the five tested abiotic stresses:UV-B、6-BA、SA、ABA and IAA, but non significant effect to wounding. Function analysis suggest that GbF3’H is a functional enzyme within the anthocyanidin and flavonol biosynthetic pathway.(3) Molecular cloning, characterization and expression models of EPSP synthase Gene From Ginkgo biloba L. The full-length cDNA sequences of EPSP synthase gene (designated as GbEPSPs) were isolated from G.biloba by using PCR and RACE technologies for the first time. The full-length cDNA of GbEPSPs is 1404bp and contains a 1035 bp open reading frame (ORF) encoding a 344 amino acid peptide sequence. Protein sequence analysis reveals that GbEPSPs had close relationship with other EPSPs and shares 81%-84% homology. Phylogenetic tree analysis showed that GbEPSPs as a gymnosperm and other angiosperm EPSPs are clustered to one monophyletic group, but the divergence time is early. RT-PCR analysis showed that GbEPSPs expressed in leaves, stems, roots and fruits, and had the highest expression in leaves and fruits, the next in stems, the least in roots. The expression of GbEPSPs could be induced by glyphosate and UV-B. ABA could improve the expression of GbEPSPs first, but deduce later. Different temperature treatments have different effects in the content of GbEPSP gene, and the highest expression at 42℃for 4h.(4) Molecular cloning, characterization and expression models of Cinnamoyl-CoA Reductase gene from G.biloba. Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. The full-length cDNA sequences of CCR gene (designated as GbCCR) were isolated from G.biloba for the first time. The full-length cDNA of GbCCR is 1178 bp long and contains a 972 bp open reading frame (ORF) encoding a 323 amino acid protein. The deduced GbCCR protein showed high identities to other plant CCRs, and had closer relationship with Picea abies shares 56.3% homology. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Phylogenetic tree analysis revealed that GbCCR shared the same ancestor with other CCRs, but the divergence time is early. Southern blot analysis indicated that GbCCR belonged to a multi-gene family. The expression analysis by RT-PCR showed that GbCCR had tissue specific manner in G.biloba, and the highest expression in stems and roots, the next in mature leaves, which were great difference with other flavones biosynthetic pathway gene. GbCCR was also found to be significant up-regulated by GA, but agrobacterium treatment had no significant change. The high level of GbCCR gene expression along the stalk suggests that the corresponding enzyme is probably involve in constitutive lignification.(5) Regulatory Element and Function Analysis of Chalcone Synthase Gene Promoter from Ginkgo biloba L. The regulative sequence (1711 bp) of chalcone synthase gene promoter (CHSP) from Ginkgo biloba L. was cloned by genomic walking. In bioinformatic analysis of sequence suggested that the sequence contained several typial cis-acting elements, including UV/blue light responsive elements, Phytohormone responsive elements, fungal elicitor responsive elements, MYB binding site, TATA-box and CAAT-box. A 1402 bp promoter sequence upstream 5’of translation start site of GbCHS were cloned and designated as GbCHSP, respectively. pBI121+CHSP and pBI121-35s were constructed and transformed into tobacco by LBA4404. These result showed that pBI121 and pBI121+CHSP both could drive the transient expression of GUS in tobacco and pBI121+CHSP expressed differentially in root, stem and leaf tissues of tobacco. Our discoveries will be help to understand the transcriptional regulatory mechanism on GbCHS expression and accumulation flavonoids.更多还原