【作者】 崔震海；

【导师】 张立军；

【作者基本信息】 沈阳农业大学 ， 作物学， 2013， 博士

【摘要】 C4植物玉米的第1-3叶维束鞘细胞叶绿体(BSC)有垛叠的基粒和PS II活性,主要利用Rubisco直接固定C02进行C3光合循环；而4叶以上,叶肉细胞(MC)利用PEPC固定CO2并以苹果酸形式运输到BSC叶绿体,再由Rubisco加入C3循环。这些叶片BSC叶绿体的结构特点是基粒和PS II活性基本消失,很少合成NADPH,因此,这些叶绿体中C3循环的运转需要依赖从叶肉细胞转运的还原力。苹果酸是细胞内多种代谢的联接者和调节者。在玉米C4光合作用中,苹果酸不仅是叶肉细胞向BSC叶绿体输送CO2的载体,还是转移还原力的携带者,在脱羧的同时产生NADPH。所以,苹果酸的脱羧作用影响叶绿体的氧化还原状态,氧化还原状态的变化可能与BSC叶绿体的基粒发育和PS II活性变化有关,可能参与进化和个体发育过程中C4光合途径形成的的调节。为研究PEPC通过苹果酸参与BSC叶绿体发育调节的作用及机制,本文观察了不同碳同化途径玉米叶片的PEPC活性、苹果酸含量和解剖结构,检测了外源苹果酸对玉米和烟草叶圆片及对转C4-PEPC基因水稻活体叶片光合作用的效应,并完成了抑制玉米C4-PEPC基因表达的小RNA克隆和载体构建,为阐明植物C4光合途径形成和BSC分化的机制提供新的思路和理论依据,也为利用转基因技术改造C3作物的光合作用提供参考。主要结果如下：1. PEPC活性、苹果酸代谢与维管束鞘细胞分化具有密切关系在本试验中,随着玉米叶片的发育,C4循环关键酶PEPC和NADP-ME等活性也随之增加,维管束鞘细胞叶绿体的C4结构特征越来越明显,内源苹果酸代谢增强,外源苹果酸处理玉米离体叶圆片和根系饲喂水稻抑制叶片的光反应。因此,叶片的PEPC活性和内源苹果酸代谢强度与C4解剖结构的发育具有密切的相关关系。2. PEPC和苹果酸在C4植物光合途径发育中的作用机制(1)苹果酸的供应降低叶绿体中NADP的数量,增大NADPH/NADP的比值,阻碍电子传递的进行,促进活性氧的产生;(2)苹果酸的脱羧或脱氢反应改变细胞的氧化还原电位,进而影响PS II的结构和功能；(3)苹果酸脱羧后生成的丙酮酸可能影响细胞的氧化还原电位和PS II的结构。3. C4-PEPC在转基因水稻中的作用机制在转C4-PEPC基因的水稻植株中,PEPC基因的表达使苹果酸代谢增强,OAA不均匀进入不同的叶绿体。苹果酸在高还原能力的叶绿体内合成,在低还原能力的叶绿体内脱羧。一部分叶绿体起C4植物MC叶绿体的作用,另一部分起BSC叶绿体的作用,在两种叶绿体之间可以进行C4微循环。4.玉米C4-PEPC基因的克隆、载体构建和转入北方粳型超级稻本文以国内玉米优良自交系郑58为材料,分两步克隆了C4型PEPC基因的启动子和其剩余全长序列,片段长7530bp,利用In-fusion技术成功构建了pCAMBIA-1391Z-PEPC载体,利用农杆菌介导法将其转入悬浮培养的北方粳型超级稻沈农265的愈伤组织,获得了PEPC酶活性显著提高的转基因水稻植株。5. C4-PEPC基因RNAi载体的构建本研究选择一个C4-PEPC自身的内含子序列作为RNAi干扰的间隔区域,先克隆正向片段与内含子,共475bp,然后与反向片段387bp相连接,成功构建了RNAi植物表达载体p7002-Z-F。但此RNAi载体与用其它序列作为间隔区域载体干扰效果的差异,还有待进一步的研究。根据本试验结果可以得出初步结果,PEPC通过合成苹果酸而影响玉米BSC叶绿体的分化和发育。在BSC中,苹果酸脱羧生成丙酮酸,同时产生大量NADPH,消耗NADP。 NADP的减少会抑制线性光合电子传递,并可能导致活性氧的产生,最终改变PS II反应中心的结构。因此,我们推测在植物进化过程中PEPC活性增强并引发大量苹果酸的合成,后者转运到BSC中参与调节BSC叶绿体基粒跺叠的消失及BSC与MC叶绿体分化方向的改变。更多还原

