马铃薯线粒体靶向表达载体的构建与应用  

Construction and Application of Potato Mitochondrial Targeted Expression Vector

作　　者：张震 李清 徐菁 陈凯园 张春芝 祝光涛 ZHANG Zhen;LI Qing;XU Jing;CHEN Kai-yuan;ZHANG Chun-zhi;ZHU Guang-tao(Joint Academy of Potato Sciences,Yunnan Normal University,Kunming 650500;Agricultural Genomics Institute,Chinese Academy of Agricultural Sciences,Shenzhen 518124)

机构地区：[1]云南师范大学马铃薯科学研究院,昆明650500 [2]中国农业科学院农业基因组研究所,深圳518124

出　　处：《生物技术通报》2024年第5期66-73,共8页Biotechnology Bulletin

基　　金：国家重点研发计划(2022YFF1002500);国家自然科学基金项目(32272725)。

摘　　要：【目的】植物线粒体基因变异是产生细胞质雄性不育(cytoplasmic male sterility,CMS)的主要原因,利用生物技术是创造CMS不育系的重要手段。通过比较启动子、线粒体转运信号肽(mitochondrial targeting signal peptides,MTS)的不同组合对下游eGFP的表达强弱,来探究最佳马铃薯线粒体靶向表达载体。【方法】选择3个启动子AtUBI10、StUBI10、2×35S和两个线粒体转运信号肽ATPγ与Rf1b进行试验,利用烟草叶片瞬时表达系统和PEG介导马铃薯原生质体转化体系进行靶向载体的表达验证。【结果】六个线粒体靶向表达载体中AtUBI10::ATPγ-eGFP在烟草瞬时表达效果最佳,可准确定位到线粒体,其次为StUBI10::ATPγ-eGFP和2×35S::ATPγ-eGFP;在马铃薯原生质体中载体AtUBI10::ATPγ-eGFP有最佳的表达效果,与烟草中表达结果一致,其次是StUBI10::Rf1b-eGFP和AtUBI10::Rf1b-eGFP,而2×35S启动子的两个载体在原生质体均表达较弱。【结论】启动子AtUBI10与线粒体转运信号肽ATPγ所构建的靶向线粒体载体为最佳组合,可用于马铃薯线粒体基因的相关研究中。【Objective】The genetic variations of mitochondria genome usually lead to cytoplasmic male sterility(CMS)in plants.Biotechnology is an important measure to produce variations resulting in CMS lines.In order to establish an efficient expression vector system for the research on potato mitochondrial genes,the optimal potato mitochondrial targeting expression vector was explored by comparing the expression level of eGFP,which was promoted by different combinations of promoters and mitochondrial targeting signal peptides(MTS).【Method】Three promoters AtUBI10,StUBI10,2×35S and two mitochondrial transport signal peptides ATPγand Rf1b were tested.The expression level of the target vector was verified by tobacco leaf transient expression system and PEG-mediated potato protoplast transformation system.【Result】AtUBI10::ATPγ-eGFP among 6 expressing vectors had the best expression effect in tobacco and was accurately located in mitochondria.The second best ones were StUBI10::ATPγ-eGFP and 2×35S::ATPγ-eGFP.The vector AtUBI10::ATPγ-eGFP also showed the best performance,followed by StUBI10::Rf1b-eGFP and AtUBI10::Rf1b-eGFP in the potato protoplast transformation system.The two vectors with 2×35S promoter were weakly expressed in protoplasts.【Conclusion】The comparative studies showed the mitochondrial vector constructed by the promoter AtUBI10 and the mitochondrial transport signal peptide ATPγis the best combination,and this vectors will be of great benefit to the relative study on mitochondrial genes.

关 键 词：马铃薯 线粒体转运信号肽 靶向表达载体 亚细胞定位 细胞质雄性不育 

分 类 号：R73[医药卫生—肿瘤]