外源因子对高表达转玉米C_4型pepc水稻悬浮细胞中PEPCase及内源信号分子的影响
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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。
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.
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。
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.
引文
Apel K, Hirt L. Reactive oxygen species:Metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology,2004,55:373-399
Barna B, Fodor M, Kiraly Z. Role of active oxygen species and antioxidants in plant disease resistance. Pest Manag Sci,2003,59(4):459-464
Beligni M V, Lamattina L. Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light—inducible responses in plants. Planta,2000,210(2):215-221.
Christa Testerink, Henk L. Dekker, et al., Ktistakis and Teun Munnik. Isolation and identification of phosphatidic acid targets from plants. The Plant Journal,2004,39:527-536.
De Jong C F, Laxalt A M, Bargtrmnn B O, et al. Phosphatidie acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J,2004,39(1):1-12
Distefano A M, Luciana Lanteri M, Have A T, et al. Nitric Oxide and Phosphatidic Acid Signaling in Plants. Plant Cell Monographs,2010,16:223-242.
Fan L, Zheng S, Wang X. Antisense suppression of phospholipase D alpha retards abseisie acid and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell,1997,9(12): 2183-2196
GovIin E M, Kvine A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen. Botrytis Cinerea Curr Biol,2000,10(13):751-757
Greenberg J T, Yao N. The role and regulation of programmed cell death in plant—pathogen interactions. Cellar Microbiology,2004,6(3):201-211
Guo A. Reimers P J, Leach J E. Effect of light on incompatible interactions between Xanthomonas oryzae pv oryzae and rice. Physiological and Molecunlar Plant Pathology,1993,42:413-425
Ibaraki Y, Kenji K. Application of image analysis to plant cell suspension cultures. Computers and Electronics in Agriculture,2001,30:193
Jacob T, Ritchie S, Assmann S M, et al. Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity. Proc Natl Aead Sci USA,1999,96(21):12192-12197
Jenes B. Pauk J, Plant regeneration from protoplast derived calli in rice (Oryza sativa L.) using Dicamba. Plant Sci.1989,63:187-198
Jiang L.Q., Feng X., Zhou W., Knyazev P.G., Ullrich A. and Chen Z.,2006,Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation. Oncogene,2006,25:5495-5560
Jose'A. Monreal, Fionn McLoughlin, Cristina Echevarrla, et al. Phosphoenolpyruvate Carboxylase from C4 Leaves Is Selectively Targeted for Inhibition by Anionic Phospholipids. Plant Physiology,2010, 152(2):634-638
Katagiri T, lshiyama K, Kato T, et al. An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana. Plant,2005,43(1):107-117.
Ku M S B, Agarie S, Nomura M, et al. High-level expression ofmaize phosphoenolpyruvate carboxylase in transgenic rice plants. Nature Biotechnol,1999,17:76-80
Kuai, B. K., Dalton, S. J., Bettany, A. J. E., et al. Efficient regeneration of fertile transgenic tall fescue (Festuca arundinacea Schreb) with a stable highly expressed foreign gene. Plant Cell Tissue and Organ Culture,1999,58:149-154.
Latijnhouwers M, Murmik T, Govers F. Phospholipase D in Phytophthora infestans and its role in zoospore encystment. Mol Plant Microbe Interact,2002,15(9):939-946.
Latijnhouwers M, Murmik T, Govers F. Phospholipase D in Phytophthora infestans and its role in zoospore encystment. Mol Plant Microbe Interact,2002,15(9):939-946
Lee S, Park J, Lee Y. Phosphatidic acid induces actin polymerization by activating protein kinases in soybean cells. Mol Cells,2003,15(3):313-319.
