毛竹紫黄质脱环氧化酶基因的克隆与功能研究
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摘要
适应各种光照环境是植物生存的本能,叶黄素循环在植物适应强光环境、光破坏防御和适应低光环境、提高光能利用效率过程中都发挥着重要作用。叶黄素循环含有三种组分,即紫黄质、花药黄质和玉米黄质。当植物吸收的光能超过其转化能力时,类囊体腔被过度酸化,低pH能够激活紫黄质脱环氧化酶,催化紫黄质生成玉米黄质的反应。玉米黄质能够将过量光能以热能的形式耗散掉,而且能够清除一些氧自由基,以减轻过量光能对植物产生的伤害。而在低光照下,玉米黄质在玉米黄质环氧化酶的作用下转化为紫黄质,减少光能的热耗散,从而提高植物的光能利用率。毛竹(Phyllostachys edulis)是分布于我国亚热带地区的散生竹种,具有较高的经济价值。本论文以毛竹实生苗为材料,在研究叶片叶绿素荧光动力学参数的基础上,开展了毛竹叶黄素循环关键酶-紫黄质脱环氧化酶的研究,以期为认识竹子叶黄素循环在不同光照环境中的作用提供分子生物学基础,主要研究结果如下:
第一,毛竹叶片叶绿素荧光动力学研究。利用叶绿素荧光技术,研究了毛竹实生苗叶片叶绿素荧光参数非光化学猝灭(NPQ)和电子传递速率(ETR)的变化规律。结果表明,充分暗适应后,随着光合有效辐射(PAR)逐渐增强,NPQ也随之增加,而ETR则呈现先上升,后下降的趋势。其表明NPQ在过量光能热耗散过程中发挥重要作用。
第二,基因克隆。将不同物种的紫黄质脱环氧化酶(VDE)同源蛋白序列进行比对,设计简并引物,扩增得到毛竹VDE基因的部分cDNA序列,再通过RACE方法,获得5′和3′端序列,经拼接获得了基因全长cDNA(1723 bp),编码区为1356 bp,共编码451个氨基酸(其中N端的103个氨基酸为转运肽),该基因命名为PeVDE。根据PeVDE的编码区序列设计引物,以基因组DNA为模板进行扩增,经测序结果表明毛竹PeVDE基因编码区基因组序列含有4个内含子和5个外显子。
第三,基因的原核表达。将编码PeVDE成熟蛋白的基因序列克隆入原核表达载体pET-32b中,得到含有目的基因的重组表达载体。将该表达载体转入大肠杆菌表达菌株(Rosetta-gamin B (DE3))感受态细胞,进行原核诱导表达。通过优化诱导条件,在30℃条件下,0.4 mmol·L-1 IPTG诱导4 h,获得了表达丰度较高的可溶蛋白。
第四,酶活性测定。通过超声破碎细菌,提取PeVDE重组总蛋白,以紫黄质为反应底物,在25℃、pH 5.1条件下暗处反应15 min,应用HPLC法检测反应产物。结果显示,反应产物中除了有紫黄质外,还有花药黄质和玉米黄质组分,由此证明重组蛋白具有生物学活性,能够将紫黄质依次转化为花药黄质和玉米黄质。
第五,Western blotting分析。用原核表达所得的重组蛋白免疫新西兰大白兔,制备多克隆抗体。分别提取毛竹实生苗的根、茎、叶鞘和叶片组织的总蛋白,并通过SDS-PAGE进行分离,以PeVDE的多克隆抗体为探针进行检测。Western Blotting结果表明,在茎、叶鞘和叶片中都能检测到PeVDE,其中以叶片中含量最高,其分子量约为50 kDa,而在根中没有检测到。
It is a potential ability for plants to survive under naturally varying light conditions. The xanthophyll cycle plays an important role both in protecting plants from photo-oxidative damage under strong light and in improving photosynthetic efficiency under inadequate light. The cycle consists of three components, i.e. violaxanthin, antheraxanthin and zeaxanthin. Under strong light the energy input exceeds the photosynthetic capacity and leads to overacidification of the thylakoid lumen. This drop in lumen pH leads to activation of the violaxanthin deepoxidase (VDE), thus shifting the xanthophyll balance from violaxanthin (V) toward zeaxanthin (Z) through antheraxanthin (A). Z is able to enhance energy dissipation as heat and efficiently scavenges reactive oxygen species which are harmful to organisms. Under low light Z is converted back to V through A by zeaxanthin epoxidase (ZE), which can increase light-harvesting efficiency by avoiding unnecessary quenching of excitation energy. Moso bamboo (Phyllostachys edulis), an economic species with great value located in subtropical regions of southern China, was selected as material for experiment. Based on the chlorophyll fluorescence parameters analysis of P. edulis, research on violaxanthin de-epoxidase was carried out, which was expected to understand the role of the xanthophyll cycle in photoprotection. The main results are as follows:
First, chlorophyll fluorescence parameter measurement. The chlorophyll fluorescence parameters such as nonphotochemical quenching (NPQ) and apparent electron transport rate (ETR) characteristics of Moso bamboo leaves were measured with a portable pulse amplitude modulation fluorometer (Imaging PAM). The results showed that NPQ enhanced with the increasing of photosynthetically active radiation (PAR) after dark adaptation adequately, while ETR increased firstly, then decreased. It indicated that NPQ played an important role in the dissipation of excess energy.
