【作者】 李文凤；

【导师】 季静；

【作者基本信息】 天津大学 ， 生物分子工程， 2010， 博士

【摘要】 选择标记基因的安全性问题是制约转基因植物产业化的主要瓶颈之一。开发生物安全标记基因替代抗生素标记基因成为解决转基因植物安全性和促进转基因植物应用的重要策略。本研究利用体外定向分子进化技术对乳酸克鲁维酵母(Kluyveromyces lactis) Cu/Zn-SOD进行改造,并对以获得的高活力突变体基因为安全标记基因,百草枯(PQ)为选择剂建立新型安全筛选体系的可行性进行了较为系统的研究。通过克隆乳酸克鲁维酵母Cu/Zn-SOD基因,并在PGK1启动子驱动下与GFP基因融合,构建YEplac195-PSGA,转化酿酒酵母W303菌株。通过菌落PCR和荧光显微观察及添加PQ进行发酵检测,结果表明乳酸克鲁维酵母Cu/Zn-SOD基因在酿酒酵母中成功表达。通过一步基因置换法构建酿酒酵母sod1缺失菌株LN303。首次采用酿酒酵母sod1缺失菌株作为表达宿主,通过在CM-Ura培养基中添加1 mmol/L PQ制造高氧压力,建立了Cu/Zn-SOD的高效筛选模型。通过优化易错PCR体系中Mn2+浓度,确定其最适浓度为0.5 mmol/L。经易错PCR建立突变文库进行筛选,得到三个酶活力提高的突变体S1、S2和S3(S99R/A92G,S135P,D126N),其酶活力分别是野生酶的2.6、1.5和1.2倍。运用计算机三维结构模拟,对氨基酸置换所导致的酶分子结构变化和活力提高分子机制进行了初步分析,结果表明局部静电环境、内部氢键相互作用及活性通道的大小和形状对酶活力提高产生了重要影响。将乳酸克鲁维酵母来源的Cu/Zn-SOD和突变体S1(mSOD)基因与拟南芥谷胱甘肽还原酶叶绿体信号肽(Chl)融合,构建植物表达载体pBI121-Chl-SOD和pBI121-Chl-mSOD,并通过农杆菌介导法成功转化烟草,分别获得再生植株35和46个株系。通过PCR、RT-PCR、叶绿体SOD活性测定证明目的基因已经整合到烟草基因组中并正常定向进入叶绿体表达。转Chl-SOD和Chl-mSOD植株的SOD活性分别是野生型植株的1.8倍和5.3倍。在PQ和200 mmol/L NaCl处理下,考察其对转基因植株的表观伤害、质膜透性、叶绿素a荧光动力学、抗氧化酶活性的影响,结果表明转基因植株抗氧化能力和耐盐能力有不同程度的提高,且提高大小顺序为转Chl-mSOD植株>转Chl-SOD植株>野生型植株。因此在烟草叶绿体中过表达乳酸克鲁维酵母来源的Cu/Zn-SOD和突变体S1基因能够提高转基因植株的抗氧化能力和耐盐能力。建立了以乳酸克鲁维酵母来源的Cu/Zn-SOD突变体S1(mSOD)基因作为选择标记基因,PQ作为选择剂的新型安全筛选体系。将植物表达载体pBI121-Chl-mSOD中去除nptⅡ标记基因表达盒,得到pBI121’。确定烟草遗传转化体系中PQ作为选择剂的适宜浓度为6 ?mol/L。在农杆菌介导的叶盘法转化烟草过程中,以mSOD基因作为选择标记基因,以6 ?mol/L PQ为选择剂,筛选得到96株表型正常的T0代转基因植株。通过PCR、Southern blot和RT-PCR对转基因植株进行检测,结果表明mSOD基因已整合到烟草基因组中,并获得转录水平上的表达。转化频率最高为39.26%,与以nptⅡ为选择标记基因,100 mg/L Kan为选择剂的传统筛选体系进行比较,转pBI121-Chl-mSOD植株和转pBI121’植株的转化频率分别是其2.16倍和2.1倍。对其遗传稳定性分析结果表明大多数T0代转基因植株为单拷贝,且表现出3:1分离方式,符合孟德尔分离定律。通过测定种子发芽率、株高、叶片数等指标,考察T0代和T1代转基因植株对PQ诱导的氧化胁迫和盐胁迫的耐受性。结果表明T0代和T1代转基因植株对PQ诱导的氧化胁迫和盐胁迫的耐受性明显高于野生型植株。此新型安全筛选体系不仅避免了使用抗生素抗性标记基因和抗除草剂标记基因,对生态环境和人类相对安全,同时具有以下优势:转化效率大大提高;获得转基因植株的周期缩短;选择标记基因可以同时为目的基因,提高转基因植物对氧化胁迫、盐胁迫的耐受性。这将对加快转基因商品化和产业化进程具有重要意义。更多还原