Leticia Chee-Gonzalez A.J., et al., Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells. Journal of Inorganic Biochemistry,2009, 103:1497-1503
Nathalie Giglioli-Guivarc'h, Jean-Noel Pierre, Spencer Brown, et al. the Light-Dependent transduction Pathway Controlling the Regulatory phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis. The Plant Cell,1996,8:573-586
Ok Ran Lee, Soo Jin Kim, Hae Jin Kim, et al. Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root. The Plant Cell Preview,2010.1-15
Ozawa K. Konamine A. Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice. Theor Appl Genet.1989,77:205-211
Park J, Gu Y, Lee Y, et al. Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-rolated small G protein GTPase mediated pathway of reactive oxygen species generation. Plant Physiol,2004,134(1):129-136
Potocky M, Elias M, Profotova B, et al. Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth. Planta,2003,217(1):122-130.
Sandermann H Jr. Active oxygen species as mediators of plant immunity:three case studies. Biol Chem, 2000,381(8):649-653
Sang Y, Cui D, W ang X. Phospholipase D and phosphatidic acid-mediated generation of superoxide in Arabidopsis. Plant Physiol,2001,126(4):1449-1458
Sherift A, Meyer H, Rieddel E, et al. Theflutnce of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzymes. Plant Sci,1998,136:43-57
Song Y., Lu S.H., Yuan Y.J. Cerium elicitor-induced phosphatidic acid triggers apoptotic signaling development in Taxus cuspidata cell suspension cultures. Chemistry and Physics of Lipids,2009, 159:13-20
Spangenberg G, Wang ZY, Wu X.L, et al. Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. Plant Physiol. 1995,145:693-701.
Tae J. L., Randall W.S., Linda H.B. et al. Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis. Plant molecular biology reporter,2004,22(9):259-267
Testerink C, Munnik T. Phosphatidic acid:a multifunctional stress signaling lipid in plants. Trends Plant Sci,2005,10(8):368-375.
Teun Munnik. Phosphatidic acid:an emerging plant lipid second messenger. TRENDS in Plant Science, 2001,6(5):227-233
Wang Z.Y., Ye X.D, Nagel J. Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue plants. Plant Cell Report,2001,20:213-219.
Yamada A, Shibuya N, Kodama O. et al. Induction of phytoalexin formation in suspension-cultured rice cells by n-acetylchitooligo saccharides. Biotech Biochen,1993,57(3):405-409
Yoshio M., Yoshioka H., Noriyuki D. An autophotographic determination of the active oxygen generation in potato tuber discs during hypersensitive response to fungal infection or elicitor. Plant Science,1995,105:45-52
Zhang H.K., Zhang X, Li Q, et al. Alpha-picolinic acid activates diverse defense responses of salicylic acid-jasmonic acid/ethylene-and Ca2+-dependent pathways in arabidopsis and rice suspension cells.Acta Botanica Sinica,2004,46(10):1200-1205
Zhang W, Wang C, Qin C, et al., The oleate-stimulated phospholipase D, PLDδ, and phosphatidic acid decrease H202- induced cell death in Arabidopsis. Plant Cell,2003,15(10):2285-2295.
曾富华,王永刚,胡木林,等.水稻病程相关蛋白的诱导和测试.西北大学学报(自然科学版)2002,32：17-21
曾富华,吴岳轩,罗泽民,等.活性氧及其清除剂诱导抗病作用与影响膜脂过氧化作用的关系.中国农业科学,2000,33(4)：103-105
曾富华,易克,徐向丽,等.不同诱导处理后水稻悬浮细胞的活性氧变化与有关酶系的关系.作物学报.2005,31(1)：18-23
陈志,陈金慧,边黎明,等.杂交鹅掌楸胚性细胞悬浮系的建立.分子植物育种,2007,5(1)：137-140
仇晴川,杨代常.分子医药农业研究进展.武汉植物学研究,2009,27(1)：83-89.
方文娟.日本结缕草(Zoysia japonica Steud.)悬浮再生体系的建立与超低温保存研究.北京：北京林业大学,2008
方文娟,韩烈保.曾会明.植物细胞悬浮培养影响因子研究.生物技术通报,2005,(5)：1]15
古桥胜久,谷田靛道彦.稻节愈伤组织的无限生长培养.水稻的组织培养.上海科学技术出版社,1981,1-3.
