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甘蔗茎秆是利用转基因方法生产重组药用蛋白或有价值的化合物的理想器官,构建能在甘蔗茎秆中高水平表达异源蛋白质的表达载体是非常有意义的。而一个高效表达的载体,启动子则是其最重要元件之一,因此,茎秆特异性启动子的获得是甘蔗作为生物反应器的前提。利用染色体步移法克隆到甘蔗己糖转运蛋白基因PST2a5′端上游的一段长1968bp的序列(Ppst2a),经序列测定及软件分析表明,该序列具有典型的启动子结构。此序列置换植物表达载体pCAMBIA1301上的CaMV35S启动子,构建植物表达载体,命名为pCAMBIA1900,该启动子下游为gus基因。利用基因枪法转化甘蔗的茎和叶,对gus基因的瞬时表达进行测定,结果表明所获得的己糖转运蛋白基因启动子只在甘蔗茎中驱动gus基因瞬时表达,该启动子具有茎秆特异性。
Sugarcane stalk is the ideal organ for the production of recombinant pharmaceutical proteins or valuable compounds by transgenic methods. It is of great significance to construct an expression vector capable of expressing heterologous proteins at high levels in sugarcane stalks. However, a highly efficient expression vector, the promoter is one of its most important elements, therefore, the stem-specific promoter is the premise of sugarcane as a bioreactor. A 1968 bp sequence (Ppst2a) upstream of the 5 ’end of sugarcane hexose transporter gene PST2a was cloned by chromosome walking and sequenced and analyzed by software. The results showed that this sequence has a typical promoter structure. This sequence replaces the CaMV35S promoter on the plant expression vector pCAMBIA1301, and constructs a plant expression vector named pCAMBIA1900. The downstream of this promoter is the gus gene. Transient transformation of sugarcane stems and leaves with particle bombardment indicated that the promoter of hexose transporter gene was transiently expressed in sugarcane stems, which led to the transient expression of gus gene. The promoter has stem-specific .