Second, gene isolation. Degenerate primers for the conserved domain of VDE were designed based on the homologous protein sequence of different species and the RACE primers were designed according to the gained conserved domain sequence. A full length cDNA encoding violaxanthin de-epoxidase was obtained by the methods of RT-PCR and RACE and named as PeVDE. The cDNA was 1723 bp which contained an open reading frame (ORF) encoding 451 amino acids. The protein structure analysis showed that the putative protein consisted of transit peptides (103 amino acids at the N-terminal end) and mature protein of VDE. Homology analysis showed that the deduced mature protein was highly homologous to other VDE proteins from different species. Based on the ORF sequence of PeVDE, primers were designed to clone the genomic sequence. Sequence analysis showed that the genomic sequence was 1921 bp containing four introns and five exons.
Third, prokaryotic expression of PeVDE. The sequence encoding the mature protein of PeVDE was subcloned into pET-32b and transformed into Rosetta-gamin B (DE3) induced by IPTG. Analysis by SDS-PAGE of cell extracts indicated that the recombinant protein was expressed effectively at 30℃induced by 0.4 mmol·L-1 IPTG for 4 h.
Fourth, the enzymatic activity assay. The bacterial pellet was lysed using an ultrasonic cell disruptor, and the crude protein was used for activity assay. The enzymatic reaction was performed with pH 5.1 at 25℃in dark for 15 min. The mixture of reaction was extracted and analyzed by HPLC. The result showed that it contained not only V but also A and Z, which indicated that the recombinant protein exhibited enzymatic activity and could catalyze V into Z through A.
Fifth, western blotting. The recombinant protein was purified and used to immunize white rabbit to obtain polyclonal antibody. The total protein from the roots, stems, leaf sheaths and leaves were extracted respectively. The polyclonal antibody of PeVDE was used as probe for western blotting. The result showed that the violaxanthin de-epoxidase was only detected in Moso bamboo stems, leaf sheaths and leaves which was richest, and the molecular weight was about 50 kDa.
第一,毛竹叶片叶绿素荧光动力学研究。利用叶绿素荧光技术,研究了毛竹实生苗叶片叶绿素荧光参数非光化学猝灭(NPQ)和电子传递速率(ETR)的变化规律。结果表明,充分暗适应后,随着光合有效辐射(PAR)逐渐增强,NPQ也随之增加,而ETR则呈现先上升,后下降的趋势。其表明NPQ在过量光能热耗散过程中发挥重要作用。
第二,基因克隆。将不同物种的紫黄质脱环氧化酶(VDE)同源蛋白序列进行比对,设计简并引物,扩增得到毛竹VDE基因的部分cDNA序列,再通过RACE方法,获得5′和3′端序列,经拼接获得了基因全长cDNA(1723 bp),编码区为1356 bp,共编码451个氨基酸(其中N端的103个氨基酸为转运肽),该基因命名为PeVDE。根据PeVDE的编码区序列设计引物,以基因组DNA为模板进行扩增,经测序结果表明毛竹PeVDE基因编码区基因组序列含有4个内含子和5个外显子。
第三,基因的原核表达。将编码PeVDE成熟蛋白的基因序列克隆入原核表达载体pET-32b中,得到含有目的基因的重组表达载体。将该表达载体转入大肠杆菌表达菌株(Rosetta-gamin B (DE3))感受态细胞,进行原核诱导表达。通过优化诱导条件,在30℃条件下,0.4 mmol·L-1 IPTG诱导4 h,获得了表达丰度较高的可溶蛋白。
第四,酶活性测定。通过超声破碎细菌,提取PeVDE重组总蛋白,以紫黄质为反应底物,在25℃、pH 5.1条件下暗处反应15 min,应用HPLC法检测反应产物。结果显示,反应产物中除了有紫黄质外,还有花药黄质和玉米黄质组分,由此证明重组蛋白具有生物学活性,能够将紫黄质依次转化为花药黄质和玉米黄质。
第五,Western blotting分析。用原核表达所得的重组蛋白免疫新西兰大白兔,制备多克隆抗体。分别提取毛竹实生苗的根、茎、叶鞘和叶片组织的总蛋白,并通过SDS-PAGE进行分离,以PeVDE的多克隆抗体为探针进行检测。Western Blotting结果表明,在茎、叶鞘和叶片中都能检测到PeVDE,其中以叶片中含量最高,其分子量约为50 kDa,而在根中没有检测到。
It is a potential ability for plants to survive under naturally varying light conditions. The xanthophyll cycle plays an important role both in protecting plants from photo-oxidative damage under strong light and in improving photosynthetic efficiency under inadequate light. The cycle consists of three components, i.e. violaxanthin, antheraxanthin and zeaxanthin. Under strong light the energy input exceeds the photosynthetic capacity and leads to overacidification of the thylakoid lumen. This drop in lumen pH leads to activation of the violaxanthin deepoxidase (VDE), thus shifting the xanthophyll balance from violaxanthin (V) toward zeaxanthin (Z) through antheraxanthin (A). Z is able to enhance energy dissipation as heat and efficiently scavenges reactive oxygen