Use of Xenopus Oocyte/mRNA Injection System for the Study of Plant Hormone Receptors

非洲爪蟾卵母细胞/mRNA注射系统为各种动物神经递质受体cdna的表征和克隆提供了关键的关键。萨顿博士提出，该系统可以以类似的方式用于植物激素受体的研究。本研究的具体目的是验证将植物mRNA注入爪蟾卵母细胞后，会导致植物激素受体合成并结合到爪蟾卵母细胞的质膜中，并与爪蟾卵母细胞第二信使系统偶联的假设。RNA将从大麦糊粉层中分离出来，这是已知的对各种植物激素的反应。将RNA注射到爪蟾卵母细胞中，卵母细胞孵育3-5天。将通过双电极电压钳技术监测卵母细胞膜中植物激素诱导的氯化物电流，并通过放射性标记激素结合研究，来测试受体是否成功合成并结合到卵母细胞膜中。如果该方法成功，本研究结果将促进植物激素受体的克隆和表征，这些受体被证明是传统生化方法难以实现的。植物激素受体在该动物细胞系统中的表达或缺乏表达也应提供有关蛋白对质膜的分选的重要信息。

The Xenopus oocyte/mRNA injection system has provided a critical key to the characterization and cloning of cDNAs for various animal neurotransmitter receptors. Dr. Sutton proposes that this system can be used in an analogous manner for the study of plant hormone (phytohormone) receptors. The specific aim of this proposal is to test the hypothesis that plant mRNA injected into Xenopus oocytes will result in the synthesis and incorporation of phytohormone receptors into the Xenopus oocyte plasma membrane, and that these receptors will couple to the Xenopus oocyte second messenger systems. RNA will be isolated from the barley aleurone layer which is known to be responsive to various phytohormones. The RNA will be injected into Xenopus oocytes and the oocytes allowed to incubate for 3-5 days. The successful synthesis and incorporation of the receptors into the oocyte membrane will be tested by monitoring for phytohormone-induced chloride currents in the membrane of the oocytes by the two-electrode voltage clamp technique and by radio-labelled hormone binding studies. If this approach is successful, the results of this research will facilitate the cloning and characterization of plant hormone receptors which have proven elusive to more traditional biochemical approaches. The expression or lack of expression of plant hormone receptors in this animal cell system should also provide significant information about protein sorting to the plasma membrane.