柱花草叶肉原生质体提取与瞬时表达体系构建  

Establishment of Isolation and Transient Expression System of Mesophyll Protoplast of Stylosanthes guianensis

作　　者：高静[1] 杨丽云 张世子 蒋凌雁 GAO Jing;YANG Li-yun;ZHANG Shi-zi;JIANG Ling-yan(College of Tropical Crops,Hainan University/Hainan Key Laboratory of Sustainable Utilization of Tropical Biological Resources,Haikou,Hainan Province 570228,China)

机构地区：[1]海南大学热带作物学院/海南省热带生物资源可持续利用重点实验室,海南海口570228

出　　处：《草地学报》2023年第3期893-902,共10页Acta Agrestia Sinica

基　　金：国家自然科学基金项目(31960342);中国科协青年人才托举工程第六届项目(2020QNRC001);海南省科协青年科技英才创新计划项目(QCXM202001)资助。

摘　　要：柱花草(Stylosanthes guianensis)是一种重要的热区豆科牧草,为利用原生质体瞬时表达体系开展柱花草基因功能研究,以20 d龄期‘热研5号’柱花草子叶为试验材料,通过正交试验考察不同因素对原生质体分离及转化效率的影响。结果表明:采用1.5%纤维素酶、1.25%离析酶、0.5 mol·L^(-1)甘露醇和4 mmol·L^(-1)MES酶解液组合,酶解时间为16 h时,原生质体的产量和活性均达到最佳,分别为(4.567×10^(6))个·g^(-1)和92.271%;原生质体浓度为(6×10^(5))个·mL^(-1)、质粒浓度为0.6μg·μL^(-1),PEG-4000浓度为50%、转化时间为5 min时,转化效率最高,可达52.524%。构建了目标蛋白SgRKL1表达载体pA7-BIUTNT::SgRKL1-GFP,利用PEG介导法将其转入柱花草原生质体,激光扫描共聚焦显微镜检测结果显示SgRKL1蛋白定位于细胞膜上,说明所建立的柱花草叶肉原生质体瞬时表达体系可应用于目标基因的瞬时表达和亚细胞定位研究。Stylosanthes guianensis is an important leguminous forage in tropical area.In order to carry out gene function studies using the transient expression system of protoplasts,the cotyledons of 20-day-old seedlings of S.guianensis were used as the experimental material,and the influences of different factors on protoplast separation and the efficiency of transformation into plasmid were investigated by orthogonal experiments.The results showed that the optimal emzymolysis condition was 1.5%Cellulase R-10,1.25%Macerozyme R-10,0.5 mol·L^(-1)mannitol,4 mmol·L^(-1)MES,and 16 h digestion time,which obtained the yield of(4.567×10^(6))per·g^(-1)protoplasts with 92.271%activity.The optimal transformation condition was(6×10^(5))per·mL^(-1)protoplast,0.6μg·μL^(-1)plasmid,50%PEG-4000 and 5 min transformation time,which gave up to 52.524%transformation efficiency.The expression vector of pA7-BIUTNT::SgRKL1-GFP was constructed and transferred into mesophyll protoplasts by PEG-mediated method.The detection of confocal microscopy showed that SgRKL1 protein was localized on the plasma membrane,indicating that the transient expression system of S.guianensis mesophyll protoplasts could be applied to the transient expression of target genes and the studies on the subcellular localization of S.guianensis.

关 键 词：柱花草 叶肉原生质体 酶解 PEG介导 瞬时表达 

分 类 号：Q943.2[生物学—植物学]