摘要
光合作用是植物生物学产量的源泉,通过基因工程手段人为加强“C4特性”在C3遗传背景下的发挥,实现C3植物光合途径的C4化,对于提高作物光能利用率和产量具有重要意义。本试验从酶活和光合均高的玉米自交系中克隆了玉米C4型丙酮酸磷酸二激酶(PPDK)和依赖于NADP的苹果酸酶(NADP-ME)基因,并利用PPDK和本实验室先前克隆的PEPC基因(GeneBank:FJ415327)转化拟南芥,研究了这两个基因的单独及协同作用,通过其生理生化方面的变化,为C3作物光合效率的改善提供了候选基因,为C4途径关键酶导入小麦等C3作物提供了依据。
主要结论如下:
1.利用同源克隆技术,从玉米中克隆了PPDK(GenBank:GU363532)和NADP-ME基因的cDNA,并插入双元表达载体pCAMBIA3301.pCAMBIA1391中,构建了p3301-PPDK、 p3301-ME和p1391-ME融合表达载体。序列测定结果表明,PPDK基因长度为3004Bp,包含一个起始密码子和971个氨基酸的开放阅读框,与己登陆的玉米PPDK基因(GeneBank: BT054438)相比,氨基酸序列同源性可达99.4%;NADP-ME基因长度为2127Bp,包含一个起始密码子和636个氨基酸的开放阅读框,与已登陆的玉米NADP-ME基因(GenBank: NM_001111843.1)相比,氨基酸序列同源性可达99.6%。
2.Southern blot表明玉米PPDK、PEPC基因已整合到拟南芥基因组中,拟南芥转化率为0.69%,双基因共转化率为0.07%,66.7%的双基因拟南芥符合Mendel单基因的遗传规律。利用农杆菌介导法将p3301-PPDK和本实验室曾构建的p3301-PEPC高效表达载体导入拟南芥中,筛选到327株抗性转基因拟南芥,其中PEPC株系125株,PPDK株系175株,PPDK、 PEPC共表达株系12株,转p3301株系15株,筛选到PPDK、PEPC、PPDK+PEPC、p330.纯系各24、26、8、3个。
3.RT-PCR、荧光定量PCR、Western blot分析表明外源基因在拟南芥基因组中得到正确表达,且转PPDK、PEPC基因植株的表达量存在较大差异。荧光定量PCR分析现蕾期PPDK、 PEPC基因的相对表达量分别在1.0-4.2倍和1.0-13.3倍;用Quantity One软件对Western blot进行定量分析表明,PPDK、PEPC基因的蛋白相对表达量分别在1.9-2.5倍和1.6-3.7倍。
4.转单、双C4关键酶基因均可以提高拟南芥光合速率,双基因的平均光合速率高于转PEPC基因的平均光合速率,转PEPC基因的平均光合速率高于转PPDK基因的平均光合速率。T3代转基因拟南芥酶活和光合速率测定表明:T3代转基因拟南芥PPDK、PEPC酶活分别比对照增加0.61-3.63倍和0.77-4.81倍,平均增加1.89和1.48倍;转PPDK基因拟南芥光合速率增加3.3-25.3%,平均增加12.2%,转PEPC基因拟南芥光合速率增加6.5-35.1%,平均增加18.9%,双基因(PPDK+PEPC)拟南芥光合速率增加12.6-45.7%,平均增加26.3%。
5.转单个C4关键酶基因加速了C4微循环。T4代转基因拟南芥酶活测定表明:PK17-1-2、 PK26-3-2的PEPC酶活分别比对照增加18.65%和46.37%,与对照差异极显显著;PC65-4-6、PC73-1-3的PPDK酶活分别比对照增加12.96%和26.54%,与对照差异极显著,说明转PPDK基因拟南芥可引起PEPC活性增加,反之亦然;T4代转基因拟南芥光合测定表明:PK17-1-2、 PK26-3-2、PC65-4-6、PC73-1-3的光合速率分别比对照增加了13.14%、23.29%、18.14%、36.44%,达极显著水平。
6.PPDK、PEPC共表达有协同效应。双基因株系PKC6-5-1的光合速率比PK26-3-2和PC73-1-3的高,但是PKC6-5-1的PEPC酶活比PC73-1-3的低,PKC6-5-1的PPDK酶活比PK26-3-2的低;双基因株系PKC17-1-3的光合速率比PK17-1-2和PC65-4-6的高,但PKC6-5-1的PPDK和PEPC酶活偏低。
ABSTRACT:The efficiency of photosynthesis is related with the crop biomass, since the harvest index and leaf area index for many crops, such as wheat and rice, is approaching a ceiling value, it has been a focus that an increase in yield potential should come from improved photosynthesis. According to differences of mechanism in carbon assimilation pathways in photosynthesis, three major photosynthetic types may be distinguished among green plants:C3, C4and Crassulacean acid metabolism plants. C4plants were evolved from C3plants, they adapt to high light, high tempretaure, lower CO2concentration and achieve higher photosynthetic capacity compared with C3plants(Black,1973). Consequently, the transfer of C4enzyme to C3plants such as rice and wheat is one strategy being adopted for improving their yield. In this experiment, PPDK with an ORF of971amino acids was isolated from maize self lines Z1194and was introduced to Arabidopsis with PEPC, systematical studies on their transcription, translation, eneyzme activities and photosynthetic performences were conducted, which could offer candidate gene and theory for engineering the C4photosynthetic pathway into C3crops such as wheat.
