Dowson Day M J, Ashurst J L, Mathias S F, Watts J W, Wilson T M, Dixon R A
AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, UK.
Plant Mol Biol. 1993 Oct;23(1):97-109. doi: 10.1007/BF00021423.
In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.
为了优化外源蛋白在转基因植物中的表达，我们研究了在植物转化载体中包含病毒非翻译前导序列的潜在益处。比较了多种5′前导序列，包括烟草花叶病毒（TMV）前导序列和花椰菜花叶病毒（CaMV）35S RNA前导序列的31个核苷酸。采用35S启动子和报告基因β-葡萄糖醛酸酶（GUS）构建的病毒前导序列通过电穿孔导入烟草叶肉原生质体进行测试，并在转基因烟草叶片中与共导入的氯霉素乙酰转移酶（CAT）基因进行对比。在瞬时分析系统中，将病毒前导序列的GUS活性与短的随机前导序列或CaMV 19S RNA ORF VI与GUS的翻译融合体的GUS活性进行比较。当这些前导序列被35S RNA或TMV前导序列取代时，表达水平提高了两到三倍。通过在TMV前导序列旁包含35S转录起始位点的四个或七个碱基，这种增强进一步提高到四到五倍。在转基因烟草中，包含TMV前导序列（八倍）或添加了35S转录起始位点碱基的该序列（十倍）的构建体保持了提高的GUS水平。以CAT基因作为内标，比较GUS酶量和GUS mRNA量，结果显示携带TMV前导序列的mRNA的翻译效率比随机前导序列对照高四到六倍。
