活性氧介导的柑橘原生质体再生能力差异的细胞与分子机制

Protoplast technology has been extensively used in plant genetic improvement and fundamental research. But a major obstacle to the application of protoplast technology is recalcitrance of plant protoplasts. With the support by NSFC, we found that oxidative stresses due to ROS accumulation may be responsible for the recalcitrance of citrus mesophyll protoplast regeneration, and the callus protoplasts possessed high antioxidant system, and can easily regenerate; in addition， NADPH oxidases were correlated with ROS generation in protoplasts. However, the underlying cellular and molecular mechanisms are not well known. First, after citrus mesophyll and callus protoplasts from the same cultivar are isolated and cultured, the localization and duration of the ROS signals are determined by fluorescent probes under laser scanning confocal microscope. Then, protoplast-specific expression of NADPH oxidase genes is detected with qRT-PCR analysis, and overexpression and RNAi vectors containing the obtained gene were transferred into seedling epicotyls and calli of citurs. Finally, RNA-Seq based on Solexa Genome Analyzer Platform is applied to investigate differential expression genes, and key transcription factors relevant to regeneration potential are also identified. The work provides a novel theory basis for a comprehensive understanding of protoplast totipotency, and also can provide a theoretical and technical basis for genetic improvement and germplasm innovation in citrus and other fruit trees involved in the use of protoplasts.

原生质体技术已广泛地应用于植物遗传改良与基础研究，而原生质体再生难是限制其应用的瓶颈问题。我们前期研究发现，活性氧引起的氧化胁迫是造成柑橘叶肉原生质体不能再生的重要原因，而愈伤组织原生质体因激活抗氧化系统可分裂再生，另外NADPH氧化酶参与了活性氧产生。但是，两者再生能力差异的细胞与分子机制尚不清楚。本项目拟以同一基因型的叶肉原生质体和愈伤组织原生质体为试材，使用荧光探针结合激光共聚焦显微镜观察活性氧产生、分布及其时空变化；采用qRT-PCR技术筛选原生质体中特异表达的NADPH氧化酶基因，并构建超表达载体与RNAi载体，转化柑橘实生苗上胚轴和愈伤组织，鉴定该基因功能；通过数字基因表达谱测序，分析差异基因表达谱，筛选出与再生能力差异相关的关键转录因子。项目的开展可为植物原生质体全能性调控提供新的科学依据，同时为促进利用原生质体再生体系进行柑橘及其他果树遗传改良和种质创新奠定理论与技术基础。

活性氧引起的氧化胁迫是造成柑橘叶肉原生质体不能再生的重要原因，而愈伤组织原生质体因激活抗氧化系统可分裂再生。但是，两者再生能力差异的细胞与分子机制尚不清楚。本项目以椪柑的叶肉原生质体和愈伤组织原生质体为试材，使用荧光探针结合激光共聚焦显微镜观察了活性氧分布和变化，同时采用RT-PCR技术分析了NADPH氧化酶家族基因的表达。结果表明，活性氧主要分布在原生质体的质膜、线粒体和胞质中；在原生质体分离过程，叶肉原生质体的活性氧含量较高，而在原生质体培养过程，愈伤组织原生质体的活性氧含量较高；Cs7g19320.1和Cs8g12000.1基因在原生质体分离与培养过程表达量较高。