The results were as follows:
1. The full-length cDNA for PPDK and NADP-ME were isolated from Zea mays by homology-based cloning technology. The full-length PPDK and NADP-ME cDNA replaced the (3-glucuronidse(GUS) coding region of the pCAMBIA3301(p3301) or pCAMBIA1391(p1391). The positive clone was named p3301-PPDK, p3301-ME and p1391-ME, respectively. In this study, we have obtained a3004bp cDNA fragment of PPDK from maize Z1194(Zea mays L.), PPDK has a2916bp open reading frame (ORF) and encodes971amino acids with a calculated molecular mass of105kDa polypeptide(GenBank accession number:GU363532). Comparison of GU363532to the C4PPDK gene (GenBank accession number:BT054438) by DNAMAN indicated99.4%similarity between the amino acid sequences. We have also obtained a2127bp cDNA fragment of NADP-ME from maize Zl194, containing one ORF with1911bp which coded a peotide of636amino acids. The identity of the cloned fragment with NADP-ME gene from NM_001111843.1was99.4%by homology alignment with software DNAMAN.
2. The integration of maize PPDK and PEPC gene in transgenic Arabidopsis plants was confirmed by Southern blotting analysis, the transgentic rate and co-transgentic rate was0.69%and9.5%, the ratio of linked-inheritance was66.7%. Arabidopsis plants were transformed by the floral dipping method with tumefaciens GV3101, including p3301-PPDK, p3301-PEPC, p3301-PPDK+p3301-PEPC(PPDK:PEPC was3:2), and p3301. Based on the segregation pattern of the transgenic seedlings by Basta-tolerant and PCR assay. A total of327transgentic plants were obtained, plants containing the PPDK gene, the PEPC gene, the PPDK+PEPC gene, or GUS gene were separated into their respective groups, indicating the following distribution of lines:twenty-four homogenous PPDK lines from175PPDK transgentic lines, twenty-six homogenous PEPC lines from125PPDK transgentic lines, eight homogenous lines linked inheritance with both PPDK and PEPC from12double transgentic lines, and three homogenous p3301lines from15transgentic lines of p3301.
3. Analyses by RT-PCR, quantitative real-time PCR and Western blotting all confirmed the effective expression of maize C4-PPDK and C4-PEPC in Arabidopsis. The expression levels of PPDK or PEPC among different transgenic lines varied, which ranged from1-to4.24-fold and1-to13.33-fold by quantitative real-time PCR, respectively. Volume analysis by software Quantity One indicated that the expression levels of PPDK were ranged from1.9-to2.5-fold relevant to the NegCtrl, the expression levels of PEPC were ranged from1.6-to3.7-fold relevant to the NegCtrl, respectively.
4. The photosynthetic performance could be improved by PPDK or PEPC alone, and what's more, Pn (PKC)> Pn (PC)> Pn(PK). In this study, the enzyme activities and the Pn of T3transgentic plants were determined.The enzyme activity of C4-PPDK and C4-PEPC were elevated by0.61-3.63and0.77-4.81times relevant to the controls, the average value was1.89and1.48folds, respectively. The Pn of Ca-PPDK, C4-PEPC and PPDK+PEPC transgenic lines were higher than those of the controls by3.3-25.3%,6.5-35.1%and12.6-45.7%.
5. Tthe introduction of C4-PPDK or C4-PEPC into Arabidopsis could contribute to the strengthen of C4-mini cycle. In this study, the enzyme activities of T4transgentic Arabidopsis were determined. The PEPC enzyme activities of PK17-1-2and PK26-3-2were18.65%and46.37%higher than those of the controls, respectively; the PPDK enzyme activities of PC65-4-6and PC73-1-3were12.96%and26.54%higher than those of the controls, respectively, the PPDK and PEPC enzyme activities could significantly increased by transgentic PPDK and PEPC Arabidopsis. The Pn of PK17-1-2, PK26-3-2, PC65-4-6and PC73-1-3were13.14%,23.29%,18.14%and36.44%higher than those of the controls, respectively.
6. The coexpression of PPDK and PEPC had synergistic effect on photosynthesis rate. To our interest, the Pn of the line PKC6-5-1were the highest among the T4transgentic Arabidopsis, however, its PPDK enzyme activities or its PEPC enzyme activities were not the highest, although the PPDK or PEPC eneyzme activities of the line PKC17-1-3was the lowest among the T4transgentic Arabidopsis, the Pn of the line PKC17-1-3were higher than those of the line PK17-1-2and PC65-4-6.
The results show that both are candidate functional genes in the improvement of Pn of C3Arabidopsis and coexpression of C4-PPDK and C4-PEPC genes had synergistic effects on Pn, which could be of great importance in the agricultural field, especially when considering the limited methods that we currently have in improving crop yields.
引文
1. Agarie S, Miura A, Sumikura R, Tsukamoto S, Nose A, Arima S, Matsuoka M, Miyao M (2002) Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plant. Plant Sci,162:257-265
2. Amsterdam (1985) Photosynthetic Mechanisms and the Environment, New York/Oxford, Barber J and Baker,329-387
3. Andrews M(1986) The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ.9:511-519
4. Aoyagi K, Bassham JA (1984) Pyruvate orthophosphate dikinase of C3 seeds and leaves as compared to the enzyme from maize. Plant Physiol,75:387-392
5. Badger MR, Price GD (1992) The CO2 concentrating mechanism in cyanobacteria and microalgae. Physiologia Plantarum,84,606-615
6. Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S, Miyao M, Datta SK (2007) Enhanced photosynthesis rate in genetically engineered indica rice expressing PEPC gene cloned from maize. Plant Science,172:1204-1209
7. Bemhard J, Eikmanns, Max T et al (1989) The phosphoenolpyruvate carboxlase gene of Corynebacterium glutamicum:Molecular cloning, nucleotide seguence, and expression. Mol Gen Genet,218:330-339.
