甘蔗茎秆特异表达基因启动子的克隆及初步分析

Cloning and Preliminary Analysis of the Stem Specific Promoter of Sugarcane

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 stem is an ideal organ for producing foreign pharmaceutical proteins and chemicals by genetic engineering. A perfect promoter driving foreign gene to express strongly and specifically in sugarcane stem is necessary for this purpose. In order to isolate a Sugarcane stem-specific promoter , a fragment of 1968bp nucleotide sequence （Ppsa,）upstream 5＇ of sugarcane pst2a gene, which was demonstrated to express specifically in sugarcane stem previously was isolated by using chromosomal walking. Bioinformatical analysis of this sequence shows that the sequence contains some typical elements of a promoter. To identify the stem-specific of this promoter, a construct was derived from pCAMBIA1301, which original CaMV 35S promoter was replaced by the 1968bp nucleotide sequence, and named as pCAMBIAI900. Transformations of pCAMBIAI900 and pCAMBIA1301 to leaves and stem pieces of sugarcane were carried out by using particle bombardment. The transient expression of gus showed that the gus expressed specifically in sugarcane with a little higher level compared with CaMV 35S. It is the first report that pst2a promoter is a potential stem-specific promoter which can further be used in transgenes into sugarcane.