species which are harmful to organisms. Under low light Z is converted back to V through A by zeaxanthin epoxidase (ZE), which can increase light-harvesting efficiency by avoiding unnecessary quenching of excitation energy. Moso bamboo (Phyllostachys edulis), an economic species with great value located in subtropical regions of southern China, was selected as material for experiment. Based on the chlorophyll fluorescence parameters analysis of P. edulis, research on violaxanthin de-epoxidase was carried out, which was expected to understand the role of the xanthophyll cycle in photoprotection. The main results are as follows:
First, chlorophyll fluorescence parameter measurement. The chlorophyll fluorescence parameters such as nonphotochemical quenching (NPQ) and apparent electron transport rate (ETR) characteristics of Moso bamboo leaves were measured with a portable pulse amplitude modulation fluorometer (Imaging PAM). The results showed that NPQ enhanced with the increasing of photosynthetically active radiation (PAR) after dark adaptation adequately, while ETR increased firstly, then decreased. It indicated that NPQ played an important role in the dissipation of excess energy.
Second, gene isolation. Degenerate primers for the conserved domain of VDE were designed based on the homologous protein sequence of different species and the RACE primers were designed according to the gained conserved domain sequence. A full length cDNA encoding violaxanthin de-epoxidase was obtained by the methods of RT-PCR and RACE and named as PeVDE. The cDNA was 1723 bp which contained an open reading frame (ORF) encoding 451 amino acids. The protein structure analysis showed that the putative protein consisted of transit peptides (103 amino acids at the N-terminal end) and mature protein of VDE. Homology analysis showed that the deduced mature protein was highly homologous to other VDE proteins from different species. Based on the ORF sequence of PeVDE, primers were designed to clone the genomic sequence. Sequence analysis showed that the genomic sequence was 1921 bp containing four introns and five exons.
Third, prokaryotic expression of PeVDE. The sequence encoding the mature protein of PeVDE was subcloned into pET-32b and transformed into Rosetta-gamin B (DE3) induced by IPTG. Analysis by SDS-PAGE of cell extracts indicated that the recombinant protein was expressed effectively at 30℃induced by 0.4 mmol·L-1 IPTG for 4 h.
Fourth, the enzymatic activity assay. The bacterial pellet was lysed using an ultrasonic cell disruptor, and the crude protein was used for activity assay. The enzymatic reaction was performed with pH 5.1 at 25℃in dark for 15 min. The mixture of reaction was extracted and analyzed by HPLC. The result showed that it contained not only V but also A and Z, which indicated that the recombinant protein exhibited enzymatic activity and could catalyze V into Z through A.
Fifth, western blotting. The recombinant protein was purified and used to immunize white rabbit to obtain polyclonal antibody. The total protein from the roots, stems, leaf sheaths and leaves were extracted respectively. The polyclonal antibody of PeVDE was used as probe for western blotting. The result showed that the violaxanthin de-epoxidase was only detected in Moso bamboo stems, leaf sheaths and leaves which was richest, and the molecular weight was about 50 kDa.
引文
Akerlund H. E., Aridsson P. O., Bratt C. E.. Partial purification of the violaxanthin de-epoxidase in photosynthesis from light to Biosphere. Kluwer Academic Publishers,Dordrecht, 1995, 103-106.
Alan B., Christian T.. Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library. Journal of Experimental Botany, 1997, 48 (9):1749-1750.
Arnoux P., Morosinotto T., Saga G., et al.. Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana. Plant Cell, 2009, 21 (7): 2036-2044.