8. Boller T and Meins F (1992) Plant Gene research:genes involved in plant defense, New York, Springer-Verlag,327-352
9. Bowes G, Salvucci ME (1989) Plasticity in the photosynthetic carbon metabolism of submerged aquatic macrophytes. Aquatic Botany,34:233-266.
10. Calvin M, Benson AA(1948) The path of carbon in photosynthesis. Science,107: 476-480
11. Casati P, Lara MV, Andreo CS (2000) Induction of a C4-like mechanism of CO2 fixation in Egeria densa, a submerged aquatic species. Plant Physiol,123:1611-1621
12. Casati Paula, Andreo Carlos S, Edwards GE (1999) Characterization of NADP-malic enzyme from two species of Chenopodiaceae:Haloxylon persicum (C4) and Chenopodium album (C3), Phytochemistry,52:985-992
13. Cheng SH, Keefe D, Mets LJ, Ku MSB (1988) Metabolism of carbon-14 carbon dioxide and intercellular compartmentation of photosynthetic enzymes in reciprocal F1 hybrids between C3-C4 intermediate and C4-like flaveria species. Plant Physiology (Rockville):86
14. Chi Wei, Zhou JingSong, Zhang Fang, Wu Naihu (2004) Photosynthetic features of transgenic rice expressing sorghum C4 type NADP-ME. Acta Botanica Sinica,46 (7): 873-882
15. Chollet R, Vidal J and O'Leary MH (1996) Phosphoenolpyruvate carboxylase:a ubiquitous, highly regulated enzyme in plants. Annu. Rev. Plant Biol,47:273-298
16. Chung MH, Chen MK, Pan SM (2000) Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res.9 (6):471-6.
17. Cotelle V, Pierre JN, Vavasseur A (1999) Potential strong regulation of guard cell phosphoenolpyruvate carboxylase through phosphorylation Journal of Experimental Botany 50:777-783
18. Cousins AB, Adam NR, Wall G W, Kimball BA, Pinter PJ, Ottman MJ(2003). Development of C4 photosynthesis in sorghum leaves grown under free-air CO2 enrichment (FACE), Journal of Experimental Botany,54(389):1969-1975
19. Cretin C, Keryer E, Tagu D, Lepiniec L, Vidal J and Gadal P (1990) Complete cDNA sequence of sorghum phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleic Acids Res,18:658
20. Cretin C, Santi S, Keryer E, Lepiniec L, Tagu D, Vidal J and Gadal P (1991) The phosphoenolpyruvate carboxylase gene family of sorghum:Promoter structures, amino acid sequences and expression of genes. Gene,99:87-94
21. Dai Z, Ku MSB, Edwards GE (1993) C4 photosynthesis (the CO2-concentrating mechanism and photorespiration) Plant Physiol,103:83-90.
22. Donald CM. In search of yield. J AusAgri Sci,1962,28:171-17
23. Drincovich Maria F., Casati Paula, Andreo Carlos S(2001) NADP-malic enzyme from plants:aubiquitous enzyme involved in different metabolic pathways, FEBS letters, 490,1-6
24. Drincovich MF, Casati P, Andreo CS, Chessin SJ, Franceschi, VR, Edwards GE, Ku MSB. (1998) Evolution of C4 photosynthesis in Flaveria species:isoforms of NADP-malic enzyme. Plant physiology,117:733-744
25. Duffus CM, Rosie R (1973) Some enzyme activities associated with the chlorophy Ⅱ containing layers of the immature barely pericarp. Planta,114:219-226
26. Edwards G. E et al. (1985) Pymvate, Pi dikinase and NADP-malate dehydrogenase in C4 photosynthesis:properties and mechanism of light/dark regulation. Plant Physiol, 36:255-286
27. Fahnenstich H, Saigo M, Niessen M, Zanor MI., Andreo CS, Fernie AR, Drincovich MF, Flugge UI, Maurino VG (2007) Alteration of organic acid metabolism in Arabidopsis overexpressing the maize C4 NADP-malic enzyme causes accelerated senescence during extended darkness. Plant Physiology,145:640-652.
