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      2. 油棕葉肉原生質體分離及瞬時轉化體系的建立
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        作者單位:

        1.海南大學;2.海南大學熱帶作物學院

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        基金項目:

        國家自然科學基金項目(面上項目,重點項目,重大項目)


        The isolation and transient transformation of oil palm mesophyll protoplasts
        Author:
        Affiliation:

        HAINAN UNIVERSITY

        Fund Project:

        The National Natural Science Foundation of China

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          摘要:

          以油棕試管苗幼嫩葉片(發芽后25天)為材料,建立了油棕葉片原生質體分離及瞬時轉化體系,為油棕功能基因的驗證提供了一個有效的驗證平臺。取幼嫩油棕葉片在30 g/L的纖維素酶和8 g/L的離析酶的作用下,酶解3.5 h后,2000 rpm/min、4℃離心5 min得到油棕葉肉原生質體。借助綠色熒光蛋白和紅色熒光蛋白雙質粒瞬時共轉化,對油棕葉肉原生質體瞬時體系的相關參數進行了優化。結果表明:瞬時轉化過程中當質粒含量比為8 : 1,PEG/Mg2+溶液中的PEG 4000的質量濃度為200 g/L、氯化鎂的質量濃度為100 g/L,熱激20 min,再經過20 min室溫孵育,最后加入的洗液的體積為2 mL時,原生質體的轉化效率最高。

          Abstract:

          In this paper, the method of extraction and transient transformation of oil palm leaf protoplasts was established. Protoplasts were isolated from leaves of oil palm by 30 g/L cellulase and 8 g/L macerozyme for 3.5 h, and collected by centrifugation at 2000 rpm/min for 5 min at 4°C. Moreover, the dual plasmid transient co-expression system was optimized using 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 °C for 20 min after placed on ice for 30 min, and incubated at room temperature in the dark for 30 min with equal amount of PEG/Mg2+ (200 g/L PEG 4000 and 100 g/L MgCl2). Finally, the protoplast isolation and transient expression system provides a new method for the functional verification of the genes form oil palm.

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        歷史
        • 收稿日期:2020-06-09
        • 最后修改日期:2020-06-30
        • 錄用日期:2020-09-17
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