基于绿色荧光蛋白培育线粒体形态可视的番茄

Visualization of mitochondrial dynamics in tomato based on green fluorescent protein

摘要:

本研究旨在实现番茄线粒体形态特征和动态变化的可视化，以便于研究番茄线粒体功能。通过农杆菌(Agrobacterium)介导的遗传转化技术，获得具有线粒体定位功能的绿色荧光蛋白(mitochondria-green fluorescent protein, Mt-GFP)的转基因番茄，测定转基因Mt-GFP番茄果实在绿熟期、破色期和破色后3 d、6 d、9 d时的色差、硬度、可溶性固形物、酸度、呼吸速率和乙烯释放量，并以野生型番茄果实为对照。结果显示，Mt-GFP蛋白不影响番茄果实正常成熟，但使番茄果实酸度升高。用蛋白免疫印迹成功验证了番茄叶片和果实中Mt-GFP蛋白的表达，并在激光共聚焦显微镜下清晰地观察到花、叶片和果实细胞中线粒体的形态特征以及花细胞中线粒体的动态变化。转基因Mt-GFP番茄植株的培育实现了番茄线粒体可视化，为研究番茄发育及果实成熟过程中的线粒体功能提供了良好的研究材料。

Abstract:

This study aimed to visualize the morphological features and dynamic changes of tomato mitochondria to provide a basis for the study of its mitochondrial functions. In this study, transgenic tomatoes expressing mitochondria-localized green fluorescent protein (mitochondria-GFP, Mt-GFP) were obtained by Agrobacterium-mediated genetic transformation. The color, hardness, soluble solids, acidity content, respiration rate, and ethylene production of the transgenic Mt-GFP tomato fruits were determined at the stage of mature green, breaker, and 3, 6, 9 days after breaker, while the wild-type tomato fruits were used as a control. As expected, Mt-GFP recombinant protein did not affect the ripening process, but induced the increased acidity of tomato fruits. The accumulations of Mt-GFP protein in tomato leaves and fruits were successfully verified by Western blotting. The morphological characteristics of mitochondria in flower, leaf and fruit cells as well as the dynamic changes of mitochondria in flower cells were clearly observed and studied under confocal laser microscope. The development of transgenic Mt-GFP tomato plants helps the visualization of tomato mitochondria and provides good research materials for the study of mitochondrial function during tomato development and fruit ripening.