Arvidsson P. O., Bratt C. E., Carlsson M., et al.. Purification and identification of the violaxanthin de-epoxidase as a 43 kD protein. Photosynthesis Research, 1996, 49: 119-129.
Aslund. Efficient production of disulfide bonded proteins in the cytoplasm in“oxidizing”mutants of E. Coli. InNovations, 1999, 10: 11-12.
Bruce D., Biggins J.. Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2. Biochimica et Biophysica Acta, 1985, 810 (3): 295-301.
Bugos R. F., Yamamoto H. Y.. Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli. Plant Biology, 1996, 93: 6320-6325.
Dau H.. Short-term adaptation of plants to changing light intensities and its relation to Photosystem II photochemistry and fluorescence emission. Journal of photochemistry and photobiology. Journal of Photochemistry and Photobiology, 1994, 26: 3-27.
Demmig B.. Xanthophyll cycle and light stress in nature:uniform response to excess direct sunlight among higher plant species. Planta, 1996, 198: 460-470.
Demmig B., Winter K., Kruger A., et al.. Photoinhibition and Zeaxanthin Formation in Intact Leaves : A Possible Role of the Xanthophyll Cycle in the Dissipation of Excess Light Energy. Plant Physiology, 1987, 84: 218-224.
Demmig-Adams B., Adams W. I., Logan B., et al.. Xanthophyll Cycle-Dependent Energy Dissipation and Flexible PhotosystemⅡEfficiency in Plants Acclimated to Light Stress. Australian Journal of Plant Physiology, 1995, 22: 249-260.
Demmig-Adams B., Winter K., Kruger A., et al.. Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of PhotosystemⅡat Chilling Temperatures. Plant Physiology, 1989, 90: 894-898.
Eskling M., Akerlund H. E.. Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light. Photosynthesis Research, 1998, 57 (1): 41-50.
Frank H. A., Josue. Direct Determination of the S1 Excited-State Energies of Xanthophylls by Low-Temperature Fluorescence Spectroscopy. The journal of Biological Chemistry, 2002, 106(19): 4815-4824.
Gilmore A. M., Hazlett T. L., Govindjee. Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92: 2273-2277.
Hager A. Lichtbedingte pH-Erniedrigung in einem chloroplast ten-kompartiment als Ursache der Enzymatischen violoanthin zu zeaxanthin-Umwandlung:Beziehungen zur photophosphorylierung. Planta, 1969, 89: 224-243.
Hager A. Die reversiblen,Lichtabhangigen xanthophyll umwandlungen im chloroplasten. Ber Dtsch Bot Ges, 1975, 27-44.
Hager A. , Holocher K.. Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease. Planta, 1994, 192 (4): 581-589.
Haldrup A., Jensen P. E., Lunde C., et al.. Balance of power: a view of the mechanism of photosynthetic state transitions. Trends in Plant Science, 2001, 6: 301-305
Havaux M., Niyogi K.. The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism. Proceedings of the National of Sciences of the United States of America, 1999, 96: 8762-8767.
Havir E. A.. Purification and properties of violaxanthin de-epoxidase from spinach. Plant Science, 1997, 123: 57-66.
Hieber A. D. Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in-vitro de-epoxidation in monogalactosyldiacylglycerol micelles. Plant and cell physiology, 2004, 45 (1): 92-102.
Isagi Y., Kawahara T., Kamo K., et al.. Net production and carbon cycling in a bamboo Phyllostachys pubescens stand. Plant Ecology, 1997, 130: 41-52.
Ivanov A. G., Krol M., Maxwell D., et al.. Abscisic acid induced protection against photoinhibition of PSⅡcorrelates with enhanced activity of the xanthophyll cycle. FEBS Letters, 1995 371: 61-64.
James F. H., Liu W., Halsey C. M. R., et al.. Ni-NTA-Gold Clusters Target His-Tagged Proeins. Jounal of Structural Biology, 1999, 127 (2): 185-198.
Johnson G. The dissipation of excess excitation energy in British plant species. Plant, Cell and Environment, 1993, 16: 673-679.
Kieselbach T., Hagman A., Andersson B., et al.. The Thylakoid Lumen of Chloroplasts. Biological Chemistry, 1998, 273: 6710-6716.
Kok B.. On the inhibition of photosynthesis by intense light. Biochim and Biophysica Acta, 1956, 2: 235-244.
Latowski D.. Amino sugars: new inhibitors of zeaxanthin epoxidase, a violaxanthin cycle enzyme. Plant Physiology, 1997, 164 (3): 231-237.
Lindberg M. A., Polacek C., Johansson S.. Amplification and cloning of complete enterovirus genomes by long distance PCR. Journal of Virological Methods, 1997, 65 (2): 191-199.