【Abstract】 In C4plant maize, there are packed thylakoid lamellaes and active PS II in the bundle sheath chlorophasts of the lower position (1-3). Herein the CO2is fixed by Rubisco in C3photosynthetic pathway. But in the upper position leaves (＞4), the CO2is initially fixed by PEPC to synthesize malate in mesophyll cells and then transported to chlorophasts in the bundle sheath. Therein, malate is decarboxylated to relaese CO2and fixed by Rubisco in C3photosynthesis. In these BSC chlorophasts, the grana disappears and the activity of PS II is deficient,and NADPH is seldomly produced. Therefore, the supply of the reducing power from MC is necessary for C3cycle. As a linker and regulator in many cell metalsolisms, in the C4photosynthetic cycle of maize, malate is not only the carrier of CO2from MC to BSC but also the transporter of the reducing power to BSC from MC. As decarboxylated, malate produces NADPH in the BSC chloroplasts. As a result, the redox potential is affected by the decarboxylation of malate in these chlorophasts. It may be related to the differentiation of grana and the development of PS II activities in BSC chlorophasts and involves the regulation of C4photosynthetic pathway during the C4plant evolution and individual development. To investigate the function and mechanism of PEPC action via malate synthesis on the development of BSC chlorophasts. In this research, we measured the PEPC activities, malate contents and anatomy of leaves with different carbon assimilation pathway, observed the effect of exogenous malate on light reaction of detached leaves of maize and tobacco and on the photosynthesi of attatched leaves of transgenic PEPC rice, and constructed a plant transformation vector of RNAi for maize C4-PEPC gene. We expected that this study would provide a new thinking and theoretical basis for the exploration on the formation mechanism of C4photosynthetic pathway and the differentiation mechanism of the bundle sheath chlorophasts and also provide reference for the modification of the photosynthetic pathway in C3plants by transgenic technology. The main results are as follows:1. Close relationship between PEPC activities, malate metabolism and BSC differentiation. In this study, during the development of maize leaves, the activities of PEPC, NADP-ME, and other photosynthetic enzymes increased, C4anatomy traits were more distinctive, and malate metabolism was enhanced. The light reaction was inhibited in detached leaves of maize in exogenous malate solution and the photosynthesis was affected in attatced leaves in rice leaves fed with exogenous malate via roots. The results showed that there was a close relationship between PEPC activities, malate metabolism and development of C4anatomy.2. Mechanism of PEPC and malate action in the development of photosynthetic pathway in plants.(1) during malate treatment, in the chloroplasts, the amount of NADP decreased and the ratio of NADPH/NADP increased, resulting in the inhibition of electron transport and the production of reactive oxygen species;(2) the decarboxylation and dehydrogenation reaction of malate changed BSC redox state and then imposed effects on the differentiation of the structure and function of PS Ⅱ;(3) pyruvic acid produced by malate decarboxylation may affect cell redox state and the structure of PS Ⅱ.3. Mechanism of C4-PEPC action in transgenic rice. In the leaves of transgenic rice, the expression of PEPC gene enhanced the malate metabolism. OAA generated by PEPC was unevenly transported into different chloroplasts and impoesd a different effect on the redox state of different chloroplast.Malate produced in high reducing power chloroplasts and decarboxylated in low reducing power chloroplasts. A part of chloroplasts played the role of mesophyll chloroplasts in C4plants and the other part took a role of BSC chloroplasts.The micro cycle of C4photosynthesis run between these two types of chloroplasts.4. Cloning, plant expression vector construction of C4-PEPC gene from maize and the transformation into northern super japonica rice. In this study, the promotor and full-length sequence of C4-PEPC gene were cloned by two-step PCR from the maize inbred line Zheng58. The whole fragment length was7530bp. The pCAMBIA-1391Z-PEPC vector was attained by ligation to form linear vector using In-Fusion technology. The transformation of the maize C4-PEPC gene into northern super japonica rice was mediating by agrobacterium. The dramatic increase of PEPC activities was detected in the transgenic rice leaves.5. Construction of plant transformation vector of RNAi for maize C4-PEPC gene. A intron segments of C4-PEPC gene was selected to be RNAi sequence. At first, the forward gene segments and intron were cloned to475bp. Then it was ligated reverse gene segments (387bp). Finally, a plant transformation vector of RNAi (p7002-Z-F) was constructed succesfully. Because this interval sequence of RNAi vector was different from others, the effect of RNA interference effect needed to further explore.According to the results, we could make a preliminary conclusion. The PEPC played a role in the differentiation and development of BSC chlorophasts in maize via malate metabolism. In BSC, malate was decarboxylated to produce pyruvic acid and NADPH which consumed the NADP in cloroplasts. The deficiency of NADP might inhibit the linear electron transport and led to the generation of reactive oxygen species, which resulted in the change in the structure of PSII reaction centers. Thus we proposed that PEPC activities increased during plant evolution and synthesized more malate. Which was transported into BSC and participated in the regulation on the disappearance of grana stack and the chlorophast differentiation in BSC differring from MC chloroplasts.更多还原