小麦叶肉细胞原生质体制备参数解析及在基因瞬时表达上的应用

Factors Influencing the Preparation of Wheat Mesophyll Protoplasts and the Application of Wheat Mesophyll Protoplasts in Gene Transient Expression

为建立一套稳定高效的小麦叶肉细胞原生质体制备技术流程,以小麦幼苗叶片为材料,采用正交和随机设计试验分析了影响小麦叶肉细胞原生质体产量的制备因素。结果发现,酶解条件相关因素对制备原生质体产量的影响由大到小依次为酶解液甘露醇浓度、纤维素酶浓度、酶解时间和离析酶浓度;甘露醇浓度和纤维素酶浓度对原生质体产量的增长表现相互促进模式,延长酶解时间情况下低纤维素酶浓度产出的原生质体较高纤维素酶浓度破碎情况少,峰值过后的产量下降慢。研究还发现取材幼苗苗龄、光照条件和取材叶位等因素对原生质体产量也有影响。综合以上结果确定了一套优化的参数组合:以黑暗培养条件下的10 d苗龄小麦幼苗第2片真叶为材料,酶解液中的纤维素酶浓度为0.5%、离析酶浓度为0.6%、甘露醇浓度为0.5 mol/L,酶解时间为6 h,使用此参数组合制备的原生质体形状均一、破碎情况轻,产量可达(7.28±2.18)×106个/g,有活性原生质体的比例可达(95.06±2.66)%。采用PEG-Ca^(2+)介导的方法将目标基因导入制备的原生质体,结果表明GFP基因的转化效率可达(61.31±5.74)%,小麦基因Ta ATG8h和异源植物拟南芥基因At PIP2;1的蛋白质产物均定位于正确的亚细胞结构处。

In order to establish a stable and efficient protocol for the isolation of wheat mesophyll protoplasts,experiments of orthogonal and completely random designs were conducted to analyze the factors influencing the yield of mesophyll protoplasts from wheat seedling leaves. It was found that the mannitol concentration in the enzyme solution showed the greatest impact on protoplast yield,followed by the time of enzymatic treatment,the cellulase concentration and the macerozyme in the enzyme solution. The mannitol concentration and the cellulase concentration showed a mutual promotion effect on the yield of protoplast. If a prolonged time of enzymatic treatment was applied,low concentrations of cellulase released more round-shaped protoplasts and sustained a more stable quantity of protoplasts after the time of the peak yield value than high concentrations of cellulase. Other factors influencing protoplast yield include seedling age,light conditions and the position of leaves collected for protoplast isolation. Based on these results,an optimized set of parameters for wheat mesophyll protoplast isolation was determined,in which the second leaves of 10-day-old wheat seedlings grown under dark conditions were selected as the explants and were digested with an enzyme solution containing 0. 5% cellulose,0. 6% macerozyme and 0. 5 mol / L mannitol for 6 hours.Uniform,round-shaped protoplasts were prepared by using this set of parameters and a yield up to（ 7. 28 ± 2. 18） ×10^6per gram leaf fresh weight could be obtained. The percentage of viable protoplasts was up to（ 95. 06 ±2. 66） %. The PEG-Ca^2 ＋-mediated transformation procedure was applied to transform gene constructs into prepared mesophyll protoplasts and a transformation efficiency of（ 61. 31 ± 5. 74） % was determined by using GFP as a detecting marker. The GFP or DsRed fusion protein of wheat Ta ATG8 h and Arabidopsis At PIP2; 1,when expressed wheat mesophyll protoplasts,showed correct subcellular localization.