Logan B., Demmig-Adams B., Adams W., et al.. Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. acclimated to four growth PPFDs in the field. Journal of Experimental Botany , 1998, 49: 1869-1879.
Malkin R., Niyogi K.. Biochemistry and molecular biology of plants. American Society of Plant Physiologists, 568-628.
Marie E., Per-Ola A., Hans-Erik A.. The xanthophyll cycle, its regulation and components. Physiologia Plantarum, 1997, 100: 806-816.
Marin E., Nussaume L.. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO Journal, 1996, 15 (10): 2331-2342.
Mehler A.. Studies on reaction of illumination choloplast I:Menchanism of the reduction of oxygen and other HIll reagents. Archives of Biochemistry and Biophysics, 1952, 33: 65-77.
Morales F., Abadia A., Belkhodia R., et al.. Iron deficiency-induced changes in photosynthetic pigmentcomposition of field-grown pear leaves. Plant Cell Environ, 1994, 17: 1153-1160.
Muller-Moule P., Conklin P. L., Niyogi K. K.. Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiology, 2002, 128: 970-977.
Niyogi K. K.. Photoprotection Revised: Genetic and Molecular Approaches. Annual Review of Plant Physiology and Plant Molecular Biology, 1999, 50: 333-359.
Osmond C.. Photorespiration and photoinhibition: some implications for the energetics of photosynthesis. Biochim and Biophysica Acta, 1981, 639: 77-98.
Osmond C.. What is photoinhibition? Some insights from comparisons of shade and sun plants. Bios Scientific Publications, 1994, 1-24.
Owens T., Shreve A., Albrecht A.. Dynamics and mechanism of singlet energy transfer between carotenoids and chlorophylls:light harvesting and nonphotochemical fluorescence quenching. Research in Photosynthesis, 1992, 179-186.
Peter J., Patricia A., Linden, et al.. Excited-State Kinetics of the Carotenoid S1 State in LHCⅡand Two-Photon Excitation Spectra of Lutein andβ-Carotene in Solution:Efficient Car S1→Chl Electronic Energy Transfer via Hot S1 States? Physical Chemistry, 2002, 106(10): 1909-1916.
Pogson B. J., Niyogi K. K., Bj?rkman O., et al.. Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants. Proceedings of the National Academy of Sciences of the United States of America,1998, 95: 13324-13329.
Polívka T., Zigmantas D., Herek J. L., et al.. The Carotenoid S1 State in LH2 Complexes from Purple Bacteria Rhodobacter sphaeroides and Rhodopseudomonas acidophila:? S1 Energies, Dynamics, and Carotenoid Radical Formation. The Journal of Physical Chemistry B, 2002, 106: 11016-11025.
Powles S. B.. Photoinhibition of Photosynthesis Induced by Visible Light. Annual Review of Plant Physiology, 1984, 35: 15-44.
Rockholm D. C., Yamamoto H. Y.. Violaxanthin De-Epoxidase (Purification of a 43-Kilodalton Lumenal Protein from Lettuce by Lipid-Affinity Precipitation with Monogalactosyldiacylglyceride). Plant Physiology, 1996, 110: 697-703.
Scurlock J. M. O., Dayton D. C., Hames B.. Bamboo: an overlooked biomass resource? Biomass and Bioenergy, 2000, 19 (4): 229-244.
Spreitzer R. J., Salvucci M. E.. Rubisco: structure, regulaotry interactions, and possibilities for a betterenzyme. Annu. Rev. Annual Review of Plant Biology, 2002, 53: 449-475
Thayer, Susan S.. Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynthesis Research, 1990, 23 (3): 331-343.
Thiele A., Krause G.. Xanthophyll cycle and thermal energy dissipation in photosystemⅡ: Relationship between zeaxanthin formation, energy-dependent fluorescence quenching and photoinhibition (Spinacia oleracea). Plant Physiology, 1994, 144: 197-209.
Von H. G., Steppuhn J., Hermann R. G.. Domain structure of mitochondrial and chloroplast targeting peptides. European Journal of Biochemistry, 1989, 180: 535-545.
Yamamoto H. Y., Higashi R. M.. Violaxanthin deepoxidase,lipid composition and substrate specificity. Archives of Biochemistry and Biophysics, 1978, 190: 514-522.
董高峰.阳生植物和阴生植物的叶黄素循环与非辐射能量耗散.武汉植物学研究, 2001, 19 (2): 128-134.
高志民,李雪平,彭镇华等.竹子捕光叶绿素a/b结合蛋白基因全长的克隆和序列分析.林业科学, 2007, 43 (3): 34-38.