和七一,余晓东.胡蜂蜂毒肽Mastoparan的研究概况.重庆师范大学学报(自然科学版),2007,24(1)：76-80
胡燕梅,郭云贵,蒋细旺,等.甘蔗及天然有机物对银杏愈伤组织合成黄铜的影响.西南农业学报.2010.23(3)
贾勇炯,陈放,李英,等.从水稻根部悬浮培养细胞分离原生质体及植株再生,生物工程学报, 1994,10(2)：168-172
蒋细良,朱昌雄,王慧敏,等.中生菌素对水稻悬浮细胞苯丙氨酸解氨酶基因转录表达的影响.中国农业科学.2005,38(7)：1353-1357
金坚敏,张静兰.悬浮培养下水稻的胚状体高频诱导.植物学报,1992,34(2)：114-120
井鑫,张兴国.植物生物反应器研究进展.西南农业大学学报(社会科学版),2004,2(4)：109-112
李红玉,吴健胜,宋从凤,等.水稻IR26悬浮细胞与白叶枯病菌互作的过敏反应现象.植物病理学报.1999,29(4)：299-303
李景梅,王媛,王巍,等.三溴偶氮胂分光光度法测定钙.冶金分析,2004,24(5)：53-55
李霞,陈婷,周月兰.籼粳稻成熟胚愈伤组织培养力的比较,南京师范大学学报(自然科学版),2005,28(4)：103-108
李霞,焦德茂,戴传超.转PEPC基因水稻对光氧化逆境的适应.作物学报,2005,31(4)：408-413
李霞,焦德茂.转C4光合基因水稻及在育种中的应用.分子植物育种,2005,3(4)：550-556
李霞,赵贇涛,阎丽娜,周月兰.籼稻扬稻6号和粳稻苏沪香粳花药培养力的比较.南京师大学报(自然科学版),2008,31(4)：108-113
李霞.籼稻扬稻6号和粳稻苏沪香粳花药培养力的比较,南京师大学报(自然科学版),2008,31(4)：108-113
吕孟雨,赵和,王海波.水稻愈伤组织生长速率研究.中国农学通报.2005,21(12)
孟威,文景芝,吴茂森,等.两种黄单胞病菌诱导水稻一氧化氮产生和防卫基因表达的比较研究.中国农业科学,2007,40(6)：1159-1165
彭程,肖涛,罗远明,等.I G F-1对I L-1诱导的兔关节软骨细胞NO和PGE2的影响.中南大学学报,2008,33(3)：197-203
彭建,王丹英,徐春梅,等.钼酸铵法测定水稻过氧化氢酶活性.中国农学通报.2009,25(16)：61-64
乔建军,赵红,牛晋阳,等.过氧化氢作用下东北红豆杉悬浮培养细胞中蛋白表达差异的研究.植物病理学报,2003,33(5)：429-433
石丽君,王春安.新霉素抑制PC12细胞烟碱受体介导的反应.生理学报,2002,54(5)：441-445.
