华南象草原生质体分离及基因瞬时表达研究

Protoplast Isolation from Elephant Grass(Pennisetum purpureum'Huanan')for Transient Gene Expression

以华南象草(Pennisetum purpureum)幼叶鞘为材料,采用L_(16)(4~5)正交试验设计,对分离原生质体过程中的纤维素酶浓度、离析酶浓度、酶解液pH、甘露醇浓度、酶解时间5个因素进行筛选,建立了高效的象草叶鞘原生质体分离体系,并进行目标基因的瞬时转化,以期为象草基因功能研究奠定基础。结果表明:叶鞘在含有1.5%纤维素酶R-10,0.75%离析酶R-10,0.5 mol·L^(-1)甘露醇,pH为5.8的酶解液中酶解4 h,原生质体产量达5.11×10~6个·g FW^(-1),活力达91.08%,酶解时间对象草原生质体产量和活力的影响最大。用PEG介导法将2个分别带有绿色荧光蛋白和红色荧光蛋白的载体转入原生质体中,转化效率最高可达77.47%,共转化效率为8.79%。所分离的原生质体可用于基因的瞬时表达研究。

Elephant grass （Penniseturn purpureum） is a kind of typical C4 clonal plant, which grows well in south China. It is commonly used as forage grass, green energy grass as well as nonwood fiber. Young leaf sheaths of elephant grass were used for mesophyll protoplasts isolation in this study. 5 factors of cellu- lase concentration, macerozyme concentration, pH, mannitol concentration, enzymolysis time were stud- ied to optimize isolation conditions based on L16 （4^5） orthogonal test. And the isolated elephant grass protoplasts were used for gene transient expression assays, aiming to lay the foundation for gene functional analysis in elephant grass. The results showed that the optimum conditions for elephant grass mesophyll protoplasts isolation were in the enzyme combinations containing 1.5 % cellulase, 0.75 % macerozyme, 0.5 mol · L^-1 mannitol, pH 5.8. The digestion was conducted in the dark under 28℃ for 4 h with gentle sha- king. Under these conditions, the protoplasts yield was 5.11 ×10^6 cells · g FW^-1 （fresh weight）, and the vitality was 91.08%. Variance analysis of orthogonal test indicated that the enzymolysis time is the most important factor influencing both protoplast yield and vitality. Two plasmid vectors, carried green and red fluorescent protein respectively, were then transformed into purified mesophyll protoplasts by PEG-media- ted method. The green and red fluorescent signal were detected in the transient transformed protoplasts, obtained the highest transformation efficiency of 77.47 %, and co-transformation efficiency of 8.79 %. The isolated protoplasts could be used for gene transient assay in elephant grass.