| 摘要: |
| 【目的】探讨转基因苜蓿植株根、茎、叶和体细胞胚中外源GUS的表达情况，为外源蛋白在苜蓿体细胞胚中的积累提供理论依据。【方法】以在加拿大广泛种植的N4.4.2紫花苜蓿品种的叶柄为外植体，进行农杆菌GV3101介导的苜蓿遗传转化，将感染的苜蓿叶柄外植体置于不同的转化培养基中培养，获得了适合于农杆菌介导的转基因苜蓿植株，将其用于转基因体细胞胚的诱导。采用PCR方法筛选阳性株系，并对其进行GUS组织化学染色分析，对转基因植株根、茎、叶和体细胞胚进行荧光定量分析。【结果】以苜蓿叶柄为外植体成功获得11株阳性转基因植物，转化效率为30.6%。GUS荧光定量分析结果表明，体细胞胚中GUS外源蛋白的表达量明显高于根、茎和叶，是其表达量的3～6倍；根、茎和叶中GUS表达量较低，且三者间没有明显差异。【结论】苜蓿体细胞胚能积累相对高的外源蛋白，是外源蛋白表达的最佳材料，为外源蛋白大量工厂化生产奠定了实验基础。 |
| 关键词: 苜蓿 农杆菌 GUS表达 体细胞胚 外源蛋白 |
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| 基金项目:中国科学院重要方向资助项目（KZCX2-YW-443）；国家“十一五”科技支撑计划项目（2008BAD98B08） |
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| Exogenous protein expression in somatic embryos in alfalfa |
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| Abstract: |
| 【Objective】The study was to explore GUS expression activity in different tissues (roots,stems,leaves and somatic embryos ) in transgenic alfalfa,which would provide a theoretical evidence for exogenous protein accumulation in somatic embryos in alfalfa.【Method】N4.4.2 alfalfa variety (Medicago sativa),widely grown in Canada,was used in the experiment.Petiole explants were used for transformation materials.Agrobacterium tumefaciens strain GV3101 harboring the plasmid pCAMBIA2301.The aim is to ensure that the observed GUS activity occurring in the plant cells is not due to the presence of residual Agrobacterium cells.Infected petiole explants were put in different media.Transgenic alfalfa via Agrobacterium mediated genetic transformation were obtained.Transgenic alfalfa plantlets were used to induce transgenic somatic embryos.Positive transgenic plantlets by PCR were tested firstly.Then,GUS activity expression in different tissues in transgenic alfalfa by GUS stain were tested.Moreover GUS expression in different tissues including corresponding roots,stems,leaves,somatic embryos was analyzed by fluorometric measure.【Result】We obtained 11 positive transgenic plantlets,transformation efficient being 30.6%.Fluorometric quantitative result showed that the amount of exogenous protein GUS expression is significantly higher in somatic embryos than in other tissues (roots,stems and leaves).The amount in somatic embryos is 3-6 folds than in other tissues.However,the amount of GUS expression was low.There is not significant difference in roots,stems and leaves.【Conclusion】Somatic embryos could accumulate higher exogenous protein,which was the optimal materials of exogenous protein extraction.These would provide the base for mass exogenous protein production in industrialized process. |
| Key words: Medicago sativa Agrobacterium tumefaciens GUS expression somatic embryos exogenous protein |