28. Famiani Franco, Walker Robert P., Tecsi Laszlo, et. al (2000) An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries, Journal of Experimental Botany,51(345):675-683
29. Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S, Furbank RT, Miyao M (2003) Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosyn Res,77:227-239
30. Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H, Ono K, Lee BH, Hirose S, Toki S, Ku MSB, Makino A, Matsuoka M and Miyao M (2001) Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiol, 127:1136-1146
31. Gallardo F, Miginiac-Maslow M, Sangwan R, Decottignies P, Keryer E et al (1995) Monocotyledonous C4 NADP-malate dehydrogenase is efficiently synthesized, targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco. Planta,197:324-332
32. Gehlen J, Panstruga R, Smets H, Merkelbach S, Kleines M, Porsch P, Fladung M, Becker I, Rademacher T, Hausler RE, Hirsch HJ (1996) Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum. Plant Mol Biol.,32 (5):831-848
33. Glackin CA, Grula JW (1990) Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize. Proc Natl Acad Sci USA,87:3004-3008
34. Glackin CA, Grula JW (1990) Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize. Proceedings of the National Academy of Sciences, USA,87:3004-3008
35. Goss, NH et al. (1980) Pyruvate phosphate dikinase:sequence of the histidyl peptide, the pyro-phosphoryl and phosphoryl carrier, Biochemistry 19,5805-5809
36. Gupta S K et al. (1994) Light/dark modulation of phosphenolpyruvate carboxylase genes in Flaveria trinercia (C4) and F. pringlei (C3), Photosynthesis Research,42:133-143
37. Hatch MD and Burnell JN (1990) Carbonic anhydrase activity in leaves and its role in the first step of C4 photosynthesis. Plant Physiology 93,825-828
38. Hatch MD and Slack CR (1968) A new enzyme for the interconversion of pyruvate and phosphopyruvate and its role in the C4 dicarboxylic acid pathway of photosynthesis. Biochem. J.,106:141-146
39. Hausler RE, Hirsch HJ, Kreuzaler F, Peterhansel C(2002) Overexpression of C4-cycleenzymes in transgenic C3 plants:a biotechnological approach to improve C3-photosynthesis. J Exp Bot,53:591-607
40. Hausler RE, Rademacher T, Li J, Lipka V, Fischer KL, Schubert S, Kreuzaler F and Hirsc HJ (2001) 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. Journal of Experimental Botany,52:1785-1803
41. Heineke D, Riens B, Grosse H, Hoferichter P, Peter U, Flugge UI, Heldt HW (1991) Redox transfer across the inner chloroplast envelope membrane. Plant Physiol,95: 1131-1137
42. Hermans J and Westhoff P (1990) Analysis of expression and evolutionary relationships of phosphoenolpyruvate carboxylase gene in Flaveria trinervia (C4) and F. pringlei (C3) Mol Gen Genet,224:459-468
43. Herzberg O,Chen CCH,Kapadia G, McGuire M, Carroll LJ, NohSJ, Dunaway-Mariano D (1996). Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites. Proc Natl Acad Sci USA,93:2652-2657
44. Hisatake S,Tetsuya M,Tsutomu K(1984) Molecular cloning of the phosphorenolpyruvate carboxlase gene, ppc of Escherichia coli. Gene,31:279-283
45. Holtum J AM, Winter K(1982) Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L, Planta,1982,155:8-16
46. Honda H, Akagi H, Shimada H (2000) An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on consdfvative amino acid residues. Gene,243:85-92
47. Huang XQ, Jiao DM, Chi W, Ku MSB (2002) Characteristics of CO2 exchange and chlorophyll fluorescence of transgenic rice with C4 genes. Acta Botanica Sinica, 44:405-412
48. Hudspeth RL and Grula JW (1989) Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylase isozyme involved in C4 photosynthesis. Plant Mol Biol,12:579-589.
49. Imaizumi N, Ishihara K, Samejima M, Kaneko S, Matsuoka M (1992) Structure and expression of pyruvate, orthophosphate dikinase gene in C3 plant, In N Murata(ed), Research in Photosynthesis. Kluwer Academic, Dordrecht, The Netherlands:875-878
50. Imaizumi N, Ku MSB, Ishihara K, Samejima M, KanekoS, Matsuoka(1997) Characterization of the gene for pyruvate, orhophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein. Plant Mol Biol,34:701-716.
51. Imaizumi N, Samejima M and Ishihara K (1997)Characteristics of photosynthetic carbon metabolism of spikelets in rice. Photosynthesis Research,52:75-82
52. Ishijima S, Katagiri F, Kodaki T, Izui K, Katsuki H et al. (1985) Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from Escherichia coli (allosteric) and Anacystis nidulans (non-allosteric):identification of conserved and variable regions. Biochem Biophys Res Commun,133:436-441.
53. Ishimaru K, Ichikawa H, Matsuoka M, Ohsugi R (1997) Analysis of a C4 maize pyruvate, orthophosphate dikinase expressed in C3 transgenic Arabidopsis plants. Plant Science, 129:57-64
54. Ishimaru K, Ohkawa Y, Ishige T, Tobias DJ, Ohsugi R(1998) Elevated pyruvate, orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene. Physiologia plantarum,103:340-346
55. Izui K, Ishijima S, Yamaguchi Y, Katagiri F, Murata T, Shigesada K, Sugiyama T and Katsuki H(1986)Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize. Nucleic Acids Res, 14:1615-1628.
56. Izui K, Kawamura T, Okumura S and Toh H(1992)Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize. In:Murata N(ed) Research in Photosynthesis,1986:827-830. Kluwer Academic Publishers, Dordrecht
57. Jenkins(1989)Effect PEPCase inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl) propenoate on photosynthesis. Plant Physiol,89:1231-1237
58. Ji BH, Zhu SQ, Jiao DM (2004) Photosynthetic C4 microcycle in transgenic rice plant lines expressing the maize C4 photosynthetic enzymes. Acta Agron Sin,30:536-543
59. Jiao DM, Huang XQ, Li X et al (2001) Characteristics of carbon assimilation and tolerance to photooxidation in transgenic rice expressing C4 photosynthesis enzymes, in PS2001 Proceedings,12th international congress on photosynthesis, Brisbane, Australia: CSIRO Publishing, S33-004,1-6
60. Jiao DM, Huang XQ, Li X, Chi W, Kuang TY, Zhang QD, Ku MSB, Cho DH (2002) Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes. Photosyn Res,72:85-93
61. Jiao DM, Kuang TY, Li X et al. (2003) Physiological characteristics of the primitive CO2 concentrating mechanism in PEPC transgenic rice. Science in China (Series C),46 (4):438-446.
