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作物学报 ›› 2016, Vol. 42 ›› Issue (04): 600-608.doi: 10.3724/SP.J.1006.2016.00600

• 研究简报 • 上一篇    下一篇

导入外源玉米C4NADP-ME基因对小麦光合效能的影响

王永霞1,2,杜新华1,2,许为钢1,2,*,齐学礼2,李艳2,王会伟2,胡琳2   

  1. 1南京农业大学作物遗传与种质创新国家重点实验室, 江苏南京 210095; 2河南省农业科学院小麦研究所 / 河南省小麦生物学重点实验室, 河南郑州 450002
  • 收稿日期:2015-10-12 修回日期:2016-01-11 出版日期:2016-04-12 网络出版日期:2016-01-25
  • 通讯作者: 许为钢, E-mail: xuwg1958@163.com, Tel: 0371-65712307
  • 基金资助:

    本研究由国家转基因生物新品种培育科技重大专项(2011ZX08002-003)和国家现代农业产业技术体系建设专项(CARS-03-03B)资助。

Photosynthetic Characteristics of Transgenic Wheat Expressing Maize C4-Type NADP-ME Gene

WANG Yong-Xia1,2,DU Xin-Hua1,2,XU Wei-Gang1,2,*,QI Xue-Li2,LI Yan2,WANG Hui-Wei2,HU Lin2   

  1. 1 Nanjing Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; 2 Henan Provincial Laboratory of Wheat Biology / Wheat Research Institute , Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
  • Received:2015-10-12 Revised:2016-01-11 Published:2016-04-12 Published online:2016-01-25
  • Contact: 许为钢, E-mail: xuwg1958@163.com, Tel: 0371-65712307
  • Supported by:

    This study was supported by the National GMO Program of China (2011ZX08002-003) and the China Agriculture Research System (CARS-03-03B).

摘要:

为研究玉米C4NADP-ME基因对小麦光合特性的影响, 以T3代转NADP-ME基因小麦株系10T(9)-1-1和10T(9)-225-4及其对照周麦23为试材, 进行了分子特征鉴定、光合生理特性分析和单株产量性状调查, 并对其作用机制进行了初步解析。结果表明, 外源基因已整合到转基因小麦的基因组中, 并能正确转录和翻译;与对照周麦23相比, 两个转基因株系在4个测定时期旗叶NADP-ME酶活性均明显提高, 而旗叶净光合速率(Pn)则明显下降;尤其在花后第7天, 两个转基因株系的酶活性较对照分别提1.33倍和1.13倍, Pn分别较对照下降17.26%和10.35%;千粒重和籽粒产量较对照均显著下降。与对照周麦23相比, 转基因株系10T(9)-225-4利用强光和同化CO2能力下降, 光合效率下降;旗叶气孔张开率和气孔导度均显著下降;苹果酸含量降低了5.6%, 丙酮酸含量则提高了17.1%;外施苹果酸可恢复其光合速率。以上研究结果表明, 玉米C4型NADP-ME基因的导入降低了小麦的光合特性, 苹果酸含量降低引起的气孔导度下降可能是导致转基因小麦光合效能降低的原因。

关键词: 转基因小麦, NADP苹果酸酶, 净光合速率, 气孔导度

Abstract:

To explore the physiological characteristics of the transgenic wheat expressing maize C4-type NADP-ME, we introduced NADP-ME into the C3 crop wheat by using particle bombardment transformation. Two transgenic wheat lines (10T(9)-1-1, 10T(9)-225-4) and parental control (Zhoumai 19)were used to study molecular characteristics and photosynthesis property, to reveal the mechanism. The results showed that the NADP-ME sequence was integrated into wheat genome, and the transcription and translation were exactly same as expect. The enzyme activity of NADP-ME in flag leaf in transgenic plants were increased significantly than untransformed plants, for instance it was increased 1.33 and 1.13 times on the 7th day after flowering. Net photosynthetic rate (Pn) of flag leaf in transgenic plants obviously decreased when compared to the untransformed plants. On the 7th day after anthesis, Pn of transgenic wheat decreased by 17.26% and 10.35%. The yield and 1000-grain weight were decreased than the control. Utilization efficiency on strong light utilizing and ability of CO2 assimilation in transgenic line 10T(9)-225-4 were significantly declined, photosynthesis rate was also decreased. stomatal opening rate and stomatal conductance were significant decrease, malic acid content of transgenic wheat reducing 5.6% while pyruvate level is raised by 17.1%, and Pn of transgenic wheat can be restored by feeding with exogenous malate. Those results indicated that the transgenic wheat expressing maize NADP-ME gene showed lower photosynthetic characteristics than the control, the reason was maybe the decrease of stomatal aperture caused by decline of malic acid content.

