香蕉Actin1启动子的分离及启动活性的分析

Isolation and Characterization of Banana Actin 1 Promoter

从香蕉基因组DNA中分离香蕉Actin1启动子序列,序列测定结果表明,该片段1253bp,包含完整的G-box和TATAbox,5'非翻译区和其下游的内含子,与文献报道序列的同源性达97.53%。以全长序列取代植物表达载体pCAMBIA3300上的35S启动子序列构建成瞬间表达载体pCAMBIA-Act1,以内含子序列HindⅢ和XbaⅠ双酶切片段(584bp)取代植物表达载体pBI121上的35S启动子,构建成瞬间表达载体pBI121-Act1-1。采用基因枪法对香蕉根、叶和果实的薄片进行转化,转化材料经培养3h,采用分光光度法测定各组织GUS的活性。结果表明,Actin1启动子在所转化的3种组织中均可启动报告基因的表达,表达活性与35S启动子接近,具有组成型表达的特点。内含子部分序列(584bp)也具有启动活性,但启动活性较低。

A banana actin 1 promoter was isolated and the fragnent was sepuenced 1253 bp, comtaining a G-box,a TATA box that existed characteristically in all promoters,5＇ non-translated region and an intron in 5＇ leader sequence. Sequencing analysis of this product revealed a high homolog）T （97.53 %） to actin 1 promoter sequence as reported.Two regions of the promoter were fused to gus reporter gene to determine regions of activity of isolated actin 1 promoter. The promoter fragment including 1 235 bp was inserted into pCAMBIA3 300 which was previously inserted with gus gene and nos terminator and then digested with Pst I and XbaI, and the newly formed transient expression vector was designated as pCAMBIA-Aet1. The fragment of 584 bp in intron of 5＇ leader was inserted into pB1121 to substitute for the original 35S promoter in pBI121, and the newly formed transient expression vector was designated as pB1121-Act 1-1. A histoehemieal method was used in detecting GUS activity in microprojectile bombarded banana tissues of leaves, roots and fruits to determine the levels of transient GUS activity from the two transient expression vectors. The results showed that fragments of 1.2 kp and 584 bp were able to drive gus gene expression and constitutive expression, while the activity of 1.2 kb fragment was almost the same to 35 S when compared between pB1121 and pCAMBIA-Aetl. The fragment of 584 bp also had activity to drive gus gene expression although its activity was lower than that of 1.2 kb. This study indicated that the banana Actin 1 promoter displayed strong constitutive expression in banana and would be useful for gene transformation in banana.