62. Jiao DM, Li X, Huang XQ, Chi W, Kuang T-Y, Ku M S B (2001)The characteristics of CO2 assimilation of photosynthesis and chlorophyll fluorescence in transgenic PEPC rice. Chin Sci Bull,46:414-418
63. Jiao JA, Podesta FE, Chollet R, O'Leary MH, Andreo CS (1990) Isolation and sequence of an active-site peptide from maize leaf phosphoenolpyruvate carboxylase inactivated by pyridoxal 5'-phosphate. Biochim Biophys Acta,1041:291-295
64. Katagiri F, Tsutomu Kodaki, Katsura Izui (1985) Nucleotide sequence of the phosphoenolpyruvate carboxlase gene of the cyanobacterium Anacystis nidulans. Gene, 38:265-269.
65. Khanna R and Sinha SK (1973) Changes in the predominance from. C4 to C3 pathway in sorghum after anthesis. Biochem Biophys. Res Commun 52:121-124
66. Khush GS(1995). Modern varieties—their real contribution to food supply and equity. Geo J,35:275-284
67. Khush GS(1999). Green revolution:preparing for the 21st century. Genome,42: 646-655
68. Kisaki, T, Hirabayashi, S & Yano, N (1973) Effects of the age of tobacco leaves on photosynthesis and photorespiration. Plant. Cell Physiology.14,505-514.
69. Kogami H, Shono M, Koike T, Yanagisawa S, Izui K, Sentoku N, Tanifuji S, Uchimiya H and Toki S (1994) Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter. Transgenic Research,3:287-296
70. Kortschack HP, Hartt CE, Burr GD (1965) Carbon dioxide fixation in sugarcane leaves. Plant Physiol,40:209-213.
71. Ku MSB, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotech,17:76-80
72. Ku MSB, Cho D, Ranade U, Hsu TP, Li X, Jiao DM, Ehleringer J, Miyao M and Matsuoka M (2000) Photosynthetic performance of transgenic rice plants overexpressing maize C4 photosynthesis enzymes[M]. In:Sheehy JE, Mitchell PL, Hardy B (eds) Redesign rice photosynthesis to improve yield, Elsevier Science/International Rice Research Institute,193-204
73. Ku MSB, Kano Murakami Y, Matsuoka M (1996) Evolution and expression of C4 photosynthesis genes. Plant Physiol,111:949-957
74. Lai LB, Wang L, Nelson TM(2002) Distinct but conserved functions for two chloroplastic NADP-malic enzyme isoforms in C3 and C4 Flaveria species, Plant Physiol,128:125-139
75. Leegood R (2002) C4 photosynthesis:principles of CO2 concentration and prospects for its introduction into C3 plants. J Exp Bot,53:581-591
76. Lepiniec L,Keryer E, Philippe H et al.(1993) Sorghum phosphoenolpyruvate carboxylase gene family:structure, function and molecular evolution. Plant Mol Biol,21 (3): 487-502.
77. Lin Y, Lusin JD, Ye D, Dunaway-Mariano D, Ames JB (2006). Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase. Biochemistry,45:1702-1711
78. Lipka V, Hausler RE, Rademacher T, Li J, Hirsch HJ et al. (1999) Solanum tuberosum double transgenic expressing phosphoenolpyruvate carboxylase and NADP-malic enzyme display reduced electron requirement for CO2 fixation. Plant Sci,144:93-105
79. Logemann E, Birkenbihl RP, Ulker B and Somssich IE (2006) An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol, plant metholds.
80. Lu CG, Zou JS(2002). Super hybrid rice breeding in China. Agric Hort,77:750-775
81. Luinenburg I, Coleman JR (1992) Identification, characterization and sequence analysis of the gene encoding phosphoenolpyruvate carboxlase in Anabaena sp. PCC 7120. Journal of General Microbiology,138:685-691.
82. Makoto Matsuoka and Ei-Ichi Minami(1989) Complete structure of the gene for phosphoenolpyruvate carboxylase from maize Eur. J. Biochem.181,593-598
83. Marshall JS, Stubbs JD, Taylor WC.(1996) Two genes encode highly similar cholroplastic NADP-malic enzymes in Flaveria. Plant Physiol,111:1251-1261
84. Martinoia Enrico, Rentsch Doris(1994) Malate compartmentation:responses to a complex metabolism, Annual Review of Plant Physiology and Plant Molecular Biology, 45:447-467
85. Matsuoka M and Minami E(1989) Complete structure of the gene for phosphoenolpyruvate carboxylase from maize. Eur J Biochem,181:593-598
86. Matsuoka M, Furbank R, Fukayama H, Miyao M (2001) Molecular engineering of C4 photosynthesis. Annu Rev Plant Mol Biol,52:297-314.
87. Matsuoka Makoto (1995) The gene for pyruvate, orthophosphate dikinase in C4 plants: structure, regulation and evolution. Plant Cell Physiol,36 (6):937-943
88. Maurino VG, Drincovich MF, Andreo CS (1996). NADP-malic enzyme isoforms in maize leaves. Biochem Mol Biol Int,38:239-250
89. Merkelbach S, Johanna Gehlen, Martin Denecke et al. (1993) Cloning sequence analysis and exoression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum. Plant Molecular Biology,23:881-888.
