多年生黑麦草原生质体制备及瞬时表达体系的建立

Protoplast Isolation and Establishment of Gene Transient Expression System in Lolium perenne L.

本研究以10~12 d苗龄的多年生黑麦草品种NUI叶片为材料,通过正交实验确定原生质体分离的最佳条件。结果表明,在甘露醇浓度为0.5 mol/L、纤维素酶R10浓度为1%、离析酶R10浓度为0.3%的酶解液中,28℃黑暗条件下酶解4 h后分离的原生质体最为理想,原生质体浓度达到4.69×10^(6)个/mL,活性达到88.59%。将能表达绿色荧光蛋白(green fluorescent protein,GFP)的重组质粒pAN580利用聚乙二醇(polyethylene glycol,PEG)介导法转化原生质体后,观察到强烈的绿色荧光信号,表明分离的黑麦草原生质体可以用作基因瞬时表达体系。遗传转化正交实验结果表明,在原生质体浓度为2×10^(5)个/mL、质粒浓度为1000 ng/mL、转化时间为25 min,PEG-4000浓度为40%的条件下,转化效率可达67.51%,可以满足对CRISPR/Cas9基因编辑载体进行编辑效率快速检测的要求。综上所述,本研究建立了一套快速、高效的原生质体瞬时表达体系可为多年生黑麦草基因编辑载体的快速鉴定验证及功能基因组研究提供有效的工具,不仅为深入开展多年生黑麦草的功能基因组学研究提供了基础,也为其在种质创新领域和分子育种层面的研究能利用基因工程的方法提供了依据。

In this study,leaf protoplasts of Lolium perenne L.var.NUI were isolated using 10~12 day seedling as starting materials.Optimum conditions of protoplast isolation were determined through orthogonal experiment.Best results were obtained for protoplasts isolated in the enzyme solution with 0.5 mol/L mannitol,1%cellulase R10 and 0.3%macerozyme R10,and were incubated for 4 hours at 28℃dark condition after enzymolysis,which yielded protoplast concentration of 4.69×10^(6)cells/mL,and protoplast viability of 88.59%.The recombinant plasmid pAN580 containing GFP fusion protein was transformed into protoplasts through PEG mediated method with strong green fluorescence detected,indicating that the protoplasts obtained by this method could be used to establish a gene transient expression system.The results of genetic transformation orthogonal experiment showed that the transformation efficiency could reach 67.51%when the protoplast concentration of 2×10^(5)cells/mL,plasmid concentration of 1000 ng/L,transformation time of 25 min and PEG-4000 concentration of 40%were used,which could meet the requirements for rapid detection of editing efficiency of CRISPR/Cas9 gene editing vector.In summary,we established a rapid and efficient protoplast gene transient expression system,which could provide an effective tool for rapid identification and verification of Lolium perenne L.gene editing vector and for functional genomics research.It not only provides the basis for the further study of the functional genomics of Lolium perenne L.,but also provides the basis for the research of genetic engineering in the field of germplasm innovation and molecular breeding.