利用植物的光合作用同化甲醛的机理研究

【作者】 潘正波；

【导师】 陈丽梅；

【作者基本信息】 昆明理工大学 ， 生物化工， 2007， 硕士

【摘要】 不管是原核生物还是真核生物，凡是能利用一碳（C1）化合物（甲烷，甲醇，甲醛，甲酰胺等）的微生物，在它们代谢作用的初期阶段都产生甲醛，它们的细胞中都有能把从各种C1化合物产生的甲醛变成为细胞组成成分的同化途径以及从甲醛获得细胞所需能量的氧化途径。甲基营养酵母如Candida boidinii，HansenuLa polymorpha和Pichia pastoris具有利用一碳化合物甲醇的高效代谢机制。在甲醇的代谢途径中，甲醇首先被乙醇氧化酶氧化为甲醛，甲醛是甲醇代谢过程中的一个关键性的中间产物，它处于甲醇同化和异化途径的分支点上，因此在甲基营养型酵母菌中也存在甲醛代谢途径，其中一部分甲醛依靠二羟基丙酮合成酶（DAS）直接和五磷酸木酮糖结合生成二羟基丙酮和三磷酸甘油醛。DAS是一种依赖焦磷酸硫胺素的转酮醇酶，催化甲醛同化途径中的第一个反应。目前已从甲基营养型酵母菌C．boidinii和H．polymorpha中克隆到编码DAS的基因（DAS1）。二羟基丙酮对酵母细胞来说是种有毒的化合物，在酵母菌中二羟基丙酮激酶（DAK）参与二羟基丙酮的脱毒作用，DAK催化的反应使二羟基丙酮磷酸化，形成无毒性的磷酸二羟丙酮，可为酵母细胞所利用。该酶普遍存在于大多数的生物体中，结合遗传学和生物化学方法，已从酵母菌Saccharomyces cerevisiae和P．pastoris中克隆到编码DAK的基因，它们所编码的蛋白质是同型二聚体。甲醛目前已成为一种无处不在的空气污染气体，几乎对所有的生物来说都有毒性，它对植物来说也是一种毒性很强的气体。自然界中的大多数植物主要以CO₂为碳源，它们吸收甲醛的能力非常有限。因为DAS和DAK催化反应的底物5-磷酸木酮糖和产物磷酸二羟丙酮及甘油醛3-磷酸也都是开尔文循环的中间产物，因此本研究尝试在植物的叶绿体中表达来自酵母菌的DAS和DAK，利用DAS和DAK构建一条甲醛固定途径，把来自甲基营养型酵母菌的甲醛固定途径整合成开尔文循环的一个支路，由此可以开创一条利用植物进行甲醛污染治理的新策略和新方法。为了在将来能尽快将这种新策略应用于提高观赏植物代谢甲醛能力的分子育种，创造有功能的观赏植物新品种，本研究也开展了一些重要观赏植物如百合新品种的组织培养研究，为新成果的应用积累研究材料。为了获得具有自主知识产权的基因，用于进行植物甲醛代谢途径的遗传工程操作，本研究也尝试利用国内的资源和条件，分离能代谢一碳化合物的酵母菌株，为将来开展甲基营养型酵母菌的分子生物学研究，克隆C1代谢途径相关基因奠定研究基础。研究结果如下：（1）根据已克隆的C．boidinii酵母菌DAS和P．pastoris DAK基因的序列设计特异性引物，通过PCR分别从这两种酵母菌的基因组中扩增DAS、DAK基因，利用gateway技术构建DAS、DAK基因的植物表达载体pK2-Rbes-DAS、pn2-Rbcs-DAK，在构建好的植物表达载体中，DAS和DAK基因的表达都在番茄Rubisco小亚基rbcS-3C启动子（光诱导型）的控制之下。在这两个表达载体中的启动子含有叶绿体定位序列，使DAS和DAK基因表达的蛋白质能定位到叶绿体中。通过农杆菌介导法用pK2-Rbcs-DAS、pH2-Rbcs-DAK转化矮牵牛和烟草，通过抗性筛选和PCR检测获得转DAS基因的烟草株系17个和矮牵牛株系21个，转DAK基因的烟草株系20个和矮牵牛株系29个。用RT-PCR检测确定DAS、DAK基因在转基因烟草和矮牵牛中均能成功地转录之后，又用pH2-Rbcs-DAK转化DAS的转基因烟草和矮牵牛株系，通过抗性筛选和PCR检测获得转DAS、DAK双基因的烟草株系17个和矮牵牛株系24个。对液体甲醛吸收速率测定结果表明转DAS、DAK双基因的矮牵牛对甲醛的吸收速率明显快于对照植物和转单个基因DAS或DAK的植物，说明转基因表达产生的DAS、DAK可能构成一条甲醛固定途径，在转双基因植物中发挥作用。（2）以百合新品种木门和罗曼蒂的子房为材料，开展组织培养研究。结果表明，木门诱导愈伤组织的最适培养基为：MS+BA 1.0 mg．L^-1+NAA 0.5 mg．L^-1，诱导率为76％，芽分化最适培养基为：MS+BA 1.0 mg．L^-1+NAA 0.1 mg．L^-1，分化率达到82.5％。生根培养基为MS，生根率：100％。诱导罗曼帝百合愈伤组织的最适培养基为MS+KT4.0 mg．L^-1+2，4-D 4.0 mg．L^-1，诱导率为84％，芽分化最适培养基为：MS+BAKT4.0 mg．L^-1+2，4-D 4.0 mg．L^-1，分化率达到70％。生根培养基为MS或1／2MS，生根率：96％。（3）从云南保山啤酒厂采集的水样中分离得到2株具有甲醛抗性的酵母菌，通过对其形态学特征、生理学特性的初步鉴定，确定为甲基营养型酵母菌并命名为flj3。更多还原