90. Meyer AO, Kelly GJ and Latzko E (1982) Pyruvate Orthophosphate Dikinase from the Immature Grains of Cereal Grasses Plant Physiol.69:7-10
91. Miyao M (2003) Molecular evolution and genetic engineering of C4 photosynthetic enzymes. J Exp Bot,54:179-189
92. Miyao M., Masumoto C, Miyazawa SI. and Fukayama H (2011) Lessons from engineering a single-cell C4 photosynthetic pathway into rice. Exp. Bot. doi:10. 1093/jxb/err023
93. Moing A; Rothan C; Svanella L et al. (2000) Role of phosphoenolpyruvate carboxylase in organic acid accumulation during peach fruit development. Physiol. Plant.108:1-10
94. Nutbeam AR and Duffus CM (1976) Evidence for C4 photosynthesis in barley pericarp tissue, Biochemical and Biophysical Research Communications,70:1198-1203.
95. Ohta S, Ishida Y, Usami S(2004). Expression of cold-tolerant pyruvate, orthophosphate dikinase cDNA, and heterotetramer formation in transgenic maize plants. Transgenic Res, 13:475-485
96. Parsley K(2006). The Arabidopsis PPDK gene is transcribed from two promoters to produce differentially expressed transcripts responsible for cytosolic and plastidic proteins. Plant Mol Biol,62:339-349
97. Pinto Manuel E, Casati Paula, Hsu Tsui-Ping et al(1999) Effects of ultraviolet-B light on growth, photosynthesis, flavonoids and NADP-Malic enzyme in bean (Phaseolus vulgaris L.) grown under different nitrogen conditions, Journal of Photochemistry and Photobiology B:Biology,48:200-209
98. Pocalyko D J, Carroll L J, Martin B M, Babbitt P C, Dunaway-Mariano D (1990) Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme of the bacterial phosphoenolpyruvate:sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs. Biochemistry.29 (48):10757-10765
99. Poetsch W, Hermans J, Westhoff P (1991) Multiple cDNAs of phosphoenolpyruvate carboxylase in the C4 dicot Flaveria trinervia. FEBS Lett.292 (1-2):133-136
100. Preiss M, Koopman E, Meye RG., et al(1994) Malate as additional substrate for fatty acid synthesis in a C4-plant type developed by salt stress from a C3 plant type maize:a screening for malate as substrate for fatty acid synthesis in chloroplasts, Journal of plant physiology,143:544-549
101. Rajagopalan AV, Devi MT, Raghavendra AS(1994) Molecular biology of C4 phosphoenolpyruvate carboxylase:structure,regulation and genetic engineering. Photosynthesis Research,39 (2):115-135.
102. Rao SK, Magnin NC, Reiskind JB, George B (2002) Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C4 system of Hydrilla verticillata. Plant Physiol.130:876-886.
103. Reiskind JB, Madsen TV,VanGinkel LC, Bowes G (1997) Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submersed monocot, Plant Cell and Environment,20 (2):211-220
104. Richard L (1989) Hudspeth. Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxlase isozyme involved in C4 photosynthesis. Plant Molecular Biology,12:579-589.
105. Richard RA (2000) Selectable traits to increase crop photosynthesis and yield of grain crops. Journal of Experimental Botany,51:447-458
106. Rickers J, John C, Cushman, Christine B, Michalowaki (1989) Expression of the CAM form of phosphoenolpyruvate carboxylase and nucleotide sequence of a full length cDNA from Mesembryanthemum crystallinum. Mol. Gen. Genet,215:447-454
107. Robin G. Walters, Freya Shephard, Jennifer JM Rogers, Stephen A. Rolfe, and Peter Horton (2003) Identification of mutants of Arabidopsis defective in acclimation of photosynthesis to the light environment. Plant Physiol,131:472-481.
108. Rosendal, L, Vance, CP, and Pedersen, WB (1990). Products of dark. CO2 fixation in pea root nodules support bacteroid metabolism. Plant Physiol.93:12-19
109. Rothermel B A, Nelson T. (1989) Primary structure of the maize NADPH-dependent malic enzyme. J Biol Che,264:19587-19592
110. Saavedra-Lira E and Perez-Montfort R (1994) Cloning and sequence determination of the gene coding for the pyruvate phosphate dikinase of Entamoeba histolytica. Gene, 142,249-251
111. Schroeder Julian I., Kwak June M., Allen Gethyn J (2001) Guard cell abscisic acid signaling and engineering drought hardiness in plants, Nature,2001,410:327-330
112. Shearer Heather L, Turpin David H, Dennis DT(2004) Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm, Archives of Biochemistry and Biophysics,429:134-144
113. Sheen J (1991) Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. The Plant Cell,3:225-245
114. Sheriff A, Meyer H, Riedel E, Schmitt JM, Lapke C (1998) The influence of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzyme. Plant Science,136:43-57
115. Sugimoto T, Tsutomu Kawasaki, Tomohiko Kato (1992) DNA sequence and expression of a phospho-enolpyruvate carboxlase gene from soybean. Molecular Biology,20: 743-747
116. Sun SB, Shen QR, Wan JM, et al (2003) Induced Expression of the Gene for NADP-malic Enzyme in Leaves of Aloe vera L. under Salt Stress, Acta Biochimica et Biophysica Sinica,35(5):423-429
117. Suzuki S, Murai N, Burnell JN, Arai M (2000) Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides. Plant Physiol,124:163-72
118. Takeuchi Y, Akagi H, Kamasawa Net al. (2000) Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme. Planta,211: 265-267
119. Tanabe M, Izui K, Toriyama K (2000) Production and analysis of transgenic C3-C4, intermediate Moricandia arvensis expressing a maize C4 phosphoenolpytuvate carboxylase gene. Plant Biotechnology,17 (2):93-98
120. Taniguchi Y, Ohkawa H, Masumoto CF, Takuya TT, Lee K, Sudoh S, Tsuchida H, Sasaki H, Fukayama H, Miyao M (2008) Overproduction of C4 photosynthetic enzymes in transgenic rice plants:an approach to introduce the C4-like photosynthetic path way into rice, J. Exp. Bot.59:1799-1809
121.Tausta SL, Nelson T. (1997) The root and leaf isoforms of NADP-dependent malic enzyme are encoded by distinct genes. Maiza Genetics Cooperation Newsletter,71: 65-66
122. Teese P (1995) Intraspecific variation for CO2 compensation point and differential growth among variants in a C3-C4 intermediate plant. Oecologia 102:371-376.