宋颖琦,杨谦.磷脂酶在植物逆境胁迫信号传导中的作用.哈尔滨工业大学学报,2006,38(2)：243-246
孙亮庆,王春连,冯斗,等.利用水稻悬浮细胞系检测白叶枯菌致病菌株的研究.作物杂志, 2008,5：24-27
覃建兵,汪越胜,何光源.水稻悬浮培养诱导生成植保素的研究.应用与环境生物学报,2006,12(3)：322-324
田敏,饶龙兵,李纪元.植物细胞中的活性氧及其生理作用.植物生理学通讯,2005,41(2)：235-241
王超,侍福梅,活性氧与一氧化氮在逆境胁迫下的相互关系.安徽农业科学,2009,37(5)：1903-1904,1908
王睿辉,陈耀锋,高秀武,等.激素对小麦幼胚胚性无性系高频率诱导的影响.西北农林科技大学学报,2001,29(1)：33-36
王亚馥,崔凯荣,陈克民,等.小麦幼胚培养中体细胞胚发生和植株再生.植物学通报,1992,9(增刊)：2
王炎钦,董海涛.磷脂酸在植物中的第二信使功能.中国生物化学与分子生物学报,2006,22(9)：697-703
王忠安.水稻佳辐占成熟种胚培养及其悬浮细胞系的建立.厦门大学学报(自然科学版),2005,6(44)：180-181
吴春霞.植物细胞悬浮培养的影响因素.安徽农业科学,2009,37(1)：36-38
吴茂森,田峰,齐放军,等.水稻与白叶枯病菌互作的基因表达谱的分析和差异性表达基因的识别.中国农业科学,2007,40(2)：277-282
吴学友,孙立平,刘丽霞,等PLC/PLD抑制剂对盐胁迫下玉米幼苗根系新的69.5 kD热稳定蛋白表达调控的影响.植物生态学报.2005,29(3)：510-515
向太和,梁海曼,钟华鑫,等.水稻胚性悬浮细胞系建立过程中生理生化变化Ⅱ氨基酸、多胺及内源激素的变化.作物学报.1997,23(3)：353-359
向太和,杨剑波,吴家道.水稻、玉米胚性悬浮细胞系的有效建立.安徽农业科学,1996,24(1)：1-8
向太和,杨剑波,吴李君,等.水稻胚性悬浮细胞系建立的细胞学研究.安徽农业科学.1993,21(1)：1-6
向太和,钟花鑫,梁海曼,等.水稻胚性悬浮细胞系建立过程中的生理生化变化.作物学报,1995,21(2)：223-229
邢登辉,吴琴生,刘大钧,禾谷类作物细胞培养——体细胞胚性潜力的诱导与表达.生物学通报,1994,29(7)：1-3
徐林林,芦笛,陆巍,等.水稻悬浮细胞系的建立及培养条件对生物产量的影响.植物生理学通讯.2006,42(4)：612-616
徐勤虎,董树亭,高荣岐.外源果糖、葡萄糖与一氧化氮供体(SNP)对玉米种子萌发的影响.玉米科学,2008,16(3)：74-77,
杨跃生,简玉瑜,郑迎冬,铜在水稻愈伤组织培养再生植株中的促进作用.中国水稻科学.1999,13：95-98.
叶玲娟.相思树的细胞培养及体胚发生研究.福建,福建农林大学,2008.
尹庆良,刘世强.水稻细胞悬浮系及其单细胞培养的研究.沈阳农业大学学报,1994,25(4)：366-372
於凤安,申宗坦.水稻悬浮培养细胞在固体和液体培养下的形态发生.安徽农业大学学报,1994,21(2)：157-169
于惠敏,侯丙凯,番茄基因工程及生物反应器研究与开发进展.山东农业科学,2007,2：21-25
于晓红,朱祯,付志明,等.提高小麦愈伤组织分化频率的因素.植物生理学报,1999,25(4)：388-394.
袁金玲,张朵,顾小平,等.孝顺竹种胚愈伤组织悬浮培养条件优化.分子植物育种,2009,7(4)：839-844
袁力勇,李绍清,李阳生,等.水稻悬浮细胞系的建立.云南大学学报,2003,25(4)：373-376
袁苑,武玉叶,李德全.植物甘油二酯激酶(DGK)信号转导作用.植物生理学通讯,2007,43(6)：1009-1014
章文华,王学敏.植物体内磷脂代谢和信号转导Chinese bulletin of life sciences.2000.12(3): 100-104
赵建聪,江婷,李维.利用植物生物反应器表达胸腺素α1的研究.四川师范大学学(自然科学版),2009,32(1)：112-117
朱根发,余毓君.爪哇稻原生质体培养与植株再生.遗传,1995,17(4)：21-24
Barna B, Fodor M, Kiraly Z. Role of active oxygen species and antioxidants in plant disease resistance. Pest Manag Sci,2003,59(4):459-464
Beligni M V, Lamattina L. Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light—inducible responses in plants. Planta,2000,210(2):215-221.