高志民,刘成,刘颖丽等.毛竹捕光叶绿素a/b结合蛋白基因cab-PhE1的克隆与表达分析.林业科学, 2009, 45 (3): 145-149.
黄承才.中亚热带东部毛竹叶片光合及呼吸的研究.浙江林业科技, 2000, 20 (5): 14-16.
黄启民,杨迪蝶.不同条件下毛竹光合作用的研究.竹类研究, 1989, 8 (2): 8-16.
J.萨姆布鲁克, D.W.拉塞尔著.分子克隆实验指南(第三版).科学出版社, 2002, 540-544.
江泽慧.世界竹藤.辽宁:辽宁科学技术出版社, 2002, 1-10.
匡廷云.光合作用原初光能转化过程的原理与调控.江苏科学出版社, 2003, 50-60.
李善春. NaCl盐胁迫下5种地被观赏竹生理特性的研究.南京林业大学硕士学位论文, 2005, 11-21.
李雪平,彭镇华,高志民等.绿竹光系统Ⅰ捕光叶绿素a/b结合蛋白基因的cDNA全长克隆及分析.安徽农业大学学报, 2010, 37 (4): 643-648.
欧阳海.论竹类在中国园林建设中的作用.广东园林, 2005, 28 (2): 29-32.
唐文莉,彭镇华,高健等.刚竹属3种重要散生竹光系统Ⅰ基因(Lhca 1)的克隆、序列分析和蛋白结构预测.北京林业大学学报, 2008, 30 (4): 109-115.
王强.光合作用光抑制的研究进展.植物学通报, 2003, 20 (5): 1003-2266.
韦朝领,江昌俊,陶汉之等.茶树鲜叶片叶黄素循环组分的高效液相色谱法测定研究及其光保护功能鉴定.茶叶科学, 2004, 24 (1): 60-64.
许大全.植物光胁迫研究中的几个问题.植物生理学通讯, 2003, 39 (5): 493-495.
叶志敏.竹子在现代城市景观设计中的应用.世界竹藤通讯, 2009, 7 (4): 29-32.
郑容妹.盐分胁迫对沿海绿竹光合作用及叶绿素的影响.竹子研究汇刊, 2002, 21 (4):76-80.
Alan B., Christian T.. Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library. Journal of Experimental Botany, 1997, 48 (9):1749-1750.
Arnoux P., Morosinotto T., Saga G., et al.. Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana. Plant Cell, 2009, 21 (7): 2036-2044.
Arvidsson P. O., Bratt C. E., Carlsson M., et al.. Purification and identification of the violaxanthin de-epoxidase as a 43 kD protein. Photosynthesis Research, 1996, 49: 119-129.
Aslund. Efficient production of disulfide bonded proteins in the cytoplasm in“oxidizing”mutants of E. Coli. InNovations, 1999, 10: 11-12.
Bruce D., Biggins J.. Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2. Biochimica et Biophysica Acta, 1985, 810 (3): 295-301.
Bugos R. F., Yamamoto H. Y.. Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli. Plant Biology, 1996, 93: 6320-6325.
Dau H.. Short-term adaptation of plants to changing light intensities and its relation to Photosystem II photochemistry and fluorescence emission. Journal of photochemistry and photobiology. Journal of Photochemistry and Photobiology, 1994, 26: 3-27.
Demmig B.. Xanthophyll cycle and light stress in nature:uniform response to excess direct sunlight among higher plant species. Planta, 1996, 198: 460-470.
Demmig B., Winter K., Kruger A., et al.. Photoinhibition and Zeaxanthin Formation in Intact Leaves : A Possible Role of the Xanthophyll Cycle in the Dissipation of Excess Light Energy. Plant Physiology, 1987, 84: 218-224.
Demmig-Adams B., Adams W. I., Logan B., et al.. Xanthophyll Cycle-Dependent Energy Dissipation and Flexible PhotosystemⅡEfficiency in Plants Acclimated to Light Stress. Australian Journal of Plant Physiology, 1995, 22: 249-260.
Demmig-Adams B., Winter K., Kruger A., et al.. Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of PhotosystemⅡat Chilling Temperatures. Plant Physiology, 1989, 90: 894-898.
Eskling M., Akerlund H. E.. Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light. Photosynthesis Research, 1998, 57 (1): 41-50.
Frank H. A., Josue. Direct Determination of the S1 Excited-State Energies of Xanthophylls by Low-Temperature Fluorescence Spectroscopy. The journal of Biological Chemistry, 2002, 106(19): 4815-4824.
Gilmore A. M., Hazlett T. L., Govindjee. Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92: 2273-2277.