123. Thomas M, Cretin C, Keryer E, Vidal J and Gadal P (1987) Photocontrol of sorghum leaf phosphoenolpyruvate carboxylase characterization of messenger RNA and of photoreceptor. Plant Physiol,85:243-246
124. Tjaden B, Plagens A, Dorr C, Siebers B, Hensel R (2006) Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax:key pieces in the puzzle of archaeal carbohydrate metabolism. Mol Microbiol,60:287-298
125. TrejoT & Stenzel R et al (2009) Transgenic tobacco plants overexpressing pyruvate phosphate dikinase increase exudation of organic acids and decrease accumulation of aluminum in the roots. Plant Soil
126. Tsuchida H, Tamai T, Fukayama Het al. (2001) High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth in a C3 plant, rice. Molecular engineering of C4 photosynthesis. Plant Cell Physiol,42:138-145
127. Ueno O and Takeda T (1992) Photosynthetic pathways, ecological characteristics and the geographical distribution of the Cyperaceae in Japan. Oecologia 89:195-203.
128. Ueno O, Samejima M, Muto Set al. (1988) Photosynthetic characteristics of an mphibious plant, Eleocharis vivipara:expression of C4 and C3 modes in contrasting environments. Proc. Natl. Acad Sci.,85:6733-6737
129. Usuda H, Ku MSB and Edwards GE (1984) Activation of NADP-malate dehydrogenase, pyruvate Pi dikinase and fructose 1,6-bisphosphatase in relation to photosynthetic rate in maize. Plant Physiol,76:238-243
130. Voznesenskaya EV, Franceschi VR, Kiirats O, Artyusheva EG, Freitag H, Edwards GE (2002) Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae) Plant J; 31:649-662
131. Voznesenskaya EV, Franceschi VR, Kiirats O, Freitag H, Edwards GE (2001) Kranz anatomy is not essential for terrestrial C4 plant photosynthesis. Nature,414:543-546
132. Wang JM and Li RZH (2008) Integration of C4-specific PPDK gene of Echinochloa to C3 upland rice and its photosynthesis characteristics analysis. African Journal of Biotechnology,7:783-787
133. Weng H B, PAN A H, YANG L Tet al. (2004) Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Molecular Biology Reporter,22:289-300
134. Westhoff P, Gowik U(2004) Evolution of C4 phosphoenolpyruvate carboxylase. Genes and proteins:a case study with the genus Flaveria. Annals of Botany,93:13-23
135. Westhoff P, Svensson P, Emst Ket al. (1997) Molecular evolution of C4 phosphoenolpyruvate carboxylase in the gunus Flaveria. Australian. Plant Physiol,24 (4): 429-436.
136. Winter K, Foster J G, Edwards G E, et al (1982) Intracellular localization of enzymes of carbon metabolism in mesembryanthemum crystailinum exhibiting C3 photosynthetic characteristics or performing crassulacean acid metabolism, Plant Physiol.,69:300-307
137. Xiong L, Schumaker KS, Zhu JK(2002) Cell signaling during cold, drought, and salt stress, The Plant Cell,2002,14:165-183
138. Yuan LP(2000). Super hybrid rice. Chin Rice Res New Lett,8(1):13-15
139. Zhang JF, Bandyopadhyay A, Sellappan K, Wang GY, Xie HA, Datta K and Datta SK (2010) Characterization of a C4 maize pyruvate orthophosphate dikinase expressed in C3 transgenic rice plants. African Journal of Biotechnology,9:234-242
140. Zhu XG, Shan L, Wang Y, Quick WP (2010) C4 rice—an ideal arena for systems biology research.. Integr. Plant Biol.52 (8),762-770
141.常丽丽,(2009),玉米光周期敏感相关基因ZmTFL1的克隆及功能验证,河南农业大学硕士论文
142.陈绪清,张晓东,梁荣奇等(2004).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,49(19):1976-1982.