Key words: Transgenic wheat, NADP-dependent malic enzyme, Net photosynthetic rate, Stomata conductance

[1]Hatch M D. C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophy Acta, 1987, 895: 81–106

[2]Agarie S, Miura A, Sumikura R, Tsukamoto S, Nose A, Arima S, Matsuoka M, Miyao-Tokutomi M. Overexpression of C4 PEPC caused 2-insensitive photosynthesis in transgenic rice plants. Plant Sci, 2002, 162: 257–265

[3]Miyao M, Masumoto C, Miyazawa S I, Fukayama H. Lessons from engineering a single-cell C4 photosynthetic pathway into rice. J Exp Bot, 2011, 62: 3021–3029

[4]Ruan C J, Shao H B, Teixeira da Silva J A. A critical review on the improvement of photosynthetic carbon assimilation in C3 plants using genetic engineering. Crit Rev Biotechnol, 2012, 32: 1–21

[5]Ku M S, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Matsuoka M. High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol, 1999, 17: 76–80

[6]Ku M S, Ranade U, Hsu T P, Cho D, Li X, Jiao D M, Ehleringer J, Miyao M, Matsuoka M.. Photosynthetic performance of transgenic rice plants overexpressing maize C4 photosynthesis enzymes. Stud Plant Sci, 2000, 7: 193–204

[7]Fukayama H, Hatch M D, Tamai T, Tsuchida H, Sudoh S, Furbank R T, Miyao M. Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosyn Res, 2003, 77: 227–239

[8]陈绪清, 张晓东, 梁荣奇, 张立全, 杨凤萍, 曹鸣庆. 玉米C4型pepc基因的分子克隆及其在小麦的转基因研究. 科学通报, 2005, 49: 1976–1982

Chen C Q, Zhang X D, Liang R Q, Zhang L Q, Yang F P, Cao M Q. Cloning maize C4 phosphoenolpyruvate carboxylase gene and transformation in wheat. Chin Sci Bull, 2005, 49: 1976–1982 (in Chinese with English abstract)

[9]Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S, Miyao M, Datta S K. Enhanced photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize. Plant Sci, 2007, 172: 1204–1209

[10]张彬, 丁在松, 张桂芳, 石云鹭, 王金明, 方立锋, 郭志江, 赵明. 根癌农杆菌介导获得稗草 Ecppc转基因小麦的研究. 作物学报, 2007, 33: 356–362

Zhang B, Ding Z S, Zhang G F, Shi Y L, Wang J M, Fang L F, Guo Z J, Zhao M. Introduction of phosphoenolpyruvate carboxylase gene from Echinochloa crusgalli into wheat mediated by Agrobacterium tumefaciens. Acta Agron Sin, 2007, 33: 356–362 (in Chinese with English abstract)

[11]张庆琛, 许为钢, 胡琳, 李艳, 张磊, 齐学礼. 玉米C4型全长pepc基因导入普通小麦的研究. 麦类作物学报, 2010, 30: 194–197

Zhang Q C, Xu W G, Hu L, Li Y, Zhang L, Qi X L. Development of transgenic wheat plants with maize C4-specific pepc gene by particle bombardment. J Triticeae Crops, 2010, 30: 194–197 (in Chinese with English abstract)

[12]李艳, 许为钢, 胡琳, 张磊, 齐学礼, 张庆琛, 王根松. 玉米磷酸烯醇式丙酮酸羧化酶基因高效表达载体构建及其导入小麦的研究. 麦类作物学报, 2009, 29: 741–746

Li Y, Xu W G, Hu L, Zhang L, Qi X L, Zhang Q C, Wang G S. Construction of a high efficient expression vector for maize phosphoenolpyruvate carboxylase gene and its transformation in wheat. J Triticeae Crops, 2009, 29: 741–746 (in Chinese with English abstract)

[13]Hu L, Li Y, Xu W, Zhang Q, Zhang L, Qi X, Dong H. Improvement of the photosynthetic characteristics of transgenic wheat plants by transformation with the maize C4 phosphoenolpyruvate carboxylase gene. Plant Breed, 2012, 131: 385–391

[14]Sheriff A, Meyer H, Riedel E, Schmitt J M, Lapke C. The influence of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzyme. Plant Sci, 1998: 136: 43–57

[15]Ding Z S, Huang S H, Zhou B Y, Sun X F, Zhao M. Overexpression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields. Plant Biotechnol Rep, 2013, 7: 155–163

[16]Zhang H F, Xu W G, Wang H W, Hu L, Li Y, Qi X L, Zhang L, Li C X, Hua X. Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat. Protoplasma, 2014, 251: 1163–1173

