Dai Ziyu, Hooker Brian S, Quesenberry Ryan D, Thomas Steven R
Chemical and Biological Processing Development Group, Process Science and Engineering Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Transgenic Res. 2005 Oct;14(5):627-43. doi: 10.1007/s11248-005-5695-5.
An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.
人们尝试在转基因烟草植株中高水平生产完整的嗜热栖热放线菌内切葡聚糖酶（E1）。在组成型强启动子Mac或光诱导型番茄核酮糖-1,5-二磷酸羧化酶小亚基（RbcS-3C）启动子的控制下，利用其原始的或苜蓿花叶病毒（AMV）RNA4 5'-非翻译前导序列（UTL）对E1进行表达研究，并通过转运肽将其靶向到不同的亚细胞区室。转运肽包括天然E1、内质网、液泡、质外体和叶绿体的转运肽。通过E1活性、蛋白质免疫印迹和RNA凝胶印迹分析来确定转基因植株中E1的表达及其稳定性。在携带六种转基因表达载体之一的转基因烟草植株中，确定了亚细胞区室对E1产生及其稳定性的影响，其中E1受Mac启动子、甘露碱合酶转录终止子和五种转运肽之一的控制。带有质外体转运肽的转基因烟草植株具有最高的平均E1活性和蛋白质积累量，通过E1比活性和蛋白质凝胶印迹估计，其约占叶片总可溶性蛋白质的0.25%。细胞间液分析证实，E1信号肽在烟草细胞中功能正常，可将E1蛋白分泌到质外体中。用AMV RNA4 UTL替代RbcS-3C UTL可使E1产量提高两倍多，但效果不如甘露碱合酶UTL。据观察，RbcS-3C启动子比Mac启动子更有利于转基因植株中E1的表达。在室温下储存一年的干烟草种子中的E1活性比收获后立即观察到的高45%，这表明E1蛋白可以在室温下长期储存。本文讨论了E1在不同亚细胞区室中的稳定性以及转基因植物中异源蛋白生产的启动子、5'-UTL和亚细胞区室化的最佳组合。