Christa Testerink, Henk L. Dekker, et al., Ktistakis and Teun Munnik. Isolation and identification of phosphatidic acid targets from plants. The Plant Journal,2004,39:527-536.
De Jong C F, Laxalt A M, Bargtrmnn B O, et al. Phosphatidie acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J,2004,39(1):1-12
Distefano A M, Luciana Lanteri M, Have A T, et al. Nitric Oxide and Phosphatidic Acid Signaling in Plants. Plant Cell Monographs,2010,16:223-242.
Fan L, Zheng S, Wang X. Antisense suppression of phospholipase D alpha retards abseisie acid and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell,1997,9(12): 2183-2196
GovIin E M, Kvine A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen. Botrytis Cinerea Curr Biol,2000,10(13):751-757
Greenberg J T, Yao N. The role and regulation of programmed cell death in plant—pathogen interactions. Cellar Microbiology,2004,6(3):201-211
Guo A. Reimers P J, Leach J E. Effect of light on incompatible interactions between Xanthomonas oryzae pv oryzae and rice. Physiological and Molecunlar Plant Pathology,1993,42:413-425
Ibaraki Y, Kenji K. Application of image analysis to plant cell suspension cultures. Computers and Electronics in Agriculture,2001,30:193
Jacob T, Ritchie S, Assmann S M, et al. Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity. Proc Natl Aead Sci USA,1999,96(21):12192-12197
Jenes B. Pauk J, Plant regeneration from protoplast derived calli in rice (Oryza sativa L.) using Dicamba. Plant Sci.1989,63:187-198
Jiang L.Q., Feng X., Zhou W., Knyazev P.G., Ullrich A. and Chen Z.,2006,Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation. Oncogene,2006,25:5495-5560
Jose'A. Monreal, Fionn McLoughlin, Cristina Echevarrla, et al. Phosphoenolpyruvate Carboxylase from C4 Leaves Is Selectively Targeted for Inhibition by Anionic Phospholipids. Plant Physiology,2010, 152(2):634-638
Katagiri T, lshiyama K, Kato T, et al. An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana. Plant,2005,43(1):107-117.
Ku M S B, Agarie S, Nomura M, et al. High-level expression ofmaize phosphoenolpyruvate carboxylase in transgenic rice plants. Nature Biotechnol,1999,17:76-80
Kuai, B. K., Dalton, S. J., Bettany, A. J. E., et al. Efficient regeneration of fertile transgenic tall fescue (Festuca arundinacea Schreb) with a stable highly expressed foreign gene. Plant Cell Tissue and Organ Culture,1999,58:149-154.
Latijnhouwers M, Murmik T, Govers F. Phospholipase D in Phytophthora infestans and its role in zoospore encystment. Mol Plant Microbe Interact,2002,15(9):939-946.
Latijnhouwers M, Murmik T, Govers F. Phospholipase D in Phytophthora infestans and its role in zoospore encystment. Mol Plant Microbe Interact,2002,15(9):939-946
Lee S, Park J, Lee Y. Phosphatidic acid induces actin polymerization by activating protein kinases in soybean cells. Mol Cells,2003,15(3):313-319.
