蒺藜苜蓿叶绿素酸酯a加氧酶(MtCAO)基因的克隆与功能分析

doi:10.11686/cyxb2019447

摘要/Abstract

摘要： 叶绿素含量直接影响植物光合效率,从而决定其生物量。叶绿素酸酯a加氧酶(CAO)催化叶绿素酸酯a转化成叶绿素酸酯b,是叶绿素合成的限速酶之一。本研究克隆了蒺藜苜蓿MtCAO,该基因编码531个氨基酸。进化分析表明,MtCAO与双子叶植物的CAO亲缘关系较近。高等植物CAO基因的结构保守,均含有9个外显子。MtCAO主要在绿色组织尤其是叶片中高丰度表达。瞬时表达拟南芥原生质体试验显示,MtCAO-GFP融合蛋白定位于叶绿体。表达模式分析表明,茉莉酸甲酯(MeJA,100 μmol·L^-1)及黑暗处理抑制MtCAO的表达,而24 h内NaCl (100 mmol·L^-1)及聚乙二醇(PEG,5%) 处理先诱导后抑制其表达,且该基因受昼夜节律调控。这与对MtCAO启动子区顺式作用元件的预测结果一致。类似于超表达AtCAO的拟南芥,超表达MtCAO的拟南芥株系叶色正常,叶绿素含量及鲜重与野生型差异不显著,推测是由于MtCAO高度保守的A结构域中的降解子导致的。

关键词: 蒺藜苜蓿, 叶绿素酸酯a加氧酶, 叶绿体定位, 昼夜节律, 转基因拟南芥

Abstract: Plant biomass is determined by photosynthesis efficiency, which in turn is affected directly by the chlorophyll concentration. Chlorophyllide a oxygenase (CAO) is one of the three speed-limiting enzymes in the chlorophyll synthesis pathway, and catalyzes chlorophyllide a into chlorophyllide b. In this study, the open reading frame of Medicago truncatula CAO (MtCAO), which encodes a protein of 531 amino acid residues, was cloned. Phylogenetic analysis demonstrated that MtCAO was closer to its dicot homologs than to those in more distantly related plant species. Different from CAOs in lower plants, CAOs in higher plants contain nine exons. MtCAO was preferentially expressed in green tissues, especially leaves. When transiently expressed, the MtCAO-GFP recombinant protein was localized in the chloroplast of Arabidopsis protoplast cells. The transcript of MtCAO was repressed by the treatment with methyl jasmonate (100 μmol·L^-1) or darkness, but induced by NaCl (100 mmol·L^-1) or PEG (5%) within 4 h then repressed until 12 h from application. Also, the expression of MtCAO fluctuated with the circadian clock. These results are consistent with the cis-element analysis of the MtCAO promoter. MtCAO was ecotopically expressed in Arabidopsis. The analysis of two independent transgenic lines revealed no significant difference in the content of chlorophyll, the chlorophyll a∶b ratio or shoot weight from the wild type. The results are similar to those from AtCAO overexpression Arabidopsis. Therefore, we postulate that the non-abnormal phenotype of the transgenic Arabidopsis constitutively expressing MtCAO probably results from the fact that the conserved A domain contains a “degron” motif, which was proven to hamper the elevation of AtCAO in Arabidopsis overexpressing AtCAO.

Key words: Medicago truncatula, chlorophyllide a oxygenase, chloroplast-localization, circadian clock, transgenic Arabidopsis thaliana

刘文文, 崔会婷, 尉春雪, 龙瑞才, 康俊梅, 杨青川, 王珍. 蒺藜苜蓿叶绿素酸酯a加氧酶(MtCAO)基因的克隆与功能分析[J]. 草业学报, 2020, 29(5): 171-181.

LIU Wen-wen, CUI Hui-ting, WEI Chun-xue, LONG Rui-cai, KANG Jun-mei, YANG Qing-chuan, WANG Zhen. Cloning and functional analysis of chlorophyllide a oxygenase encoding gene MtCAO in Medicago truncatula[J]. Acta Prataculturae Sinica, 2020, 29(5): 171-181.

