皇竹草原生质体分离及瞬时表达系统的建立  

Development of a System for Protoplast Isolation from Pennisetum sinese Roxb and for Transient Expression

作　　者：陈梓涵 彭睿 相雨欣 颜朗 赖先军 CHEN Zihan;PENG Rui;XIANG Yuxin;YAN Lang;LAI Xianjun(Panxi Crops Research and Utilization Key Laboratory of Sichuan Province,Xichang University,Xichang 615013,Sichuan,China;School of Agricultural Science,Xichang University,Xichang 615013,Sichuan,China)

机构地区：[1]西昌学院厅州共建攀西特色作物研究与利用四川省重点实验室,四川西昌615013 [2]西昌学院农业科学学院,四川西昌615013

出　　处：《西昌学院学报（自然科学版）》2024年第3期17-24,共8页Journal of Xichang University(Natural Science Edition)

基　　金：国家自然科学基金地区科学基金项目(32160329);四川省科技厅重点研发项目(2021YFN0132)。

摘　　要：[目的]建立皇竹草高效原生质体分离体系和瞬时表达系统。[方法]通过CRISPR-Cpf1系统构建了针对皇竹草PsC3H53基因的pCAMBIA1304-LbCas12a-PsC3H53cr表达载体,对纤维素酶R-10的酶解液质量分数设置了4组处理(1.5%、2.0%、2.5%、3.0%)和7组处理的酶解时间(3、4、5、6、7、8、9 h),并采用聚乙二醇(PEG)介导原生质体转化。[结果]酶解7 h,2.5%纤维素酶、0.5%果胶酶与0.4%离析酶组合的酶解条件最佳,原生质体的产量可达1.712×10^(6) cell/(g·FW),PEG法可成功介导质粒转化皇竹草原生质体并在荧光显微镜下观察到绿色荧光蛋白。[结论]在皇竹草中成功建立瞬时表达系统和遗传转化体系,经优化最佳酶解组合和时间后即能通过PEG介导实现皇竹草原生质体的瞬时转化,为后续皇竹草的再生及品种培育奠定了基础。[Objective]It is important to develop an efficient protoplast isolation and transient expression system in high-quality forage grass,Pennisetum sinese Roxb.[Method]In this study,the pCAMBIA1304-LbCas12a-PsC3H53cr vector was constructed through the CRISPR-Cpf1 system.The plasmid was mixed with protoplast solution and transformation so-lution.The protoplasts were cultured under weak light conditions.Four treatments(1.5%,2.0%,2.5%,3.0%)and seven sets of enzyme digest times(3,4,5,6,7,8,9 h)were set for the enzymatic hydrolysis solution of cellulase R-10.The enzymatic hydrolysis conditions suitable for P.sinese were optimized to improve its yield and activity.Finally,polyethyl-ene glycol(PEG)was used to mediate protoplast transformation.[Result]The enzyme digestion for 7 h was optimal with the combination of 2.5%cellulase,0.5%pectinase and 0.4%dissociative enzyme,and the yield of protoplasts was up to 1.712×10^(6) cells/(g·FW).PEG method can successfully mediate plasmid transformation into protoplasts of P.sinese and green fluorescence protein could be observed under fluorescence microscope.[Conclusion]A transient expression system and genetic transformation system were successfully established in P.sinese.After optimizing the best enzymatic hydroly-sis combination and time,the transient transformation of P.sinese bioplastids could be achieved through PEG mediation,laying the foundation for the subsequent regeneration and variety breeding of P.sinese.

关 键 词：皇竹草 载体构建 原生质体 PEG瞬时转化 

分 类 号：Q943.2[生物学—植物学] S543.9[农业科学—作物学]