叶绿体中过表达乙醇酸氧化酶对水稻光合作用与生长的影响

doi:10.13560/j.cnki.biotech.bull.1985.2025-0550

摘要/Abstract

摘要：

目的验证植物叶绿体具有代谢乙醛酸生成CO₂的能力。方法克隆了水稻的乙醇酸氧化酶1（OsGLO1）、乙醇酸氧化酶3（OsGLO3）与过氧化氢酶C（OsCATC）以及来源于大肠杆菌的过氧化氢酶（EcKAT）基因，并在其前端融合了叶绿体定位信号编码序列RC2；后续以不同的组合方式将GLO与CAT/KAT进行组合以构建不同多基因表达载体，并将其在水稻叶绿体中定向表达，从而催化叶绿体中的乙醇酸生成乙醛酸。结果转录水平、蛋白水平和酶活水平检测结果都表明上述目的基因可在水稻叶绿体中高效表达并行使正常的催化功能；但是获得的转基因水稻植株均呈现植株生长缓慢，分蘖数减少，光合速率下降等表型。结论仅在水稻叶绿体中将乙醇酸转化为乙醛酸不能提高水稻的光合固碳效率，水稻叶绿体可能没有催化乙醛酸生成CO₂进入光合碳同化代谢的能力。

关键词: 乙醇酸氧化酶, 过氧化氢酶, 乙醛酸, 叶绿体, 光合作用, CO₂, 水稻, 光呼吸

Abstract:

Objective Validate the hypothesis that plant chloroplasts possess the ability to metabolize glyoxylate into CO₂. Method In this study, the rice glycolate oxidase 1 (OsGLO1), glycolate oxidase 3 (OsGLO3) and catalase C (OsCATC) genes and the catalase (EcKAT) gene from Escherichia coli were cloned, and the chloroplast localization signal coding sequence RC2 was fused to their front ends; subsequently, GLO and CAT/KAT were combined in different combinations to construct different multi-gene expression vectors, which were directed its expression in rice chloroplasts, thereby catalyzing the production of glyoxylic acid from glycolic acid in chloroplasts. Result Results from transcript-level, protein-level, and enzyme activity analyses confirmed the high-efficiency expressions of target genes in rice and having normal catalytic function. However, all transgenic rice plants had phenotypes such as dwarfism, reduced tiller number, and decreased photosynthesis. Conclusion These findings indicate that only converting glycolate into glyoxylate in chloroplasts fails to enhance photosynthetic carbon fixation efficiency in rice, suggesting that rice chloroplasts may lack the capacity to catalyze glyoxylate into CO₂.

Key words: glycolate oxidase, catalase, glyoxylate, chloroplasts, photosynthesis, CO₂, rice, photorespiration

李波娣, 李志超, 朱国辉, 彭新湘, 张智胜. 叶绿体中过表达乙醇酸氧化酶对水稻光合作用与生长的影响[J]. 生物技术通报, 2025, 41(10): 87-97.

LI Bo-di, LI Zhi-chao, ZHU Guo-hui, PENG Xin-xiang, ZHANG Zhi-sheng. Expression of Glycolate Oxidase in Rice Chloroplasts and Its Effects on Photosynthesis and Growth[J]. Biotechnology Bulletin, 2025, 41(10): 87-97.

图/表 8

表1 GCMS法测定光呼吸相关代谢物时的柱温变化

Table 1 Variation of column temperature during the determination of photorespiration-related metabolites by GCMS method

项目 Item

eating rate （℃/min）

升温速率 H

inal temperature （℃）

最终温度 F

保持时间 Holding time （min）

变化范围

Range of variation

表2 光呼吸相关代谢物的保留时间及其特征碎片离子

Table 2 Retention times of photorespiration-related metabolites and their characteristic fragment ions

组分 Component	特征碎片离子大小 Characteristic fragment ion size	etention time （s）保留时间 R
乙醇酸 Glycollic acid	177	7.455
丙氨酸 Alanine	116	8.045
草酸 Oxalic acid	219	8.740
甘氨酸 Glycine	174	12.000
琥珀酸 Succinic acid	147	12.140
甘油酸 Glyceric acid	189	12.380
丝氨酸 Serine	204	12.870
苹果酸 Malic acid	117	14.743
α-酮戊二酸 α-ketoglutarate	198	15.910
核糖醇 Ribitol	73	17.760
柠檬酸 Citric acid	273	18.845

图1 水稻叶绿体光呼吸代谢改造GLO3-CAT途径示意图GLO：乙醇酸氧化酶3；CAT：过氧化氢酶

Fig. 1 Schematic diagram of GLO3-CAT pathway by the chloroplast photorespiratory metabolism in riceGLO: Glycolate oxidase 3. CAT: Catalase

