油棕叶肉原生质体分离及瞬时转化体系的建立
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国家自然科学基金项目(31660222);海南省自然科学基金创新团队项目(2019CXTD397)


Isolation of oil palm mesophyll protoplasts and establishment of transient transformation system
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    摘要:

    以油棕试管苗幼嫩叶片(发芽后25 d)为材料,建立油棕叶肉原生质体分离及瞬时转化体系。取幼嫩油棕叶片在30 g/L纤维素酶和8 g/L离析酶的作用下,酶解3.5 h后,2 000 r/min、4℃离心5 min得到油棕叶肉原生质体。借助绿色荧光蛋白和红色荧光蛋白双质粒瞬时共转化,对油棕叶肉原生质体瞬时体系的相关参数进行优化,结果显示:当瞬时转化过程中质粒含量比为8∶1,PEG/Mg2+溶液中PEG4000的质量浓度为200 g/L、Mg2+溶液质量浓度为100 g/L,热激20 min,再经过20 min室温孵育,最后加入的洗液体积为2 mL时,原生质体的转化效率最高。

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    The method of isolating and transiently transforming oil palm leaf protoplasts was established. Protoplasts were isolated from leaves of oil palm with 30 g/L cellulase and 8 g/L macerozyme for 3.5 h,and collected by centrifugating at 2 000 r/min for 5 min at 4℃. The dual plasmid transient coexpression system was optimized with green fluorescent protein (GFP) and red fluorescent protein (RFP). The best transformation efficiency was the obtained when the plasmids were mixed with oil palm protoplasts with the mass ratio of 8∶1,heat shocked at 45℃ for 20 min after placed on ice for 30 min,and incubated at room temperature under the dark condition for 30 min with equal amount of PEG/Mg2+ (200 g/L PEG 4000 and 100 g/L MgCl2). It will provide a novel method for further identifying the function of the genes from oil palm.

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王一菲,刘新星,张青,郑育声,李东栋.油棕叶肉原生质体分离及瞬时转化体系的建立[J].华中农业大学学报,2021,(1):154-159

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  • 收稿日期:2020-06-09
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  • 在线发布日期: 2021-02-10
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