过量表达分泌型MT3对植物镉富集的影响
Effect of Overexpression of Secretory MT3 in Transgenic Plants on Enrichment of Cadmium in Plants
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摘要: 金属硫蛋白(metallothionein, MT)MT3是人体中参与重金属解毒的主要蛋白，前期研究表明啤酒酵母(Saccharomyces cerevisiae)α因子信号肽(MF-α)介导重组蛋白EGFP分泌到植物体外。但是目前还没有研究报道转基因植物中过量表达分泌型MT3对植物重金属镉(Cd)的富集能力是否有影响。本研究通过人工方法合成MF-α信号肽、增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)和MT3的融合基因MF-α-EGFP-MT3，构建该融合基因的植物表达载体pK-35S-MF-α-EGFP-MT3，转化野生型(WT)烟草和天竺葵获得转基因植物。通过电化学方法检测转基因植物MT3转录水平、转基因植物根系分泌液中EGFP-MT3蛋白的水平。用Cd溶液处理转基因植物，通过表型观察和电化学方法检测根、茎和叶中Cd的含量。结果表明，转基因烟草和天竺葵中都有MT3基因的转录；且根系分泌EGFP-MT3蛋白的量大约为0.45～0.68 mg·g-1(以鲜质量计)。100 μmol·L-1的Cd溶液处理转基因烟草植株，表型变化分析发现转基因植株受损情况低于WT，近根部叶片叶绿素含量显著高于WT，说明EGFP-MT3的分泌可降低Cd的毒害作用。转基因烟草植株根、茎和叶片对Cd的富集量比WT高约40%。用50 μmol·L-1的Cd溶液处理转基因天竺葵植株，结果表明转基因植株根对Cd的富集量比WT高约30%，茎对Cd的富集量比WT高约4倍。以上结果证明过量表达EGFP-MT3可以提高转基因烟草和天竺葵对Cd的富集能力，可能是EGFP-MT3分泌根系表面增加转基因植物根系对Cd的吸附作用，同时在转基因植物组织细胞内积累的EGFP-MT3也可增加植物组织对Cd的富集作用。Abstract: Metallothionein (MT), especially MT3, is the main protein involved in the detoxification of heavy metals within the human body. Previous studies have demonstrated that the alpha factor signal peptide (MF-α) from Saccharomyces cerevisiae mediates the secretion of recombinant protein EGFP outside plants. However, there is currently no available research on whether overexpression secretory MT3 in genetically modified plants affects their ability to accumulate cadmium (Cd), a heavy metal. In this study, we synthesized the fusion gene MF-α-EGFP-MT3 using artificial methods, which combines the signal peptide MF-α with enhanced green fluorescent protein (EGFP) and MT3. Subsequently, we constructed a plant expression vector pK-35S-MF-α-EGFP-MT3 carrying this fusion gene and transformed it into wild-type (WT) tobacco and Geranium plants to obtain transgenic plants. Using electrochemical methods, we detected the transcription level of MT3 in genetically modified plants and measured the level of EGFP-MT3 protein in the root exudates. After treating genetically modified plants with a Cd solution, we analyzed measured Cd content in roots, stems, and leaves through phenotype observation and electrochemical methods. Results showed that both genetically modified tobacco and Geranium transcribed the MT3 gene. Furthermore, approximately 0.45～0.68 mg·g-1 (based on fresh mass) of EGFP-MT3 protein was secreted by the root system. The transgenic tobacco plants treated with a 100 μmol·L-1 Cd exhibited a reduced damage phenotype and significantly higher chlorophyll content compared to the WT, indicating that the secretion of EGFP-MT3 can mitigate the toxic effects of cadmium. Genetically modified tobacco plants had approximately 40% higher accumulation of Cd in their roots, stems, and leaves compared to the WT. Transgenic Geranium plants were exposed to a 50 μmol·L-1 Cd. The results revealed a 30% increase in Cd accumulation in the roots and a four-fold higher accumulation in the stems of transgenic plants compared to those of WT. These findings provide evidence that the overexpression of EGFP-MT3 enhances the capacity of transgenic tobacco and Geranium plants to accumulate Cd, potentially due to an increased adsorption capacity resulting from the secretion of EGFP-MT3 on root surfaces and its accumulation within plant tissues.
