| 摘要: |
| 【目的】研究紫薇叶片原生质体的分离方法，并对制备的原生质体进行瞬时转化，为紫薇本源物种的基因功能分析提供技术支持。【方法】以F1株系S047（以屋久岛紫薇（L. fauriei）为母本、紫薇品种‘Pocomoke’为父本杂交获得）及‘Ebony Embers’和‘Pocomoke’3个紫薇品种幼嫩叶片为材料，对其进行酶解，筛选最适紫薇品种，在此基础上，采用单因素试验初步筛选纤维素酶、离析酶质量浓度（5，10，15，20，25，30 g/L）及甘露醇浓度(0.4,0.6,0.8 mol/L)，之后采用3因素3水平正交试验筛选纤维素酶（10,20,30 g/L）、离析酶(3,5,7 g/L)和果胶酶(2,4,6 g/L)的最适质量浓度，确定分离紫薇原生质体的最优条件。用聚乙二醇（PEG）介导法将质粒pSuper1300::GFP转化紫薇原生质体。【结果】分离原生质体的最适紫薇品种为S047株系，其在酶解过程中无褐化现象，分离液呈明亮绿色，可得到完整的原生质体；其他2个品种未分离到原生质体。单因素试验结果显示，最适的纤维素酶质量浓度为15~20 g/L，离析酶质量浓度为5~10 g/L，甘露醇浓度为0.6 mol/L。正交试验结果表明，10 g/L纤维素酶、5 g/L离析酶、4 g/L果胶酶和0.6 mol/L甘露醇混合液为最佳酶解溶液，在黑暗条件下酶解紫薇幼嫩叶片5 h，可成功分离出紫薇原生质体，其产量达28×105 g-1，破损率为14%。将纯化的原生质体进行PEG介导的转化后，在激光共聚焦荧光显微镜下观察到绿色荧光信号，表明基因转化成功。【结论】获得了紫薇叶片原生质体分离体系，并成功进行了基因瞬时转化。 |
| 关键词: 紫薇 酶解法 叶片原生质体 瞬时转化 |
| DOI： |
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| 基金项目:国家重点研发计划项目（2019YFD1001004）；江西省林业局林业科技创新专项（［2019］14号） |
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| Protoplast isolation and transient transformation of Lagerstroemia indica leaves |
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LIU Jieru,FENG Lu,LIN Qifang,et al
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| Abstract: |
| 【Objective】To provide technology support for gene function analysis of original species of Lagerstroemia indica,the method of separating leaf protoplasts of L. indica was studied and the protoplasts were transiently transformed.【Method】The most suitable crape myrtle cultivars were selected by using the young leaves of three crape myrtle varieties as materials.On this basis,single factor test was used to screen the concentrations of cellulase/macerozyme (5,10,15,20,25,30 g/L) and mannitol (0.4,0.6,0.8 mol/L).Then,the effects of cellulase (10,20,30 g/L),macerozyme (3,5,7 g/L) and pectinase (2,4,6 g/L) concentrations on protoplast separation were analyzed by three factor and three level orthogonal experimental design to determine the optimum conditions for protoplasts isolation,and the pSuper1300::GFP vector was transformed into protoplasts of L. indica using PEG.【Result】S047 strain was the best cultivar for isolation of protoplasts. There was no browning during enzymatic hydrolysis.The separation solution was bright green,and complete protoplasts could be obtained.Protoplasts were not obtained from other two cultivars.The single factor test showed that the optimum concentrations of cellulase,segregation enzyme and mannitol were 15-20 g/L,5-10 g/L,and 0.6 mol/L,respectively.The result of orthogonal experimental showed that the mixture of 10 g/L cellulase,5 g/L maceronase,4 g/L pectin and 0.6 mol/L mannitol was the best enzymatic hydrolysis solution.The protoplasts of young leaves of L. indica were successfully isolated under dark condition for 5 h,the yield was 28×105 g-1 and the damage rate was 14%.After PEG mediated transformation of purified protoplasts, green fluorescence signal was observed under laser confocal fluorescence microscope.【Conclusion】The protoplast isolation system of crape myrtle leaves was successfully obtained,and the gene transient transformation was successfully conducted. |
| Key words: Lagerstroemia indica enzymatic hydrolysis leaf protoplast transient transformation |
