结缕草叶肉细胞原生质体瞬时基因表达系统的构建  

An efficient protocol for Zoysia japonica mesophyll protoplast isolation and transformation,and its application in subcellular localization and protein interaction analysis

作　　者：管瑾 郭一荻 刘凌云 尹淑霞[1] 滕珂 GUAN Jin;GUO Yi-di;LIU Ling-yun;YIN Shu-xia;TENG Ke(School of Grassland Science,Beijing Forestry University,Beijing 100083,China;Institute of Grassland,Flowers and Ecology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China)

机构地区：[1]北京林业大学草业与草原学院,北京100083 [2]北京市农林科学院草业花卉与景观生态研究所,北京100097

出　　处：《草业学报》2023年第7期61-71,共11页Acta Prataculturae Sinica

基　　金：国家自然科学基金(31901397);国家林业和草原局委托项目(2021045001);北京市自然科学基金(6204039)资助。

摘　　要：为了结合转基因方法探索结缕草基因功能,建立高效、快速的结缕草原生质体制备及瞬时基因表达系统,利用正交试验对影响原生质体制备的主要因素进行优化,同时利用原生质体进行亚细胞定位和蛋白互作研究。结果表明,当酶配比为2%(w/v) cellulase R10和1.5%(w/v) macerozyme R10,酶解时间为7 h时,原生质体产量和活性均达到最高,分别为1.23×107个·mL^(-1)和98%以上,满足后续转化所需。多次重复试验表明,加入5~10μg质粒,原生质体转化率可达75%以上。利用原生质体转化体系进行了亚细胞定位检测,结果发现结缕草ZjNOL和ZjNYC1定位在叶绿体中,ZjZFN定位在细胞核中。双分子荧光互补分析证明ZjNOL和ZjNYC1在叶绿体中相互作用。原生质体制备及转化方法与遗传学和组学技术相结合,能够为结缕草基因功能研究和基因编辑提供支持。An efficient leaf mesophyll protoplast isolation and transient gene expression system in Zoysia japonica is highly desirable for in vivo gene functional studies in its homologous system.The main factors influencing the isolation of protoplasts from Z.japonica were optimized by orthogonal design.The transformation protocol was used for protein subcellular localization and protein-protein interaction.In this study,a highly efficient protoplast(1.23×10^(7) protoplasts·mL^(−1))was generated using 2%(w/v)cellulase R10 and 1.5%(w/v)macerozyme R10 for digestion over 7 h.FDA staining results showed that approximately 99% of protoplasts exhibited high activity.Repeated experiments showed that by adding 5-10μg plasmid,the transformation efficiency reached more than 75%.Using this protocol to identify subcellular locations,ZjNOL and ZjNYC1 were proved to be localized in chloroplasts,and ZjZFN was visualized in nuclei.The bimolecular fluorescence complementation assay showed that ZjNOL and ZjNYC1 interacted with each other in the chloroplast.The protoplast isolation and transformation protocol,when combined with genetics and omics techniques,will allow further study of gene functions in Z.japonica.

关 键 词：结缕草 原生质体 瞬时基因表达 亚细胞定位 蛋白互作 

分 类 号：Q942[生物学—植物学]