Leticia Chee-Gonzalez A.J., et al., Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells. Journal of Inorganic Biochemistry,2009, 103:1497-1503
Nathalie Giglioli-Guivarc'h, Jean-Noel Pierre, Spencer Brown, et al. the Light-Dependent transduction Pathway Controlling the Regulatory phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis. The Plant Cell,1996,8:573-586
Ok Ran Lee, Soo Jin Kim, Hae Jin Kim, et al. Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root. The Plant Cell Preview,2010.1-15
Ozawa K. Konamine A. Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice. Theor Appl Genet.1989,77:205-211
Park J, Gu Y, Lee Y, et al. Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-rolated small G protein GTPase mediated pathway of reactive oxygen species generation. Plant Physiol,2004,134(1):129-136
Potocky M, Elias M, Profotova B, et al. Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth. Planta,2003,217(1):122-130.
Sandermann H Jr. Active oxygen species as mediators of plant immunity:three case studies. Biol Chem, 2000,381(8):649-653
Sang Y, Cui D, W ang X. Phospholipase D and phosphatidic acid-mediated generation of superoxide in Arabidopsis. Plant Physiol,2001,126(4):1449-1458
Sherift A, Meyer H, Rieddel E, et al. Theflutnce of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzymes. Plant Sci,1998,136:43-57
Song Y., Lu S.H., Yuan Y.J. Cerium elicitor-induced phosphatidic acid triggers apoptotic signaling development in Taxus cuspidata cell suspension cultures. Chemistry and Physics of Lipids,2009, 159:13-20
Spangenberg G, Wang ZY, Wu X.L, et al. Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. Plant Physiol. 1995,145:693-701.
Tae J. L., Randall W.S., Linda H.B. et al. Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis. Plant molecular biology reporter,2004,22(9):259-267
Testerink C, Munnik T. Phosphatidic acid:a multifunctional stress signaling lipid in plants. Trends Plant Sci,2005,10(8):368-375.
Teun Munnik. Phosphatidic acid:an emerging plant lipid second messenger. TRENDS in Plant Science, 2001,6(5):227-233
Wang Z.Y., Ye X.D, Nagel J. Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue plants. Plant Cell Report,2001,20:213-219.
Yamada A, Shibuya N, Kodama O. et al. Induction of phytoalexin formation in suspension-cultured rice cells by n-acetylchitooligo saccharides. Biotech Biochen,1993,57(3):405-409
Yoshio M., Yoshioka H., Noriyuki D. An autophotographic determination of the active oxygen generation in potato tuber discs during hypersensitive response to fungal infection or elicitor. Plant Science,1995,105:45-52
Zhang H.K., Zhang X, Li Q, et al. Alpha-picolinic acid activates diverse defense responses of salicylic acid-jasmonic acid/ethylene-and Ca2+-dependent pathways in arabidopsis and rice suspension cells.Acta Botanica Sinica,2004,46(10):1200-1205
Zhang W, Wang C, Qin C, et al., The oleate-stimulated phospholipase D, PLDδ, and phosphatidic acid decrease H202- induced cell death in Arabidopsis. Plant Cell,2003,15(10):2285-2295.
曾富华,王永刚,胡木林,等.水稻病程相关蛋白的诱导和测试.西北大学学报(自然科学版)2002,32：17-21
曾富华,吴岳轩,罗泽民,等.活性氧及其清除剂诱导抗病作用与影响膜脂过氧化作用的关系.中国农业科学,2000,33(4)：103-105
曾富华,易克,徐向丽,等.不同诱导处理后水稻悬浮细胞的活性氧变化与有关酶系的关系.作物学报.2005,31(1)：18-23
陈志,陈金慧,边黎明,等.杂交鹅掌楸胚性细胞悬浮系的建立.分子植物育种,2007,5(1)：137-140
仇晴川,杨代常.分子医药农业研究进展.武汉植物学研究,2009,27(1)：83-89.
方文娟.日本结缕草(Zoysia japonica Steud.)悬浮再生体系的建立与超低温保存研究.北京：北京林业大学,2008
方文娟,韩烈保.曾会明.植物细胞悬浮培养影响因子研究.生物技术通报,2005,(5)：1]15
古桥胜久,谷田靛道彦.稻节愈伤组织的无限生长培养.水稻的组织培养.上海科学技术出版社,1981,1-3.
