| 摘要: |
| 【目的】研究高效分离连翘叶片原生质体的条件，并以PEG介导进行瞬时转化，为连翘基因的同源表达提供技术。【方法】以金叶连翘（Forsythia koreana‘Suwon Gold’）组培苗幼嫩叶片为材料，采用4因素3水平L9(34)正交试验，分析纤维素酶质量浓度（15，20，25 g/L）、离析酶质量浓度（3，5，7 g/L）、甘露醇浓度（0.2，0.4，0.6 mol/L）及酶解时间（5，7，9 h）对原生质体分离的影响，探索最优酶解条件，并在此条件下分离原生质体，之后以PEG介导转化pSuper1300::GFP质粒，研究其遗传转化效果。【结果】L9(34)正交试验结果显示，不同处理的金叶连翘叶片原生质体产量为1.17×106~5.71×106 g-1，影响最大的因素为离析酶质量浓度，且4个因素对产量均有极显著影响；原生质体活力为81.19%~90.69%，影响最大的因素为离析酶质量浓度，且其对活力的影响达到显著水平。最佳酶解条件为：在20 g/L纤维素酶、5 g/L离析酶、0.2 mol/L甘露醇的混合液中酶解9 h，在此条件下原生质体产量为5.57×106 g-1，活力为86.79%。PEG介导法结果显示，以GFP为报告基因，在转化后的金叶连翘原生质体中可以检测到绿色荧光信号。【结论】获得金叶连翘叶片原生质体分离的最佳条件，且在此条件下原生质体产量、活力较高，能够成功进行瞬时转化。 |
| 关键词: 原生质体分离 连翘叶片 瞬时转化 GFP |
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| 基金项目:北京市科技计划项目(Z181100002418006)；北京林业大学建设世界一流学科和特色发展引导专项资金项目（2019X-KJS0323） |
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| Protoplast isolation and transient transformation of Forsythia leaves |
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ZHAO Zhengtian,ZHANG Xiaolu,CHENG Tangren,,et al
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| Abstract: |
| 【Objective】To provide technology for homologous expression of Forsythia gene,conditions for efficient isolation protoplasts from Forsythia leaves were studied and transient transformation in PEG mediated was conducted.【Method】Young leaves of F. koreana ‘Suwon Gold’ tissue cultured seedlings were selected for four-factor and three-level orthogonal test of L9(34).The effects of cellulase concentration (15,20 and 25 g/L),macerozyme concentration (3,5 and 7 g/L), mannitol concentration (0.2,0.4 and 0.6 mol/L) and enzymolysis time (5,7 and 9 h) on protoplast separation were analyzed and the optimum enzymolysis conditions were obtained.The protoplasts were isolated under the optimal conditions and transformed by pSuper1300::GFP vector in PEG-mediated to study the genetic transformation effect.【Result】The orthogonal test of L9(34) showed that the protoplast yield of F. koreana ‘Suwon Gold’ leaves under different conditions ranged from 1.17×106 g-1 to 5.71×106 g-1.All four factors had extremely significant effects on protoplast yield and the most influential factor was macerozyme concentration.Protoplast vitality of F. koreana ‘Suwon Gold’ leaves under different conditions ranged from 81.19% to 90.69%,and macerozyme concentration was the leading factor with significant effects.The enzymolysis in solution containing 20 g/L cellulase,5 g/L macerozyme,and 0.2 mol/L mannitol for 9 hours was optimal.Under these conditions,protoplast yield was 5.57×106 g-1 and protoplast vitality was 86.79%.The PEG-mediated method showed that the green fluorescent signals were detected in the transformed protoplasts of F. koreana ‘Suwon Gold’ leaves using GFP as the reporter gene.【Conclusion】Under the optimized conditions,the protoplast yield and vitality were high,and the transient transformation of protoplasts was successfully achieved. |
| Key words: protoplast isolation Forsythia leaves transient transformation GFP |