【Abstract】 Biosafety issues of selectable marker genes are the major choke points which restrict the process of genetically modified plants industrialization. The development of bio-security marker genes to substitute for antibiotics and herbicides resistance genes is viewed as an important and alternative strategy to solve the biosafety problem and promote the application of transgenic plants. In this study, Kluyveromyces lactis Cu/Zn-SOD was modified by in vitro directed molecular evolution to obtain high activity mutant gene which was used as a selectable marker gene with paraquat as selective agent. The feasibility of this new efficient screening syetem was systematically discussed.We constructed YEplac195-PSGA plasmid by cloning Kluyveromyces lactis Cu/Zn-SOD gene fused to GFP gene under PGK1 promoter. The plasmid was transformed into yeast W303 strain. The expression of Kluyveromyces lactis Cu/Zn-SOD gene in yeast was confirmed by colony PCR, fluorescence microscopic observation and fermentation added with paraquat.By the one-step gene replacement, sod1Δyeast mutant LN303 was constructed. Under high oxygen pressure produced by 1 mmol/L of paraquat added to CM-Ura medium, sod1Δyeast mutant was first used as an expression host. Hence, Cu/Zn-SOD efficient screening was established.For optimization of error-prone PCR system, the Mn2+ concentration was set of 0.5 mmol/L. By error-prone PCR, mutant library was established and screened,obtaining three mutations S1, S2, S3 (S99R/A92G, S135P, D126N) with increased enzyme activity, which were 2.6-, 1.5- and 1.2-folds compared to wild type, respectively. We used computer simulation of three-dimensional structure to primarily analyse structural changes and the possible molecular mechanism of increased activity of the mutant enzymes caused by the amino acid substitution. The result showed that, local electrostatic environment, internal hydrogen bond interaction, and the size and shape of activity channel greatly impacted on the enzyme activity enhancement.The plant expression vectors pBI121-Chl-SOD and pBI121-Chl-mSOD were constructed by fusion of Kluyveromyces lactis Cu/Zn-SOD gene, the mutant S1 (mSOD) gene to the chloroplast transit sequence from Arabidopsis thaliana glutathione reductase (Chl), respectively. Via Agrobacterium-mediated transformation of tobacco, 35 and 46 transgenic lines were successfully regenerated, respectively. PCR, RT-PCR, and chloroplast SOD activity assay demonstrated that the target gene was integrated into the tobacco genome, normally expressed and targeted into the chloroplast. The SOD activities of transgenic Chl-SOD plants and transgenic Chl-mSOD plants were respectively 1.8-and 5.3-folds of wild-type plants. Transgenic plants treated with paraquat and 200 mmol/L NaCl were investigated for apparent injury, membrane permeability, chlorophyll a fluorescence and antioxidant enzyme activities. Results showed that the transgenic lines showed different degrees of increased resistance to oxidative stress and salt stress, and the order of stress tolerance enhanced was: transgenic Chl-SOD plants>transgenic Chl-mSOD plants>wild-type plants. Therefore, over-expression of Kluyveromyces lactis Cu/Zn-SOD and mutant S1 (mSOD) gene in tobacco chloroplasts can enhance oxidative stress tolerance and salt stress tolerance of transgenic plants.A new security screening system was established based on Kluyveromyces lactis Cu/Zn-SOD mutant S1 gene as a selectable marker gene and paraquat as the selective agent. The optimal concentration of paraquat concentration was determined to be 6 ?mol/L. 96 normal phenotype of T0 independent transgenic tobacco events were produced via Agrobacterium mediated procedure using mSOD gene as a selectable marker gene and 6 ?mol/L paraquat selection. PCR, Southern blot and RT-PCR detection in transgenic plants demonstrated that mSOD gene was integrated into the tobacco genome and expressed. The highest transformation frequency was 39.26%. Compared with traditional screening system using the standard selectable marker gene nptⅡand a selection regime of 100 mg/L kanamycin, the ratios of mSOD-positive transgenic plants were respectively 2.16-and 2.1-folds for the transformation of pBI121-Chl-mSOD and pBI121’ plants. Genetic stability analysis of transgenic tobacco demonstrated most T0 transgenic plants contained single-copy insertion, with 3:1 segregation consistent with Mendel’s law.The germination rates of T1 seeds, height, and leaf number were recorded to inspect paraquat-induced oxidative stress and salt stress tolerance of T0 and T1 generation of transgenic plants. The result showed that both of them were higher than that of wild-type plant. This new security screening system avoided the use of antibiotic or herbicide resistance genes therefor relatively safe to environment and human. It offered other benefits, for example transformation efficiency improved and the plants growing period shortened. Selectable marker gene can also be used as target gene, which confer oxidative stress tolerance and salt stress tolerance of transgenic plants.更多还原