Hager A. Lichtbedingte pH-Erniedrigung in einem chloroplast ten-kompartiment als Ursache der Enzymatischen violoanthin zu zeaxanthin-Umwandlung:Beziehungen zur photophosphorylierung. Planta, 1969, 89: 224-243.
Hager A. Die reversiblen,Lichtabhangigen xanthophyll umwandlungen im chloroplasten. Ber Dtsch Bot Ges, 1975, 27-44.
Hager A. , Holocher K.. Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease. Planta, 1994, 192 (4): 581-589.
Haldrup A., Jensen P. E., Lunde C., et al.. Balance of power: a view of the mechanism of photosynthetic state transitions. Trends in Plant Science, 2001, 6: 301-305
Havaux M., Niyogi K.. The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism. Proceedings of the National of Sciences of the United States of America, 1999, 96: 8762-8767.
Havir E. A.. Purification and properties of violaxanthin de-epoxidase from spinach. Plant Science, 1997, 123: 57-66.
Hieber A. D. Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in-vitro de-epoxidation in monogalactosyldiacylglycerol micelles. Plant and cell physiology, 2004, 45 (1): 92-102.
Isagi Y., Kawahara T., Kamo K., et al.. Net production and carbon cycling in a bamboo Phyllostachys pubescens stand. Plant Ecology, 1997, 130: 41-52.
Ivanov A. G., Krol M., Maxwell D., et al.. Abscisic acid induced protection against photoinhibition of PSⅡcorrelates with enhanced activity of the xanthophyll cycle. FEBS Letters, 1995 371: 61-64.
James F. H., Liu W., Halsey C. M. R., et al.. Ni-NTA-Gold Clusters Target His-Tagged Proeins. Jounal of Structural Biology, 1999, 127 (2): 185-198.
Johnson G. The dissipation of excess excitation energy in British plant species. Plant, Cell and Environment, 1993, 16: 673-679.
Kieselbach T., Hagman A., Andersson B., et al.. The Thylakoid Lumen of Chloroplasts. Biological Chemistry, 1998, 273: 6710-6716.
Kok B.. On the inhibition of photosynthesis by intense light. Biochim and Biophysica Acta, 1956, 2: 235-244.
Latowski D.. Amino sugars: new inhibitors of zeaxanthin epoxidase, a violaxanthin cycle enzyme. Plant Physiology, 1997, 164 (3): 231-237.
Lindberg M. A., Polacek C., Johansson S.. Amplification and cloning of complete enterovirus genomes by long distance PCR. Journal of Virological Methods, 1997, 65 (2): 191-199.
Logan B., Demmig-Adams B., Adams W., et al.. Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. acclimated to four growth PPFDs in the field. Journal of Experimental Botany , 1998, 49: 1869-1879.
Malkin R., Niyogi K.. Biochemistry and molecular biology of plants. American Society of Plant Physiologists, 568-628.
Marie E., Per-Ola A., Hans-Erik A.. The xanthophyll cycle, its regulation and components. Physiologia Plantarum, 1997, 100: 806-816.
Marin E., Nussaume L.. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO Journal, 1996, 15 (10): 2331-2342.
Mehler A.. Studies on reaction of illumination choloplast I:Menchanism of the reduction of oxygen and other HIll reagents. Archives of Biochemistry and Biophysics, 1952, 33: 65-77.
Morales F., Abadia A., Belkhodia R., et al.. Iron deficiency-induced changes in photosynthetic pigmentcomposition of field-grown pear leaves. Plant Cell Environ, 1994, 17: 1153-1160.
Muller-Moule P., Conklin P. L., Niyogi K. K.. Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiology, 2002, 128: 970-977.
Niyogi K. K.. Photoprotection Revised: Genetic and Molecular Approaches. Annual Review of Plant Physiology and Plant Molecular Biology, 1999, 50: 333-359.
Osmond C.. Photorespiration and photoinhibition: some implications for the energetics of photosynthesis. Biochim and Biophysica Acta, 1981, 639: 77-98.
Osmond C.. What is photoinhibition? Some insights from comparisons of shade and sun plants. Bios Scientific Publications, 1994, 1-24.
Owens T., Shreve A., Albrecht A.. Dynamics and mechanism of singlet energy transfer between carotenoids and chlorophylls:light harvesting and nonphotochemical fluorescence quenching. Research in Photosynthesis, 1992, 179-186.
Peter J., Patricia A., Linden, et al.. Excited-State Kinetics of the Carotenoid S1 State in LHCⅡand Two-Photon Excitation Spectra of Lutein andβ-Carotene in Solution:Efficient Car S1→Chl Electronic Energy Transfer via Hot S1 States? Physical Chemistry, 2002, 106(10): 1909-1916.
