甘蓝原生质体制备体系优化及瞬时转化体系的建立

Optimization of Protoplasts Preparation System and Establishment of Transient Transformation System in Brassica oleracea var.capitata L.

为研究甘露醇浓度、酶解时间、离心力大小和不同外植体对原生质体产量和活力的影响,采用正交试验方案对甘蓝原生质体制备体系进行了优化,并建立了甘蓝原生质体的瞬时转化体系,同时利用该体系对甘蓝CRISPR/Cas9基因编辑系统进行了初步研究。结果表明:以下胚轴为材料,酶液组成为1%纤维素酶+0.5%离析酶+0.6 mol/L甘露醇+10 mmol/L MES+1 mmol/L CaCl2+0.1%BSA+5 mmol/Lβ-巯基乙醇,26℃,60 r/min,黑暗酶解6 h后,用2 115 r/min离心5 min收集细胞,可获得高质量的原生质体,原生质体产量约为3.0×106个/g,活力约为90.9%;另外,在20%PEG4000介导下,将带有绿色荧光蛋白的载体(PYBA 1132)分别转入从甘蓝下胚轴、子叶、叶球分离获得的原生质体中,在荧光显微镜下观察到GFP绿色荧光信号,转化效率分别为43%,35%,19%,表明下胚轴分离获得的原生质体最适于瞬时转化。同时以下胚轴分离获得的原生质体为受体,分别转化线粒体特异表达载体COX和质体特异表达载体969,也观察到了荧光信号,成功建立了甘蓝原生质体瞬时转化体系,为甘蓝功能基因定位与功能分析提供了技术平台。最后,利用该体系对CRISPR/Cas9基因编辑载体(PBSE401-BoPDS)中sgRNA的靶向编辑能力进行了检测,结果显示,靶点1和靶点2附近的序列中有碱基替换和插入,表明该体系能够应用于甘蓝瞬时基因编辑,为甘蓝基因功能研究以及新种质的创制建立了重要的技术平台。

The effects of mannitol concentration,enzymatic hydrolysis time,centrifugal speeds and different explants on the yield and vitality of protoplasts were studied by orthogonal experiment to optimize the protoplasts preparation system.The transient transformation system of Brassica oleracea protoplasts from different explants was also established.The transient transformation system was also used for preliminary study of the CRISPR/Cas9 system.The results showed that the optimal enzyme solution for protoplasts isolation was 1%CelluseR-10+0.5%MacerozymeR-10+0.6 mol/L mannitol+10 mmol/L MES+1 mmol/L CaCl 2+0.1%BSA+5 mmol/Lβ-mercaptoethanol.The hypocotyl of Brassica oleracea was incubated in enzyme solution for 6 h at 26℃in a dark rotary shaker at 60 r/min.The isolated protoplasts were centrifuged at 2115 r/min for 5 minutes.The protoplasts yield amounted to 3.0×106/g fresh weight and the vitality was about 90.9%.In addition,the PYBA 1132 vector with green fluorescent protein was successfully transformed into the purified protoplasts from hypocotyls,cotyledons and head leaves of Brassica oleracea by 20%PEG4000.The fluorescence signals were detected under the microscope.The transformation efficiency was about 43%,35%and 19%,respectively.The protoplasts isolated from hypocotyls were the best suitable for transient transformation.The mitochondrion and plastid localization vectors COX and 969 were also successfully expressed in the protoplasts from hypocotyls.These results indicated that the transient transformation system had been established.It provided a platform for analyzing the gene localization and function in Brassica oleracea.At last,the editing capability of PBSE401-BoPDS was tested in the system.The results showed that the base substitution and insertion happened in the sequences near the target 1 and target 2.It meant that the CRISPR/Cas9 worked in this transient transformation system,which established an important platform for studying the gene functions and generating the germplasm resources of Brassica oleracea.