图2 转基因水稻的获得及鉴定A：水稻遗传转化过程（a1：愈伤组织筛选；a2：预分化；a3：分化；a4：生根壮苗）；B：转基因水稻目的基因的Western blot检测，GLO和CAT均使用His单克隆抗体进行检测，等蛋白上样；C‒E：转基因水稻目的基因转录水平检测（C：OsGLO1转录水平；D：OsGLO3转录水平；E：OsCATC转录水平）；F：GLO活性；G：G1K-OE和G3C-OE的CAT活性；H：转基因水稻叶片的DAB染色；I：RbOH-b相对表达量；G1-OE：叶绿体GLO1过表达株系；G1K-OE：叶绿体GLO1-KAT过表达株系；G3-OE：叶绿体GLO3过表达株系；G3C-OE：叶绿体GLO3-CAT过表达株系；n=3，*P＜0.05，**P＜0.01，***P＜0.001，t检验。下同

Fig. 2 Acquirement and identification of transgenic rice plantsA: Transfomation procedure of rice (a1: screening of callus; a2: pre-differentiation; a3: differentiation; a4: rooting and strenghening). B: Western blot analysis of transgenic plants, GLO and CAT were detected by His monoclonal antibody in Western blot analysis. C-E: mRNA levels of transgenic plants (C: mRNA levels of OsGLO1; D: mRNA levels of OsGLO3; E: mRNA levels of CAT). F: GLO activity. G: CAT activity of G1K-OE and G3C-OE transgenic plants. H: DAB-staining of transgenic rice leaves. I: Relative expression of RbOH-b. G1-OE: Overexpressed line of chloroplast GLO1. G1K-OE: Over expressed line of chloroplast GLO1-KAT. G3-OE: Overexpressed line of chloroplast GLO3. G3C-OE: Overexpressed line of chloroplast GLO3-CAT. n=, *P＜0.05, **P＜0.01, ***P＜0.001, t-test. The same below

图3 转基因水稻的表型观察A：转基因水稻幼苗期表型观察；B：株高；C：最大根长；D：G1-OE与G1K-OE转基因水稻；E：剑叶长度；F：剑叶宽度；G：G3-OE与G3C-OE转基因水稻；H：株高；I：分蘖数；n=10

Fig. 3 Phenotypic observation of transgenic rice plantsA: Phenotypic observation of transgenic plants at seedling stage. B: Plant height. C: Maximum length of root. D: Transgenic rice plants of G1-OE and G1K-OE. E: Length of flag leaf. F: Width of flag leaf. G: Transgenic rice plants of G3-OE and G3C-OE. H: Plant height. I: Number of tillers. n=10

图4 转基因植株分蘖期叶片光合相关参数和碳水化合物含量测定A， B： n=10； C， D： n=3

Fig. 4 Analysis of the photosynthetic-related parameters and carbohydrate content in the leaves of transigenic plants at tillering stage

图5 GCGT转基因植株幼苗期叶片光呼吸相关代谢物含量的测定自然光下用木村B营养液水培至第4叶完全展开，取第2片叶，取样时间为13：00‒15：00，n=3

Fig. 5 Content determination of photorespiration metabolites of GCGT tansgenic plants leaves at seedling stageTake the second leaf after hydroponic with Kimura B nutrient solution under natural light until the fourth leaf fully spreads, and the sampling time is 13:00‒15:00, n=3

图6 转基因水稻PLGG1的转录水平分析

Fig. 6 Analysis of mRNA levels of PLGG1 in transgenic rice plants (n=3)