Pogson B. J., Niyogi K. K., Bj?rkman O., et al.. Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants. Proceedings of the National Academy of Sciences of the United States of America,1998, 95: 13324-13329.
Polívka T., Zigmantas D., Herek J. L., et al.. The Carotenoid S1 State in LH2 Complexes from Purple Bacteria Rhodobacter sphaeroides and Rhodopseudomonas acidophila:? S1 Energies, Dynamics, and Carotenoid Radical Formation. The Journal of Physical Chemistry B, 2002, 106: 11016-11025.
Powles S. B.. Photoinhibition of Photosynthesis Induced by Visible Light. Annual Review of Plant Physiology, 1984, 35: 15-44.
Rockholm D. C., Yamamoto H. Y.. Violaxanthin De-Epoxidase (Purification of a 43-Kilodalton Lumenal Protein from Lettuce by Lipid-Affinity Precipitation with Monogalactosyldiacylglyceride). Plant Physiology, 1996, 110: 697-703.
Scurlock J. M. O., Dayton D. C., Hames B.. Bamboo: an overlooked biomass resource? Biomass and Bioenergy, 2000, 19 (4): 229-244.
Spreitzer R. J., Salvucci M. E.. Rubisco: structure, regulaotry interactions, and possibilities for a betterenzyme. Annu. Rev. Annual Review of Plant Biology, 2002, 53: 449-475
Thayer, Susan S.. Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynthesis Research, 1990, 23 (3): 331-343.
Thiele A., Krause G.. Xanthophyll cycle and thermal energy dissipation in photosystemⅡ: Relationship between zeaxanthin formation, energy-dependent fluorescence quenching and photoinhibition (Spinacia oleracea). Plant Physiology, 1994, 144: 197-209.
Von H. G., Steppuhn J., Hermann R. G.. Domain structure of mitochondrial and chloroplast targeting peptides. European Journal of Biochemistry, 1989, 180: 535-545.
Yamamoto H. Y., Higashi R. M.. Violaxanthin deepoxidase,lipid composition and substrate specificity. Archives of Biochemistry and Biophysics, 1978, 190: 514-522.
董高峰.阳生植物和阴生植物的叶黄素循环与非辐射能量耗散.武汉植物学研究, 2001, 19 (2): 128-134.
高志民,李雪平,彭镇华等.竹子捕光叶绿素a/b结合蛋白基因全长的克隆和序列分析.林业科学, 2007, 43 (3): 34-38.
高志民,刘成,刘颖丽等.毛竹捕光叶绿素a/b结合蛋白基因cab-PhE1的克隆与表达分析.林业科学, 2009, 45 (3): 145-149.
黄承才.中亚热带东部毛竹叶片光合及呼吸的研究.浙江林业科技, 2000, 20 (5): 14-16.
黄启民,杨迪蝶.不同条件下毛竹光合作用的研究.竹类研究, 1989, 8 (2): 8-16.
J.萨姆布鲁克, D.W.拉塞尔著.分子克隆实验指南(第三版).科学出版社, 2002, 540-544.
江泽慧.世界竹藤.辽宁:辽宁科学技术出版社, 2002, 1-10.
匡廷云.光合作用原初光能转化过程的原理与调控.江苏科学出版社, 2003, 50-60.
李善春. NaCl盐胁迫下5种地被观赏竹生理特性的研究.南京林业大学硕士学位论文, 2005, 11-21.
李雪平,彭镇华,高志民等.绿竹光系统Ⅰ捕光叶绿素a/b结合蛋白基因的cDNA全长克隆及分析.安徽农业大学学报, 2010, 37 (4): 643-648.
欧阳海.论竹类在中国园林建设中的作用.广东园林, 2005, 28 (2): 29-32.
唐文莉,彭镇华,高健等.刚竹属3种重要散生竹光系统Ⅰ基因(Lhca 1)的克隆、序列分析和蛋白结构预测.北京林业大学学报, 2008, 30 (4): 109-115.
王强.光合作用光抑制的研究进展.植物学通报, 2003, 20 (5): 1003-2266.
韦朝领,江昌俊,陶汉之等.茶树鲜叶片叶黄素循环组分的高效液相色谱法测定研究及其光保护功能鉴定.茶叶科学, 2004, 24 (1): 60-64.
许大全.植物光胁迫研究中的几个问题.植物生理学通讯, 2003, 39 (5): 493-495.
叶志敏.竹子在现代城市景观设计中的应用.世界竹藤通讯, 2009, 7 (4): 29-32.
郑容妹.盐分胁迫对沿海绿竹光合作用及叶绿素的影响.竹子研究汇刊, 2002, 21 (4):76-80.