和七一,余晓东.胡蜂蜂毒肽Mastoparan的研究概况.重庆师范大学学报(自然科学版),2007,24(1)：76-80
胡燕梅,郭云贵,蒋细旺,等.甘蔗及天然有机物对银杏愈伤组织合成黄铜的影响.西南农业学报.2010.23(3)
贾勇炯,陈放,李英,等.从水稻根部悬浮培养细胞分离原生质体及植株再生,生物工程学报, 1994,10(2)：168-172
蒋细良,朱昌雄,王慧敏,等.中生菌素对水稻悬浮细胞苯丙氨酸解氨酶基因转录表达的影响.中国农业科学.2005,38(7)：1353-1357
金坚敏,张静兰.悬浮培养下水稻的胚状体高频诱导.植物学报,1992,34(2)：114-120
井鑫,张兴国.植物生物反应器研究进展.西南农业大学学报(社会科学版),2004,2(4)：109-112
李红玉,吴健胜,宋从凤,等.水稻IR26悬浮细胞与白叶枯病菌互作的过敏反应现象.植物病理学报.1999,29(4)：299-303
李景梅,王媛,王巍,等.三溴偶氮胂分光光度法测定钙.冶金分析,2004,24(5)：53-55
李霞,陈婷,周月兰.籼粳稻成熟胚愈伤组织培养力的比较,南京师范大学学报(自然科学版),2005,28(4)：103-108
李霞,焦德茂,戴传超.转PEPC基因水稻对光氧化逆境的适应.作物学报,2005,31(4)：408-413
李霞,焦德茂.转C4光合基因水稻及在育种中的应用.分子植物育种,2005,3(4)：550-556
李霞,赵贇涛,阎丽娜,周月兰.籼稻扬稻6号和粳稻苏沪香粳花药培养力的比较.南京师大学报(自然科学版),2008,31(4)：108-113
李霞.籼稻扬稻6号和粳稻苏沪香粳花药培养力的比较,南京师大学报(自然科学版),2008,31(4)：108-113
吕孟雨,赵和,王海波.水稻愈伤组织生长速率研究.中国农学通报.2005,21(12)
孟威,文景芝,吴茂森,等.两种黄单胞病菌诱导水稻一氧化氮产生和防卫基因表达的比较研究.中国农业科学,2007,40(6)：1159-1165
彭程,肖涛,罗远明,等.I G F-1对I L-1诱导的兔关节软骨细胞NO和PGE2的影响.中南大学学报,2008,33(3)：197-203
彭建,王丹英,徐春梅,等.钼酸铵法测定水稻过氧化氢酶活性.中国农学通报.2009,25(16)：61-64
乔建军,赵红,牛晋阳,等.过氧化氢作用下东北红豆杉悬浮培养细胞中蛋白表达差异的研究.植物病理学报,2003,33(5)：429-433
石丽君,王春安.新霉素抑制PC12细胞烟碱受体介导的反应.生理学报,2002,54(5)：441-445.
