油桐叶肉细胞原生质体分离及瞬时转化体系的建立

The Establishment of Isolation and Transient Transformation Methods of Protoplasts of Vernicia fordii Mesophyll Cells

【目的】探索油桐叶肉细胞原生质体分离的最适条件,建立油桐原生质体的遗传转化体系,使在油桐体内研究自身基因的功能成为可能。【方法】以油桐成熟叶片和组培苗幼叶为材料,通过酶解法成功分离得到油桐叶肉细胞的原生质体并确定最适分离条件。在此基础上,以获得的原生质体为受体系统,建立PEG介导的油桐原生质体基因转化方法。【结果】原生质体分离结果表明,酶解时间对原生质体产量和活性的影响最大,其次是纤维素酶浓度,而离析酶浓度和甘露醇浓度对原生质体产量和活性的影响较小。以成熟叶片为材料分离原生质体的最适条件为纤维素酶浓度1.5%、离析酶浓度1%、甘露醇浓度0.6 mol·L-1、酶解时间12 h,以组培苗幼叶为材料分离原生质体的最适条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。为了建立油桐原生质体的瞬时转化体系,通过PEG介导法将拟南芥MGT6基因导入到油桐原生质体中,结果发现MGT6蛋白定位于原生质体质膜,与之前报道的研究结果一致,这表明本研究建立的油桐原生质体转化方法可成功将外源基因导入油桐原生质体并使其表达。【结论】建立油桐成熟叶片和组培苗幼叶叶肉细胞原生质体的高效分离方法,综合考虑取材的便利性和对后续原生质体培养的影响,建议以组培苗幼叶为材料分离原生质体,分离条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。在分离得到油桐叶肉细胞原生质体的基础上,本研究建立的PEG介导的原生质体遗传转化方法能以油桐叶肉细胞原生质体为受体,高效地将外源基因导入其中并使外源基因表达。本研究结果不仅可促进油桐基础研究的发展,在通过细胞融合和基因工程手段进行油桐种质创新方面也具有重要意义。

【Objective】 This study tries to explore the optimal conditions of separation of mesophyll cell protoplasts of Vernicia fordii and to establish its genetic transformation system,making it possible to study gene functions of V. fordii in vivo.【Method】 This research firstly obtained mesophyll cell protoplasts of V. fordii through enzyme hydrolysis method using mature leaves and test tube plantlet leaves respectively. Gene transformation method of V. fordii protoplast mediated by PEG was established with protoplast receptor system. 【Result】 Protoplast isolation revealed that the most significant effect on the yield and activity of protoplast was enzymatic hydrolysis time followed by the concentration of cellulase. The concentrations of macerozyme and mannitol showed less influence on the yield and activity of protoplast. The optimal conditions for the isolation of protoplasts from mature leaves were 1. 5% cellulase concentration,1% macerozyme concentration,0. 6 mol·L-1 mannitol concentration and 12 hours of enzymolysis. The optimal conditions for the isolation of protoplasts from young leaves of plants propagated by tissue culture were 2% cellulase concentration,1% macerozymeconcentration,0. 7 mol·L-1 mannitol concentration and 6 hours of enzymolysis. In order to establish transient transformation system of V. fordii protoplast,this research transformed Arobidopsis thaliana MGT6 gene into V. fordii protoplast using PEG-mediated method. The result showed that MGT6 was located in the plasma membrane of protoplast,indicating that gene transformation method of V. fordii protoplast can successfully introduce exogenous genes into protoplasts and make it express.【Conclusion】 This research established isolation and transient transformation methods of protoplasts of V. fordii mesophyll cells obtained from mature leaves and young leaves of plants propagated by tissue culture,respectively. Considering the convenience of sampling materials and the influence on the subsequent protoplast culture,we suggest that young leaves of tissue-cultured plants should be used to isolate protoplasts with conditions of 2%cellulase concentration,1% macerozyme concentration,0. 7 mol·L-1 mannitol concentration and 6 hours of enzymolysis.On the basis of isolation of mesophyll cell protoplasts of V. fordii,the PEG-mediated genetic transformation method used in this study can be used to efficiently introduce exogenous genes into protoplasts with successful expression. In summary,the results not only promote the development of the basic researches on V. fordii,but also have important significance in germplasm innovation through cell fusion and gene engineering.