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 E nrichment of Cadmium in Plants

作　　者：陈悦 黄先敏[1] 张永至 马仲炼[1] 马永翠 马沙 张玉婷 刘玉梅 周仁洪 李昆志[2] 陈玉惠 Chen Yue;Huang Xianmin;Zhang Yongzhi;Ma Zhonglian;Ma Yongcui;Ma Sha;Zhang Yuting;Liu Yumei;Zhou Renhong;Li Kunzhi;Chen Yuhui(Zhaotong College,Zhaotong 657000,China;Kunming University of Science and Technology,Kunming 650500,China;Zhaoyang District Plant Protection and Inspection Station,Zhaotong 657000,China)

机构地区：[1]昭通学院,昭通657000 [2]昆明理工大学,昆明650500 [3]昭阳区植保检植站,昭通657000

出　　处：《生态毒理学报》2024年第4期324-337,共14页Asian Journal of Ecotoxicology

基　　金：云南省天麻与真菌共生生物学重点实验室建设项目;云南省天麻绿色种植与加工工程研究中心;云南省高校重点实验室项目(X212200002);昭通市兴昭人才项目(昭党人才【2023】3号)。

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 c erevisiae)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)。100μmol·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50μmol·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。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 S accharomyces 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-αwith 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 s ystem.The transgenic tobacco plants treated with a 100μmol·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 m itigate 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 50μmol·L-1 Cd.The results revealed a 30%increase in Cd accumulation in the roots and a four-fold higher a ccumulation in the stems of transgenic plants compared to those of WT.These findings provide evidence tha

关 键 词：转基因植物 啤酒酵母-α信号肽 金属硫蛋白分泌 融合基因 Cd富集 

分 类 号：X171.5[环境科学与工程—环境科学]