宋颖琦,杨谦.磷脂酶在植物逆境胁迫信号传导中的作用.哈尔滨工业大学学报,2006,38(2)：243-246
孙亮庆,王春连,冯斗,等.利用水稻悬浮细胞系检测白叶枯菌致病菌株的研究.作物杂志, 2008,5：24-27
覃建兵,汪越胜,何光源.水稻悬浮培养诱导生成植保素的研究.应用与环境生物学报,2006,12(3)：322-324
田敏,饶龙兵,李纪元.植物细胞中的活性氧及其生理作用.植物生理学通讯,2005,41(2)：235-241
王超,侍福梅,活性氧与一氧化氮在逆境胁迫下的相互关系.安徽农业科学,2009,37(5)：1903-1904,1908
王睿辉,陈耀锋,高秀武,等.激素对小麦幼胚胚性无性系高频率诱导的影响.西北农林科技大学学报,2001,29(1)：33-36
王亚馥,崔凯荣,陈克民,等.小麦幼胚培养中体细胞胚发生和植株再生.植物学通报,1992,9(增刊)：2
王炎钦,董海涛.磷脂酸在植物中的第二信使功能.中国生物化学与分子生物学报,2006,22(9)：697-703
王忠安.水稻佳辐占成熟种胚培养及其悬浮细胞系的建立.厦门大学学报(自然科学版),2005,6(44)：180-181
吴春霞.植物细胞悬浮培养的影响因素.安徽农业科学,2009,37(1)：36-38
吴茂森,田峰,齐放军,等.水稻与白叶枯病菌互作的基因表达谱的分析和差异性表达基因的识别.中国农业科学,2007,40(2)：277-282
吴学友,孙立平,刘丽霞,等PLC/PLD抑制剂对盐胁迫下玉米幼苗根系新的69.5 kD热稳定蛋白表达调控的影响.植物生态学报.2005,29(3)：510-515
向太和,梁海曼,钟华鑫,等.水稻胚性悬浮细胞系建立过程中生理生化变化Ⅱ氨基酸、多胺及内源激素的变化.作物学报.1997,23(3)：353-359
向太和,杨剑波,吴家道.水稻、玉米胚性悬浮细胞系的有效建立.安徽农业科学,1996,24(1)：1-8
向太和,杨剑波,吴李君,等.水稻胚性悬浮细胞系建立的细胞学研究.安徽农业科学.1993,21(1)：1-6
向太和,钟花鑫,梁海曼,等.水稻胚性悬浮细胞系建立过程中的生理生化变化.作物学报,1995,21(2)：223-229
邢登辉,吴琴生,刘大钧,禾谷类作物细胞培养——体细胞胚性潜力的诱导与表达.生物学通报,1994,29(7)：1-3
徐林林,芦笛,陆巍,等.水稻悬浮细胞系的建立及培养条件对生物产量的影响.植物生理学通讯.2006,42(4)：612-616
徐勤虎,董树亭,高荣岐.外源果糖、葡萄糖与一氧化氮供体(SNP)对玉米种子萌发的影响.玉米科学,2008,16(3)：74-77,
杨跃生,简玉瑜,郑迎冬,铜在水稻愈伤组织培养再生植株中的促进作用.中国水稻科学.1999,13：95-98.
叶玲娟.相思树的细胞培养及体胚发生研究.福建,福建农林大学,2008.
尹庆良,刘世强.水稻细胞悬浮系及其单细胞培养的研究.沈阳农业大学学报,1994,25(4)：366-372
於凤安,申宗坦.水稻悬浮培养细胞在固体和液体培养下的形态发生.安徽农业大学学报,1994,21(2)：157-169
于惠敏,侯丙凯,番茄基因工程及生物反应器研究与开发进展.山东农业科学,2007,2：21-25
于晓红,朱祯,付志明,等.提高小麦愈伤组织分化频率的因素.植物生理学报,1999,25(4)：388-394.
袁金玲,张朵,顾小平,等.孝顺竹种胚愈伤组织悬浮培养条件优化.分子植物育种,2009,7(4)：839-844
袁力勇,李绍清,李阳生,等.水稻悬浮细胞系的建立.云南大学学报,2003,25(4)：373-376
袁苑,武玉叶,李德全.植物甘油二酯激酶(DGK)信号转导作用.植物生理学通讯,2007,43(6)：1009-1014
章文华,王学敏.植物体内磷脂代谢和信号转导Chinese bulletin of life sciences.2000.12(3): 100-104
赵建聪,江婷,李维.利用植物生物反应器表达胸腺素α1的研究.四川师范大学学(自然科学版),2009,32(1)：112-117
朱根发,余毓君.爪哇稻原生质体培养与植株再生.遗传,1995,17(4)：21-24