143.丁在松,赵明,荆玉祥等(2007).玉米ppc基因过表达对转基因水稻光合速率的影响.33(5):717-722
144.段美娟(2010)玉米C4光合酶基因转化超级杂交稻亲及转基因水稻生物学特性研究.博士学位论文,湖南农业大学:1-138
145.郝建军,康宗利(2005)植物生理学,北京,化学工业出版社,250-272
146.郝乃斌,谭克辉,那松青等(1991).C3植物绿色器官PEP羧化酶活性的比较研究.植物学报,33(9):692-697
147.焦德茂,李霞,黄雪清等(2001).转PEPC基因水稻的光合CO2同化和叶绿素荧光特性.科学通报,46:414-417
148.李思远,(2008),玉米光周期敏感基因类Hd6的克隆和功能分析,河南农业大学硕士论文
149.李卫华,卢庆陶,郝乃斌等(2001).大豆叶片C4循环途径酶.植物学报,43(8):805-808
150.李霞,焦德茂,戴传超(2005).转PEPC基因水稻对光氧化逆境的响应.作物学报,31(4):408-413
151.李霞,焦德茂,戴传超等(2001).转PEPC基因杂交水稻的光合生理特性.作物学报,27:137-143
152.李煦,王荣春,何影,杨爱芳(2007).多基因双T-DNA植物表达载体的构建及拟南 芥转化.山东东大学学报(理学版),42(9):1-6
153.李艳,(2009),玉米C4途径关键酶(PEPC、PPDK)基因的克隆及PEPC基因对小麦的遗传转化,河南农业大学博士论文
154.李艳,许为钢,胡琳等(2009).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,29(5):741-746
155.刘振业.作物高光效育种.北京:中国农业科学,1979,7-10
156.陆时万,徐祥生,沈敏健(2007)植物学,高等教育出版社.
157.拟南芥实验手册(2004),化学工业出版社
158.潘瑞炽,董愚得(2004).植物生理学.北京:高等教育出版社
159.潘晓华,邓强辉(2007).作物收获指数的研究进展,江西农业大学学报,1-5
160.万东石,李红玉,张立新(2003)植物体内干旱信号的传递与基因表达,西北植物学报,23(1):151-157
161.汪家政,范明(2001).蛋白质技术手册.北京:科学出版社,38-39
162.王关林,方宏筠(2002).植物基因工程(第二版).科学出版社:477-478
163.王强,卢从明,张其德,郝迺斌,戈巧英,董凤琴,白克智,匡廷云(2002).超高产杂交稻两优培九的光合作用、光抑制和C4途径酶特性,中国科学C辑.
164.王新涛,(2009),玉米光周期敏感基因ZmGI的克隆和功能分析,河南农业大学硕士论文
165.魏爱丽,王志敏,翟志席,龚元石(2002).土壤干旱对小麦旗叶和穗器官C4光合酶活性的影响.中国农业科学,200336(5):508-512
166.吴琼,许为钢,李艳,齐学礼,胡琳,张磊,韩琳琳.(2011)田间条件下转玉米C4型PEPC基因小麦光合生理特性的初步研究.作物学报:URL:http://www.cnki. net/kcms/detail/11.1809.S.20110906.1104.015.html
167.武维华(2004).植物生理学.北京:科学出版社
168.徐晓玲,王志敏,张俊平(2001).灌浆期热胁迫对小麦不同绿色器官光合性能的影响.植物学报,43(6),571-577
169.许为钢,胡琳,王根松等(2009).小麦品种郑麦9023的选育策略及对小麦产量育种的思考.河南农业科学,9:14-18
170.尹吴(2009),转C4植物光合关键基因PEPC杨树的初步分析.南京:南京林业大学,49-50.
171.袁定阳,段美娟,谭炎宁,易自力,袁隆平,辛世文(2007).共转化法获得无筛选标记的转PEPC.PPDK基因水稻恢复系纯合体.杂交水稻,22(2):57-63
172.张边江,陈全战,焦德茂(2008).构建C4水稻——一场新绿色革命的挑战.科技导报,96-98
173.张边江,华春,周峰,周泉澄,陈全战,王荣富,焦德茂等(2008).转PEPC+PPDK双基因水稻的光合特性.中国农业科学,41(10):3008-3014
174.张彬,丁在松,张桂芳等(2007).根癌农杆菌介导获得稗草Ecppc转基因小麦的研究.作物学报,33(3):356-362
175.张方,迟伟,金成哲等(2003).高粱C4型磷酸烯醇式丙酮酸羧化酶基因的分子克隆及其转基因水稻的培育.科学通报,48(14):1542-1546
176.张桂芳,赵明,丁在松,张丽,肖俊涛(2005).稗草磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的克隆与分析,作物学报,31(10):1365-1369
177.张庆琛,(2008),转玉米C4型PEPC基因小麦光合生理特性的研究,河南农业大学硕士论文
178.张庆琛,许为钢,胡琳等(2010).玉米C4型全长PEPC基因导入普通小麦的研究.麦类作物学报,30(2):194-197
179.张晓娟(2010).坛紫菜PEPCK及PEPC基因的克隆与各生活史阶段表达分析.中国科学院研究生院(海洋研究所),硕士学位论文.
180.赵福庚,何云龙,罗庆云(2004)植物逆境生理生态学,北京,化学工业出版
181.赵世杰,刘华山,董新纯等.植物生理学实验指导.北京:中国农业科技出版社,1998.98-163
182.周宝元,在松,赵明(2011)PEPC过表达可以减轻干旱胁迫对水稻光合的抑制作用.作物学报,37(1):112-118
183.朱建楚,胡银岗,奚亚军等(2005).实时荧光定量PCR技术在检测外源基因拷贝数中的应用.西北农业学报,14(6):78-82