[17]Ku M S, Wu J, Dai Z, Scott R A, Chu C, Edwards G E. Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol, 1991, 96: 518–528

[18]Häusler RE, Rademacher T, Li J, Lipka V, Fischer KL, Schubert S, Kreuzaler F, Hirsch HJ. Single and double overexpression of C4-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants. J Exp Bot, 2001, 52: 1785–1803

[19]Huang X Q, Jiao D M, Chi W, Ku M S B. Characteristics of CO2 exchange and chlorophyll fluorescence of transgenic rice with C4 genes. Acta Bot Sin, 2002, 44: 405–412

[20]Takeuchi Y, Akagi H, Kamasawa N, Osumi M, Honda H. Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme. Planta, 2000, 211: 265–274

[21]Tsuchida H, Tamai T, Fukayama H, Agarie S, Nomura M, Onodera H, Ono K, Nishizawa Y, Lee B, Hirose S. High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth in a C3 plant, rice. Plant Cell Physiol, 2001, 42: 138–145

[22]Chi W, Zhou J, Zhang F, Wu N. Photosynthetic features of transgenic rice expressing sorghum C4 type NADP-ME. Acta Bot Sin, 2004, 46: 873–882

[23]Laporte M M, Shen B, Tarczynski M C. Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function. J Exp Bot, 2002, 53: 699–705

[24]王玉民. 玉米C4途径关键酶基因(PPDK、NADP-ME)的克隆及PPDK、PEPC在拟南芥中的表达分析. 河南农业大学博士学位论文, 河南郑州, 2011. pp 22–47

Wang Y M. Molecular Cloning of Maize C4 Key Enzyme Genes (PPDK and NADP-ME) and Expression Analysis of PPDK and PEPC in Arabidopsis. PhD Dissertation of Henan Agricultural University, Zhengzhou, China, 2011. pp 22–47 (in Chinese with English abstract)

[25]杜西河. 玉米PEPC、PPDK和NADP-ME基因在拟南芥中的表达分析. 河南农业大学硕士学位论文, 2013. pp 17–21

Du X H. Expression Analysis of Maize C4 Key Enzyme (Phosphoenolpyruvate Carboxylase, Pyruvate Orthophosphate Dikinase and NADP-Malic Enzyme) Genes in Arabidopsis. MS Thesis of Henan Agricultural University, Zhengzhou, China, 2013. pp 17–21 (in Chinese with English abstract)

[26]陈昆松, 李方, 徐昌杰, 张上隆, 傅承新. 改良CTAB法用于多年生植物组织基因组DNA的大量提取. 遗传, 2004, 26: 529–531

Chen K S, Li F, Xu C J, Zhang S L, Fu C X. An efficient macro-method of genomic DNA isolation from Actinidia chinensis leaves. Hereditas (Beijing), 2004, 26: 529–531 (in Chinese with English abstract)

[27]王兰兰, 何兴元, 陈玮, 李雪梅. 大气中O3、CO2浓度升高对蒙古栎叶片生长的影响. 中国环境科学, 2011, 31: 340–345

Wang L L, He X Y, W Chen, Li X M. Effects of elevated O3 or/and CO2 on growth in leaves of Quercus mongolica. China Environ Sci, 2011, 31: 340–345 (in Chinese with English abstract)

[28]Boehringer M G. Methods of biochemical analysis and food analysis. Boehringer Mannheim GmbH, Biochemica, Mannheim, 1989

[29]李琳, 李光兴, 代庆伟. 比色法快速分析苯丙酮酸含量的研究. 化学试剂, 2002, 24(1): 22–23

Li L, Li G X, Dai Q W. Analysis of the content of phenylpyruvic acid in mixture by colorimetry. Chem Reagents, 2002, 24(1): 22–23

[30]Jiao D, Huang X, Li X, Chi W, Kuang T, Zhang Q, Ku M S, Cho D. Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes. Photosyn Res, 2002,72: 85–93

[31]Sarah C, Julian M H. Integrating C4 photosynthesis into C3 crops to increase yield potential. Plant Biotechnol, 2012, 6: 1004

[32]Chen G Y, Ye J Y. Effects of oxaloacetate and malate on photosynthesis in leaves and in intact chloroplasts from spinach. Acta Phytophysiol Sin, 2001, 27: 478–482

[33]朱素琴, 季本华, 焦德茂. 外源C4二羧酸对转玉米PEPC基因水稻C4光合途径的促进作用. 中国水稻科学, 2004, 18: 326–332

Zhu S Q, Ji B H, Jiao D M. Promotive effect of exogenous C4-bicarboxylate on photosynthetic C4 pathway in transgenic rice plant expressing maize specific PEPC gene. Chinese J Rice Sci, 2004, 18: 326–332
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