【作者】 韩梅；

【导师】 安志刚；

【作者基本信息】 东北林业大学 ， 植物学， 2007， 硕士

【摘要】 本文以长春花(Catharanthus roseus(L．)G．Don．)植物幼苗为材料，利用Gateway克隆技术对其萜类吲哚生物碱(Terpene indole alkaloids，TIAs)生物合成途径中编码关键酶的基因进行大规模克隆，获得相关基因的入门克隆和表达克隆，制备相应的抗体。并对这些基因的mRNA表达水平进行检测。所得结果如下：1．以长春花植物幼苗中提取的总RNA为模板，反转录生成cDNA，经两步Gateway-PCR法获得长春花TIAs类化合物生物合成途径中编码关键酶DXS、DXR、G10H、SLS、TDC、STR和DAT的目的基因(dxs、dxr、g10h、sls、tdc、str和dat)的全长序列。2．利用Gateway克隆技术通过BP重组反应将目的基因插入到入门载体(pDONR201)中，构建目的基因的入门质粒，筛选出阳性克隆并通过测序验证。3．通过LR重组反应将目的基因从入门载体上转移到表达载体(pETG10A／20A／30A)，导入大肠杆菌表达菌株BL21(DE3)的感受态细胞中。通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达，经SDS-PAGE检测，表明重组的分别含有dxr、sls、str和g10h基因的表达质粒的大肠杆菌菌株均有目的蛋白的表达。4．利用Ni-TED树脂分别对表达量较高的DXR、SLS和STR融合HIS标签的蛋白进行纯化，纯化后的蛋白经SDS-PAGE检测没有杂带，用Brad-ford法对纯化后的蛋白进行定量，结果表明所得蛋白已达到制备抗体的要求，目前抗体制备正在进行中。5．对这些基因在长春花幼苗中mRNA表达水平的Northern杂交实验显示dxr、sls、str和tdc基因在根、茎、叶、花和果实中均有表达，并且从总体上看根和幼叶中的表达量相对高些，老叶中的表达量相对低一些；但是没有检测到目的基因g10h、dxs和dat相应的mRNA杂交信号，原因可能是与所选的植物材料的生长阶段和生长环境有关。更多还原

【Abstract】 This thesis performed a large scale of cloning for genes encoding key enzymes in terpene indole alkaloids (TIAs) pathway from Catharanthus roseus (L.)G.Don.via Gateway technology. These genes’ mRNA expression levels in C. roseus seedlings were also detected. The results are as follows:1. The full length sequences of dxs, dxr, g10h, sls, tdc, str and dat which encoded key enzymes involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway were obtained via two-step Gateway PCR method, with the transcripted cDNA from total RNA of Catharanthus roseus seedlings as templates.2. After PCR amplification, these attB-flanked DNA fragments were inserted into attPcontaining donor vector (pDONR201) by BP clonase to generate entry clones, respectively. Positive clones were screened first by colony cracking, PCR and restriction enzyme analysis, and then these inserted genes were confirmed by gene sequencing.3. In order to get prokaryotic expression systems, ORFs of dxr, g10h, sls and str have been transferred to their destination vectors: pETG10A, pETG20A and pETG30A, and been transformed into competent cells of E.cloi host strain BL21(DE3). SDS-PAGE analysis results indicated that those recombinant expression clones which encoded enzymes of DXR, SLS and STR were overexpressed after IPTG induction.4. Fusion proteins DXR-HIS, DXR-HIS-TrxA, DXR-HIS-GST, SLS-HIS and STR-HIS were purified by Ni-TED resin. According to the results of SDS-PAGE and Bradford, these purified proteins were qualified for antisera preparation.5. Northern blot results displayed that transcript levels of dxr, sls, str and tdc in root, stem, leave, flower and fruit from C roseus were detected, in general the transcript levels in root and young leave were higher than in other tissues. But there’re no signals for Northern blot ofglOh, dxs and dat. For one reason maybe that there’re no mRNA transcripts in the seedling stage we chosed for these genes, or maybe they’re inclined to be under strict post-transcriptional regulation.更多还原