论文标题:菠菜cptpi基因和水稻cfba基因在鱼腥藻7120中的表达及其对光合作用影响的研究
Expression of cptpi Gene from Spinach and cfba Gene from Rice in Anabaena sp. PCC 7120 and Its Regulation of Photosynthesis
论文作者
论文导师 王全喜；施定基,论文学位 硕士,论文专业 水生生物学
论文单位 上海师范大学,点击次数 89,论文页数 118页File Size6926K
2006-05-01论文网 http://www.lw23.com/lunwen_110114727/
triosephosphate isomerase (TPI); fructose-1,6-bisphosphate aldolase (FBA);Anabaena sp. PCC7120; transgene; photosynthetic regulation
光合作用是地球上最重要的化学反应，是所有生命活动能量的最终来源。光合碳还原环(Calvin循环)是大多数光合生物碳固定的主要途径，是光合作用过程中的一个重要方面，同时也是光合作用调控的核心环节。光合作用研究的一个重点就是确定Calvin循环中的限速点，从而可以提高植物的生产力。 Calvin循环是一个由一系列酶共同催化的复杂串联反应系统，一共包含11个酶，催化13步反应，分为3个阶段：羧化、还原和再生。丙糖磷酸异构酶(triosephosphate isomerase，TPI，EC 5．3．1．1)和果糖-1，6-二磷酸醛缩酶(fructose-1，6-bisphosphate aldolase，FBA，EC 4．1．2．13)分别是再生阶段中的第一个和第二个酶，催化两个相继反应：TPI催化的是两个三碳——3-磷酸甘油醛(GAP)和磷酸二羟丙酮(DHAP)之间的可逆异构化反应；而FBA则可逆地催化这两个三碳糖缩化形成为六碳糖——果糖-6-磷酸(FBP)，是Calvin循环中固定CO_2后的第一个催化3C化合物转化为6C化合物的酶。两者位于第一个分支点上，是Calvin循环中控制光合碳流量的重要酶。然而，TPI和FBA催化的均是可逆反应，被认为是“非限速酶”，在光合碳流量中的调控作用一直不被重视。近年来，反义转基因植物的研究揭示了催化可逆反应的“非限速酶”在光合碳流量的调控中可能会起到意想不到的作用。 在这样的背景下，本论文利用过表达方法，将菠菜叶绿体tpi基因以及其同水稻胞质fba的串联基因转入到鱼腥藻7120中，测定了酶活、生长、光合放氧等一系列指标。以此来探讨TPI单酶水平的提高以及TPI-FBA双酶水平的提高对光合作用的影响。 本论文的主要内容及结果如下： 1．将菠菜叶绿体tpi基因连接在光诱导的高效启动子PpsbA下游，克隆入pDC-08中构建了既能在大肠杆菌又能在蓝藻中表达的穿梭表达载体pDC-TPI，并通过三亲接合转移转入鱼腥藻7120。 2．将菠菜叶绿体tpi和水稻胞质fba的串联基因连接在光诱导的高效启动子PpsbA下游，克隆入pDC-08中构建了既能在大肠杆菌又能在蓝藻中表达的穿梭表达载体pDC-AT，并通过三亲接合转移转入鱼腥藻7120。 3．测定了转基因藻和野生藻中相应酶的活性，并进行了比较。结果如下：①转pDC-TPI鱼腥藻蛋白粗提液中TPI的比活力比野生藻提高了21.8％，表明了菠菜叶绿体tpi基因转入鱼腥藻7120后成功地提高了转基因藻中TPI的活性；②转pDC-AT鱼腥藻蛋白粗提液中TPI的比活力比野生藻提高了26.2％，比转pDC-TPI鱼腥藻提高
Photosynthesis is the most important chemic reaction in the world, and it is the ultimate energy source of any living organisms. The photosynthetic carbon reduction (Calvin) cycle is the primary pathway of carbon fixation in most photosynthetic life-form, and is the core reaction of photosynthesis regulation. A major goal in photosynthetic research is to identify limiting points in the process, in order to produce plants with increased yield.Calvin cycle is a complicated reaction system. It comprises 11 different enzymes, catalyzing 13 reactions, and can be divided into 3 phases: carboxylation, deoxidization and regeneration. Triosephosphate isomerase(TPI) and fructose-1,6-bisphosphate aldolase(FBA) serve as the first and the second enzymes in the regenerative phase of the Calvin cycle respectively. They catalyze two concatenated reactions. TPI catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehydes-3-phosphate. FBA catalyzes the reversible reaction that dihydroxyacetone phosphate and D-glyceraldehydes-3-phosphate are utilized to form fructose-6-phosphate. There are two branch points in Calvin cycle. The first point relates to DHAP, which can be transported into cytoplasm and gradually synthesize sucrose. Second point relates to fructose-6-phosphate, which can been gradually synthesize starch. So TPI and FBA are two important enzymes of Calvin cycle and may be exert significant control over the photosynthetic carbon flux. However, they were considered as "non-regulated enzyme", because they catalyze reversible reaction, and their regulations of carbon flux were always neglected. Over the last 10 years, antisense transgenic plants have been used as tools to address this and have revealed some unexpected findings.In this article, chloroplast tpi gene from spinach and tandem genes of chloroplast tpi gene from spinach and cytoplast fba gene from rice were transferred into filamentous, heterocystour cyanobacterium Anabaena sp. PCC 7120 using gene overexpress technology. The enzyme activity, growth parameters and photosynthesis activity were measured and compared between transgenic and wild type cells. The aim was to discuss the influence on photosynthesis as the level of TPI and FBA changed.The contents and results of this experiment were as follows:
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