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张云, 王丹媚, 王孝源, 等. 外源茉莉酸对菊芋镉胁迫下光合特性及镉积累的影响[J]. 作物学报, 2021, 47(12): 2490-2500 Zhang Y, Wang D M, Wang X Y, et al. Effects of exogenous jasmonic acid on photosynthetic characteristics and cadmium accumulation of Helianthus tuberosus L. under cadmium stress [J]. Acta Agronomica Sinica, 2021, 47(12): 2490-2500 (in Chinese)
吴博晗, 吴向阳, 李霞, 等. 镉对水稻及种植土壤影响的研究进展[J]. 江苏农业科学, 2021, 49(18): 25-33 Wu B H, Wu X Y, Li X, et al. Research progress on effects of cadmium on rice and planting soil [J]. Jiangsu Agricultural Sciences, 2021, 49(18): 25-33 (in Chinese)
Luo S L, Calderón-Urrea A, Yu J H, et al. The role of hydrogen sulfide in plant alleviates heavy metal stress [J]. Plant and Soil, 2020, 449(1): 1-10 曾希柏, 徐建明, 黄巧云, 等. 中国农田重金属问题的若干思考[J]. 土壤学报, 2013, 50(1): 186-194 Zeng X B, Xu J M, Huang Q Y, et al. Some deliberations on the issues of heavy metals in farmlands of China [J]. Acta Pedologica Sinica, 2013, 50(1): 186-194 (in Chinese)
张义贤. 汞、镉、铅胁迫对油菜的毒害效应[J]. 山西大学学报(自然科学版), 2004, 27(4): 410-413 Zhang Y X. The toxic effects of mercury, cadmium and lead to Brasica chinesis L. [J]. Journal of Shanxi University (Natural Science Edition), 2004, 27(4): 410-413 (in Chinese) 周艳. GSH缓解番茄幼苗盐胁迫的耐盐机制研究[D]. 石河子: 石河子大学, 2019: 56-68 Zhou Y. Salt-tolerant mechanism of GSH alleviates salt-induced stress in tomato seedlings [D]. Shihezi: Shihezi University, 2019: 56 -68 (in Chinese)
Wang F J, Tan H F, Zhang Y T, et al. Salicylic acid application alleviates cadmium accumulation in brown rice by modulating its shoot to grain translocation in rice [J]. Chemosphere, 2021, 263: 128034 Herbette S, Taconnat L, Hugouvieux V, et al. Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots [J]. Biochimie, 2006, 88(11): 1751-1765 Razinger J, Dermastia M, Koce J D, et al. Oxidative stress in duckweed (Lemna minor L.) caused by short-term cadmium exposure [J]. Environmental Pollution, 2008, 153(3): 687-694 车涛, 张丽萍, 金竹萍, 等. 硫化氢对镉胁迫下大白菜幼苗光合生理的影响[J]. 农业环境科学学报, 2019, 38(5): 1008-1016 Che T, Zhang L P, Jin Z P, et al. Hydrogen sulfide regulates the photosynthetic physiology of Brassica pekinensis (Lour.) Rupr. under cadmium stress [J]. Journal of Agro-Environment Science, 2019, 38(5): 1008-1016 (in Chinese)
Li Q, Wang G, Wang Y R, et al. Foliar application of salicylic acid alleviate the cadmium toxicity by modulation the reactive oxygen species in potato [J]. Ecotoxicology and Environmental Safety, 2019, 172: 317-325 Oz G, Zangger K, Armitage I M. Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: Metallothionein-3 [J]. Biochemistry, 2001, 40(38): 11433-11441 Jin S M, Xu C, Li G L, et al. Functional characterization of a type 2 metallothionein gene, SsMT2, from alkaline-tolerant Suaeda salsa [J]. Scientific Reports, 2017, 7(1): 17914 陈逸云. 芹菜金属硫蛋白基因的克隆和功能研究[D]. 南京: 南京农业大学, 2016: 28 Chen Y Y. Cloning and characterization of the gene encoding metallothionein in celery [D]. Nanjing: Nanjing Agricultural University, 2016: 28 (in Chinese) 杜佳, 杨静莉, 李成浩. 林木金属硫蛋白研究进展[J]. 植物生理学报, 2011, 47(8): 759-764 Du J, Yang J L, Li C H. Research progress in forest tree metallothioneins [J]. Plant Physiology Journal, 2011, 47(8): 759-764 (in Chinese)
Kim S H, Song W Y, Ahn Y O, et al. Enhancement of cadmium resistance by overexpression of BrMT3 in Arabidopsis [J]. Journal of Plant Biotechnology, 2009, 36(1): 68-74 Obertello M, Wall L, Laplaze L, et al. Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca [J]. Molecular Plant-Microbe Interactions, 2007, 20(10): 1231-1240 Shahpiri A, Deljoonia R. Enhancement of catalase and superoxide dismutase activities in transgenic Escherichia coli expressing rice metallothionein isoforms [J]. Journal of BioScience and Biotechnology, 2018, 7: 5-10 Patankar H V, Al-Harrasi I, Al Kharusi L, et al. Overexpression of a metallothionein 2A gene from date palm confers abiotic stress tolerance to yeast and Arabidopsis thaliana [J]. International Journal of Molecular Sciences, 2019, 20(12): 2871 Zhang H X, Lv S F, Xu H W, et al. H2O2 is involved in the metallothionein-mediated rice tolerance to copper and cadmium toxicity [J]. International Journal of Molecular Sciences, 2017, 18(10): 2083 曹文佳. 过表达分泌型植物金属硫蛋白增强转基因烟草富集重金属的机理和抗性[D]. 云南: 昆明理工大学, 2019: 35-46 Cao W J. Overexpression of secretory plant metallothionein enhances the mechanism and resistance of transgenic tobacco to heavy metal enrichment [D]. Yunnan: Kunming University of Science and Technology, 2019: 35 -46 (in Chinese)
