Gallie D R, Young T E
Department of Biochemistry, University of California, Riverside 92521- 0129.
Plant Physiol. 1994 Nov;106(3):929-39. doi: 10.1104/pp.106.3.929.
Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.
在种子发育和萌发过程中，糊粉层和胚乳中的基因表达受到高度调控。对大麦（Hordeum vulgare）糊粉层中α-淀粉酶表达的研究产生了目前关于发芽谷物种子中基因表达激素控制的范例。由于缺乏有效的转化系统，玉米（Zea mays）种子糊粉层和胚乳组织中的基因表达研究受到了阻碍。我们在此报告了从玉米糊粉层和胚乳组织中快速分离原生质体、使用聚乙二醇或电穿孔对其进行转化以及这些细胞中基因表达调控的情况。与黑墨西哥甜玉米悬浮细胞相比，在糊粉层和胚乳原生质体中，Adh1启动子活性相对于35S启动子有所降低，在黑墨西哥甜玉米悬浮细胞中Adh1启动子活性几乎与35S启动子一样强。内含子介导的表达刺激在转化的糊粉层或胚乳原生质体中比在细胞悬浮培养原生质体中要高得多，数据表明内含子的作用可能受细胞类型影响。为了研究细胞质调控，将大麦α-淀粉酶的5'和3'非翻译区与萤火虫荧光素酶编码区融合，并在将体外合成的mRNA导入糊粉层和胚乳原生质体后，检测其对翻译和mRNA稳定性的影响。α-淀粉酶非翻译区以组织特异性方式调节翻译效率，增加糊粉层或胚乳原生质体中的翻译，但在玉米或胡萝卜细胞悬浮原生质体、动物细胞或体外翻译裂解物中则不然。
