【作者】 王满；

【导师】 李霞； 蔡庆生；

【作者基本信息】 南京农业大学 ， 植物学， 2011， 硕士

【摘要】 磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)是C4光合途径中固定CO2的关键酶,对C4作物的高光效有重要作用。高表达转玉米C4型磷酸烯醇式丙酮酸羧化酶基因水稻(高表达转玉米C4型pepc水稻)无论在生理性状,光合指标及产量等性状方面都优于未转基因水稻原种。为了研究C4型PEPC在C3植物水稻中的生理机制,本文以高表达转玉米C4型pepc水稻(PC)和原种Kitaake(WT)为材料,建立了悬浮细胞系,通过外施化学试剂诸如用磷脂酶C抑制剂硫酸新霉素(Neomycin sulfate, NS)、磷脂酶D抑制剂正丁醇(1-butanol),钙及钙的鏊合剂乙二醇双(2-氨基乙醚)四乙酸Ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, EGTA),在细胞水平上研究外源因子对细胞内PEPC酶活性以及相关信号分子NO、过氧化氢及钙的影响,结果发现：1、PC和WT悬浮细胞系的建立2,4-D浓度为2mg/L,6-BA浓度为1mg/L,通过3次继代培养(2,4-D浓度2-0.5mg/L,6-BA浓度为2-0.2 mg/L, ABA浓度为1 mg/L),依次减低外源激素的施用,可获得颗粒状且分散的愈伤组织,转移到液体培养基(2,4-D浓度0.5mg/L,6-BA浓度为0.2 mg/L, ABA浓度为1 mg/L),适合的培养温度为28℃,悬浮细胞系继代时新原液的比例为3：1,6周可建立稳定的供试材料的悬浮细胞系。2、单独外施新霉素或正丁醇处理1h抑制细胞内PA的含量,PC细胞内PEPC酶活性及NO、H2O2含量下降,有浓度和时间依赖性,单独外施Mas处理1h促进细胞内PA含量,PC细胞内的酶活性及NO、H2O2含量降低,葡萄糖作用与Mas相似。3、PC细胞中,联合加入正丁醇和新霉素1h后,PEPC酶活性、NO、H2O2含量上升；加入Mas, PEPC酶活性、NO、H2O2含量下降；葡萄糖作用效果与Mas相似。同时伴随Ca2+的大量释放。4、当在PC悬浮细胞中同时加入正丁醇、新霉素和EGTA时,PC内PEPC活性、NO和H202含量比对照增加。虽然联合施加三种抑制剂的细胞中钙离子释放量比单独加入外源抑制剂EGTA降低很多,但与未施抑制剂的PC对照相比,细胞内的钙离子仍略有增加,表现PC的PEPC活性仍诱导增加,可见PC细胞内的内源钙的浓度与PEPC酶活性的调节密切有关,这可能由于本文外施的EGTA的浓度只能抑制部分PC内钙库的迸发。更多还原

【Abstract】 PEPCase is an key enzyme fixing CO2 in the C4 photosynthetic pathway, which has an important effect on the high photosynthetic efficiency of C4 crops. High expression of transgenic maize-C4-phosphoenolpyruvate carboxylase gene rice (high-expressing transgenic corn C4 pepc rice) are better than no transgenic rice original seed in physiological traits, photosynthetic indexes and yield traits. In order to research the physiological mechanism of C4 pepc in C3 plant rice, this article use high expression corn C4 pepc rice (PC) and untransformed rice plant Kitaake (WT) as material, cell suspension systems was established by adding the effects chemical reagents such as phospholipid enzyme C(PLC) inhibitor sulfate new mold pigment (Neomycin sulfate, NS), phospholipid enzyme D (PLD) inhibitor butanol (1-butanol), calcium and the calcium of a flat iron plate for making cakes mixture glycol double (2-amino ether) four acetate ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid, EGTA) and the effects were studies on cell within PEPC enzyme activity, related signal molecular NO, hydrogen peroxide(H2O2) and the calcium of effect in the cell level. The results were found as following:1.Establishment of cell suspension system PC and WT:2mg/L 2,4-D and 1mg/L 6-BA were added to induce the callus from the rice seed, then put the callus into the mediums of regeneration with hormones such as 2-0.5mg/L 2,4-D,2-0.2 mg/L 6-BA,1 mg/L ABA), however reduce outside source hormone of application step by step, finally the shaped,dispersed and particle callus can get to transfer to liquid medium adding 0.5mg/L 2,4-D,0.2 mg/L 6-BA,1 mg/L ABA at 28℃. The cell suspension system for rice material can be established stably after 6 weeks by adding generation new liquid of proportion for 3.1orriginally.2.Adding neomycin alone or n-butyl alcohol for one hour to inhibit PA in the cell, PEPC enzyme activity and NO, H2O2 concentration in PC were reduced depend on the concentration and time. After adding Mas for 1h to increase PA in the cell, the enzyme activity and NO, H2O2 in the cell of PC were reduced, so did the glucose effects in PC.3.In the cell, after adding both the n-butanol and neomycin in PC for 1h, the PEPC activities of PC were increased, while the NO, H2O2 content were decreased; When adding the Mas in PC, PEPC enzyme activity, NO, H2O2 concentration of PC were increased; the effects of glucose was similarly to that of Mas. At the same time, it appeared more Ca2+ concentration in the cell sinigicantly.4. When the 1-butanol, neomycin and EGTA were added at the same time in PC, PEPCase activities NO and H2O2 contents of PC were increased. Although the calcium ion releasing after mentioned above inhibitors in PC were reduced much lower than those only adding EGTA, the Ca2+ concentration in PC was still slightly increased as compared to PC-control without adding EGTA.The results showed that endogenous concentrations of calcium in the cell of PC was more related to regulate of PEPC activities due to the adding concentration of EGTA in this paper can partly inhibit the bursting of calcium sink.更多还原