李胜昔. 电化学免疫传感器在肿瘤标志物检测中的应用研究[D]. 上海: 复旦大学, 2012: 45-36 Li S X. Research of electrochemical immunosensor for the detection of tumor biomarker [D]. Shanghai: Fudan University, 2012: 45 -36 (in Chinese)
翁芝莹. 快速检测食品中痕量铅镉、铜的电化学传感方法的研究[D]. 上海: 上海交通大学, 2012: 23-24 Weng Z Y. Study on electrochemical sensing method for rapid detection of trace amounts of lead, cadmium, and copper in food [D]. Shanghai: Shanghai Jiao Tong University, 2012: 23 -24 (in Chinese)
Li L S, Meng Y P, Cao Q F, et al. Type 1 metallothionein (ZjMT) is responsible for heavy metal tolerance in Ziziphus jujuba [J]. Biochemistry Biokhimiia, 2016, 81(6): 565-573 许黎明, 蒋国凤, 周兴. 人金属硫蛋白基因(MT1E)在毕赤酵母中的表达及其铬抗性[J]. 环境科学学报, 2014, 34(10): 2724-2730 Xu L M, Jiang G F, Zhou X. Transgenic Pichia pastoris over-expressing human metallothionein gene (MT1E) on heavy metal chromium stress [J]. Acta Scientiae Circumstantiae, 2014, 34(10): 2724-2730 (in Chinese)
彭德莲, 刘霞, 黄亚东, 等. 人金属硫蛋白在乳酸菌中的融合表达及其预防食源性镉污染的应用[J]. 中国生物工程杂志, 2018, 38(3): 41-50 Peng D L, Liu X, Huang Y D, et al. Prevention of gastrointestinal cadmium poisoning with food-grade Lactococcus lactis strain expressing metallothionein fusion protein [J]. China Biotechnology, 2018, 38(3): 41-50 (in Chinese)
Liu Y, Kang T, Cheng J S, et al. Heterologous expression of the metallothionein PpMT2 gene from Physcomitrella patens confers enhanced tolerance to heavy metal stress on transgenic Arabidopsis plants [J]. Plant Growth Regulation, 2020, 90(1): 63-72 Chaturvedi A K, Patel M K, Mishra A, et al. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco [J]. PLoS One, 2014, 9(10): e111379 Balasundaram U, Venkataraman G, George S, et al. Metallothioneins from a hyperaccumulating plant Prosopis juliflora show difference in heavy metal accumulation in transgenic tobacco [J]. International Journal of Agriculture, Environment and Biotechnology, 2014, 7(2): 241 范仲学, 蔡利娟, 杜瑞雪, 等. 龙葵SorMT2a和SorMT2c基因的克隆及其转化烟草研究[J]. 山东农业科学, 2010, 42(12): 1-5 Fan Z X, Cai L J, Du R X, et al. Cloning of SorMT2a and SorMT2c from Solanum nigrum and study on their transformation to Nicotiana tobacum [J]. Shandong Agricultural Sciences, 2010, 42(12): 1-5 (in Chinese)
Rono J K, Le-Wang L, Wu X C, et al. Identification of a new function of metallothionein-like gene OsMT1e for cadmium detoxification and potential phytoremediation [J]. Chemosphere, 2021, 265: 129136 张威, 杜婕, 黄亚群. 植物中过表达MT转录因子调控镉胁迫响应的Meta分析[J]. 河北农业大学学报, 2022, 45(1): 30-38 Zhang W, Du J, Huang Y Q. Meta-analysis of the effects of overexpressing of MT transcription factors on plant responses to cadmium stress [J]. Journal of Hebei Agricultural University, 2022, 45(1): 30-38 (in Chinese)
卢碧霞. 转金属硫蛋白基因水稻的特异表达及其对重金属抗性的研究[D]. 杨凌: 西北农林科技大学, 2006: 86-87 Lu B X. The expression of metallothionein gene under tissue-specific promoter in rice and the tolerance to heavy metals in transgenic lines [D]. Yangling: Northwest A & F University, 2006: 86 -87 (in Chinese)
Yang J L, Wang Y C, Liu G F, et al.Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast [J]. Molecular Biology Reports, 2011, 38(3): 1567-1574 Mekawy A M M, Assaha D V M, Ueda A. Constitutive overexpression of rice metallothionein-like gene OsMT-3a enhances growth and tolerance of Arabidopsis plants to a combination of various abiotic stresses [J]. Journal of Plant Research, 2020, 133(3): 429-440 -
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