参考文献

[1] Lim H, Tanaka A, Tanaka R, et al. In vitro enzymatic activity assays implicate the existence of the chlorophyll cycle in chlorophyll b-containing cyanobacteria. Plant and Cell Physiology, 2019, 60(12): 2672-2683.
[2] Sakuraba Y, Tanaka R, Yamasato A, et al. Determination of a chloroplast degron in the regulatory domain of chlorophyllide a oxygenase. Journal of Biological Chemistry, 2009, 284(52): 36689-36699.
[3] Beale S I. Green genes gleaned. Trends in Plant Science, 2005, 10(7): 309-312.
[4] Tanaka A, Ito H, Tanaka R, et al. Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(21): 12719-12723.
[5] Oster U, Tanaka R, Tanaka A, et al. Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. The Plant Journal, 2000, 21(3): 305-310.
[6] Oster U, Bauer C E, Rüdiger W. Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli. Journal of Biological Chemistry, 1997, 272(15): 9671-9676.
[7] Espineda C E, Linford A S, Devine D, et al. The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(18): 10507-10511.
[8] Lee S, Kim J H, Yoo E S, et al. Differential regulation of chlorophyll a oxygenase genes in rice. Plant Molecular Biology, 2005, 57(6): 805-818.
[9] Mueller A H, Dockter C, Gough S P, et al. Characterization of mutations in barley fch2 encoding chlorophyllide a oxygenase. Plant and Cell Physiology, 2012, 53(7): 1232-1246.
[10] Jiang K Y, Zhou M B, Yang H Y, et al. Cloning and functional characterization of PjCAO gene involved in chlorophyll b biosynthesis in Pseudosasa japonica cv. Akebonosuji. Trees-Structure and Function, 2016, 30(4): 1303-1314.
[11] Kunugi M, Takabayashi A, Tanaka A. Evolutionary changes in chlorophyllide a oxygenase (CAO) structure contribute to the acquisition of a new light-harvesting complex in micromonas. Journal of Biological Chemistry, 2013, 288(27): 19330-19341.
[12] Liu M, Lu Y, Wang S, et al. Characterization of the leaf color mutant hy and identification of the mutated gene in Chinese cabbage. Journal of the American Society for Horticultural Science, 2018, 143(5): 363-369.
[13] Pattanayak G K, Biswal A K, Reddy V S, et al. Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants. Biochemical and Biophysical Research Communications, 2005, 326(2): 466-471.
[14] Reinbothe C, Bartsch S, Eggink L L, et al. A role for chlorophyllide a oxygenase in the regulated import and stabilization of light-harvesting chlorophyll a/b proteins. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(12): 4777-4782.
[15] Khumaida N, Kisman K, Sopandie D. Cloning and characterization of partial chlorophyll a oxygenase (CAO) gene involved in soybean shade tolerance mechanism. Journal of Tropical Crop Science, 2015, 2(2): 1-4.
[16] Wang Y X, Hu Y, Zhu Y F, et al. Transcriptional and physiological analyses of short-term iron deficiency response in apple seedlings provide insight into the regulation involved in photosynthesis. BMC Genomics, 2018, 19(1): 461.
[17] Biswal A K, Pattanayak G K, Pandey S S, et al. Light intensity-dependent modulation of chlorophyll b biosynthesis and photosynthesis by overexpression of chlorophyllide a oxygenase in tobacco. Plant Physiology, 2012, 159(1): 433-449.
[18] Yang Y, Xu J, Huang L, et al. PGL, encoding chlorophyllide a oxygenase 1, impacts leaf senescence and indirectly affects grain yield and quality in rice. Journal of Experimental Botany, 2016, 67(5): 1297-1310.
[19] Cao L Y, Qian Q, Zhu X D, et al. Breeding of a photo-thermo sensitive genic male sterile indica rice Zhongzi S with a purple-leaf marker and the hoterosis of its hybrid rice produced with it. Acta Agronomica Sinica, 1999, 25(1): 44-49.
曹立勇, 钱前, 朱旭东, 等. 紫叶标记籼型光-温敏核不育系中紫S的选育及其配组的杂种优势. 作物学报, 1999, 25(1): 44-49.
[20] Zhou C, Han L, Pislariu C, et al. From model to crop: Functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement. Plant Physiology, 2011, 157(3): 1483-1496.
[21] Clough S J, Bent A F. Floral dip: A simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal, 1998, 16(6): 735-743.
[22] Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nature Protocols, 2007, 2(7): 1565-1572.
[23] Lichtenthaler H K, Buschmann C. Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. Current Protocols in Food Analytical Chemistry, 2001, 1(1): F4.3.1-F4.3.8.
[24] Yamasato A, Nagata N, Tanaka R, et al. The N-terminal domain of chlorophyllide a oxygenase confers protein instability in response to chlorophyll b accumulation in Arabidopsis. Plant Cell, 2005, 17(5): 1585-1597.
[25] Sakuraba Y, Yamasato A, Tanaka R, et al. Analysis of the N-terminal domain of chlorophyllide a oxygenase by random mutagenesis. Photosynthesis. Dordrecht: Energy from the Sun. Springer, 2008: 1043-1046.
[26] Sakuraba Y, Ryamasato T. Determination of a chloroplast degron in the regulatory domain of chlorophyllide a oxygenase. Journal of Biological Chemistry, 2009, 284(52): 36689.
[27] Sakuraba Y, Yamasato A, Tanaka R, et al. Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase. Plant Physiology Biochemistry, 2007, 45(10/11): 740-749.
[28] Preiss S, Thornber J P. Stability of the apoproteins of light-harvesting complex I and II during biogenesis of thylakoids in the chlorophyll b-less barley mutant chlorina f2. Plant Physiology, 1995, 107(3): 709-717.
[29] Covington M F, Maloof J N, Marty S, et al. Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biology, 2008, 9(8): R13.
[30] Harmer S L, Hogenesch J B, Straume M, et al. Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science, 2000, 290: 2110-2113.
[31] Li H S, Xu Y. Effect of light on synthesis of chlorophylls. Modern Agricultural Science and Technology, 2014, (21): 161-164.
李汉生, 徐永. 光照对叶绿素合成的影响. 现代农业科技, 2014, (21): 161-164.
[32] Wasternack C. Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Annals of Botany, 2007, 100(4): 681-697.
[33] Wang P R, Zhang F T, Gao J X, et al. An overview of chlorophyll biosynthesis in higher plants. Acta Botanica Boreali-Occidentalia Sinica, 2009, 29(3): 629-636.
王平荣, 张帆涛, 高家旭, 等. 高等植物叶绿素生物合成的研究进展. 西北植物学报, 2009, 29(3): 629-636.
[34] Liang S, Zhao G X, Zhu X C. Hyperspectral estimation models of chlorophyll content in apple leaves. Spectroscopy and Spectral Analysis, 2012, 32(5): 1367-1370.
梁爽, 赵庚星, 朱西存. 苹果树叶片叶绿素含量高光谱估测模型研究. 光谱学与光谱分析, 2012, 32(5): 1367-1370.