参考文献 28

[1]	Falcon WP, Naylor RL, Shankar ND. Rethinking global food demand for 2050 [J]. Popul Dev Rev, 2022, 48(4): 921-957.
[2]	Tian ZX, Wang JW, Li JY, et al. Designing future crops: challenges and strategies for sustainable agriculture [J]. Plant J, 2021, 105(5): 1165-1178.
[3]	Croce R, Carmo-Silva E, Cho YB, et al. Perspectives on improving photosynthesis to increase crop yield [J]. Plant Cell, 2024, 36(10): 3944-3973.
[4]	Zhu XG, Long SP, Ort DR. Improving photosynthetic efficiency for greater yield [J]. Annu Rev Plant Biol, 2010, 61: 235-261.
[5]	Jin KN, Chen GX, Yang YR, et al. Strategies for manipulating rubisco and creating photorespiratory bypass to boost C₃ photosynthesis: prospects on modern crop improvement [J]. Plant Cell Environ, 2023, 46(2): 363-378.
[6]	张智胜, 朱国辉, 彭新湘. 优化碳同化实现作物高光效研究进展 [J]. 华南农业大学学报, 2022, 43(6): 69-77.
	Zhang ZS, Zhu GH, Peng XX. Advances in improvement of crop photosynthetic efficiency by optimizing the photosynthetic carbon assimilation [J]. J South China Agric Univ, 2022, 43(6): 69-77.
[7]	Zhang ZS, Zhu GH, Peng XX. Photorespiration in plant adaptation to environmental changes [J]. Crop Environ, 2024, 3(4): 203-212.
[8]	朱国辉, 张智胜, 彭新湘. 光呼吸演化、调控与遗传改良 [J]. 生命科学, 2024, 36(10): 1226-1239.
	Zhu GH, Zhang ZS, Peng XX. Photorespiration evolution, regulation, and genetic improvement [J]. Chin Bull Life Sci, 2024, 36(10): 1226-1239.
[9]	Ehlers I, Augusti A, Betson TR, et al. Detecting long-term metabolic shifts using isotopomers: CO₂-driven suppression of photorespiration in C₃ plants over the 20th century [J]. Proc Natl Acad Sci USA, 2015, 112(51): 15585-15590.
[10]	Chen GX, Li YN, Jin KN, et al. Synthetic photorespiratory bypass improves rice productivity by enhancing photosynthesis and nitrogen uptake [J]. Plant Cell, 2024, 37(1): koaf015.
[11]	Wang LM, Shen BR, Li BD, et al. A synthetic photorespiratory shortcut enhances photosynthesis to boost biomass and grain yield in rice [J]. Mol Plant, 2020, 13(12): 1802-1815.
[12]	Shen BR, Wang LM, Lin XL, et al. Engineering a new chloroplastic photorespiratory bypass to increase photosynthetic efficiency and productivity in rice [J]. Mol Plant, 2019, 12(2): 199-214.
[13]	South PF, Cavanagh AP, Liu HW, et al. Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field [J]. Science, 2019, 363(6422): eaat9077.
[14]	Kebeish R, Niessen M, Thiruveedhi K, et al. Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana [J]. Nat Biotechnol, 2007, 25(5): 593-599.
[15]	Nölke G, Houdelet M, Kreuzaler F, et al. The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield [J]. Plant Biotechnol J, 2014, 12(6): 734-742.
[16]	Dalal J, Lopez H, Vasani NB, et al. A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa [J]. Biotechnol Biofuels, 2015, 8: 175.
[17]	Li BD, Huang AY, Wang LM, et al. Increased sugar content impairs pollen fertility and reduces seed-setting in high-photosynthetic-efficiency rice [J]. Crop J, 2024, 12(6): 1547-1558.
[18]	Shen BR, Zhu CH, Yao Z, et al. An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice [J]. Sci Rep, 2017, 7: 46231.
[19]	Xu Z, Guo WD, Mo BQ, et al. Mitogen-activated protein kinase 2 specifically regulates photorespiration in rice [J]. Plant Physiol, 2023, 193(2): 1381-1394.
[20]	Mo BQ, Chen XF, Yang JJ, et al. Engineering of photorespiration-dependent glycine betaine biosynthesis improves photosynthetic carbon fixation and panicle architecture in rice [J]. J Integr Plant Biol, 2025, 67(4): 979-992.
[21]	Oliver DJ, Zelitch I. Increasing photosynthesis by inhibiting photorespiration with glyoxylate [J]. Science, 1977, 196(4297): 1450-1451.
[22]	Li XY, Chen LR, Zeng XY, et al. Wounding induces a peroxisomal H₂O₂ decrease via glycolate oxidase-catalase switch dependent on glutamate receptor-like channel-supported Ca²⁺ signaling in plants [J]. Plant J, 2023, 116(5): 1325-1341.
[23]	Li XY, Liao MM, Huang JY, et al. Dynamic and fluctuating generation of hydrogen peroxide via photorespiratory metabolic channeling in plants [J]. Plant J, 2022, 112(6): 1429-1446.
[24]	Zhang ZS, Liang X, Lu L, et al. Two glyoxylate reductase isoforms are functionally redundant but required under high photorespiration conditions in rice [J]. BMC Plant Biol, 2020, 20(1): 357.
[25]	Lu YS, Li Y, Yang QS, et al. Suppression of glycolate oxidase causes glyoxylate accumulation that inhibits photosynthesis through deactivating rubisco in rice [J]. Physiol Plant, 2014, 150(3): 463-476.
[26]	Xu HW, Zhang JJ, Zeng JW, et al. Inducible antisense suppression of glycolate oxidase reveals its strong regulation over photosynthesis in rice [J]. J Exp Bot, 2009, 60(6): 1799-1809.
[27]	Mittler R, Zandalinas SI, Fichman Y, et al. Reactive oxygen species signalling in plant stress responses [J]. Nat Rev Mol Cell Biol, 2022, 23(10): 663-679.
[28]	Aroca A, García-Díaz I, García-Calderón M, et al. Photorespiration: regulation and new insights on the potential role of persulfidation [J]. J Exp Bot, 2023, 74(19): 6023-6039.