【Abstract】 The formaldehyde （HCHO） is produced in the early steps of C1 metabolisms in all of the prokaryotes and eukaryotes which can utilize C1 compounds （methane, methanol, formaldehyde and methylated amines）. The cells of these organisms posses HCHO assimilation pathways which convert HCHO, produced from C1 compounds, into cell constituents and HCHO dissimilation pathways which oxidize HCHO to produce energy for cell growth. There is a highly efficient metabolism for the utilization of the one-carbon compound methanol in methylotrophic yeast species, such as Candida boidinii, HansenuLa polymorpha and Pichia pastoris. In methylotrophic yeasts, formaldehyde is the key intermediate in methanol metabolism since it stands at the branchpoint of pathways for methanol assimilation and dissimilation. Formaldehyde is generated from methanol via alcohol oxidase. A portion of the formaldehyde is directly fixed with xyluLose-5-phosphate （Xu5P） to produce dihydroxyacetone （DHA） and glyceraldehydes 3-phosphate （GAP）. DAS is a type of thiamine pyrophosphate-dependent transketolase and catalyzes the first reaction in the HCHO-assimilation pathway. Recently, the DAS gene （DAS1） was cloned from the methylotrophic yeasts, C. boidinii and H. polymorpha. DHA is toxic to yeast cells and dihydroxyacetone kinase （DAK） is involved in the detoxification of DHA. The reaction catalyzed by DAK converts DHA into dihydroxyacetone phosphate （DHAP） which is no toxic and can be ustilized by yeast cells. DAK presents in most organisms. DAK genes in the Saccharmyces cerevisiae and P. pastoris were cloned and characterized by a combined genetic and biochemical approach and the proteins encoded by these DAK genes are homodimer.Formaldehyde is becoming a major indoor air pollutant and also toxic to all plants. The vast majority of plants around us photosynthetically assimilate carbon dioxide via the Calvin cycle pathway and they have very limited capacity to up-take HCHO gas. Since the substrates and products of DAS and DAK, Xu5P, DHAP and GAP are the intermediates of the Calvin cycle, the present study attempted to express DAS and DAK from the yeasts into plant chloroplasts and construct a HCHO-fixing pathway with DAS and DAK. Thus theHCHO-fixation pathway from yeasts was incorporated into the Calvin cycle as a bypass in plants. This wouLd create a new strategy and method for phytoremediation of HCHO pollution. In order to apply this new strategy to ornamental for enhancement of their ability to metabolize formaldehyde and generation of functional ornamental in the future as soon as possible, the tissue cuLture for some important ornamentals such as new lily species, Conca D ’or and La Mancha was conducted in this study for accumuLation of experimental materials. This study also tried to isolate yeast strains which can use C1 compounds with local source for cloning of genes related to C1 metabolism and thus established a foundation for the molecuLar study on methylotrophic yeasts in the future. The main resuLts of this study are concluded as follows:（1） DAS and DAK genes were amplified from the genomic DNA of C.boidinii and P. pastoris , respectively. The plant expression vectors, pK2-Prbcs-DAS and pH2-Prbcs-DAK were constructed for DAS and DAK genes by gateway technology. In these two plant expression vectors, the expression of the DAS and DAK genes was under the control of tomato Rubisco-3c promoter （Prbcs）. The Prbcs promoter contains a transit peptide sequence which allows the location of the expressed DAS and DAK into chloroplasts. The DAS and DAK genes were introduced into Nicotiana tabacum and Petunia hybrida by Agrobacterium tumefaciens- mediated procedure. The transgenic plants were selected on MS medium containing antibiotics and identified by PCR. 17 lines of transgenic tobacco and 21 lines of transgenic Petunia, respectively, were obtained for DAS gene, while 20 transgenic tobacco lines and 29 transgenic Petunia lines, respectively, were obtained for DAK gene.The transcriptions of DAS and DAK genes were confirmed by RT-PCR analysis. The transgenic DAS tobacco and petunia were transformed again with pH2-Prbcs-DAK for generation of double gene （DAS/DAK） transgenic plants. The transgenic plants were selected on MS medium containing antibiotics and identified by PCR. 19 lines of transgenic tobacco and 24 lines of transgenic Petunia, respectively, were obtained. The further analysis indicated that the double gene transgenic petunia possessed stronger capacity to take up liquid formaldehyde than the control and the single gene, DAS or DAK, transgenic plants. This resuLt suggests that the expressed DAS and DAK might establish aHCHO-fixation pathway which functions in the double gene transgenic plants.（2） The tissue cuLture for Conca D’or, a new species of lily, was conducted with its ovary. The resuLts showed that the best medium for callus formation was MS+BA 1.0 mg.L^-1+NAA 0.5 mg.L^-1 and the inducing rate for callus was 76%. The best medium for bud initiation was MS+BA 1.0 mg.L^-1+NAA 0.1 mg.L^-1 and the inducing rate was 82.5%. The best medium for root formation was MS, and the rooting rate reached 100%. At the same time, the tissue cuLture for Mancha was also conducted with its ovary. The best medium for callus formation was MS +KT4.0 mg.L^-1 + 2,4-D 4.0 mg.L^-1 and the inducing rate for callus was 84%. The best medium for bud initiation was MS + KT4.0 mg.L^-1 + 2, 4-D 4.0 mg.L^-1 ,and the inducing rate was 70%. The best medium for root formation was MS, and the rooting rate reached 96%（3） Two yeast strains resistant to high HCHO were isolated from sewage samples. The morphological and physiological identification showed one of them was methylotrophic yeast and designated as Yflj3更多还原