苹果异戊烯基转移酶基因启动子调控特性研究

异戊烯基转移酶(IPT)是细胞分裂素合成途径的第一限速酶,过表达苹果MdIPT3a的转基因烟草表现出典型的细胞分裂素特异的生长表型,可作为苹果同源转基因的潜在筛选标记。为阐明MdIPT3a自主启动子对基因表达的调控作用,本试验克隆了富士苹果MdIPT3a基因上游启动子序列,分别构建不同长度的MdIPT3a启动子驱动GUS基因的植物表达载体,以CaMV35S驱动GUS基因的表达载体作为对照,遗传转化烟草W38,并对转基因植株进行GUS定性和定量分析,探讨不同长度的MdIPT3a启动子对GUS表达的影响。结果表明,转基因植株的各组织均有GUS染色,不同长度的MdIPT3a启动子均能驱动GUS基因表达,最小的有效启动子长446 bp,且含MdIPT3a启动子的转基因植株GUS表达水平显著低于含35S启动子的对照植株。本试验结果为MdIPT3a的同源转基因技术研究提供了一定的理论参考。

Regulatory Characteristics of the Apple Isopentenyl Transferase Gene Promoter

Isopentenyl transferase (IPT) is the first rate-limiting enzyme of the cytokinin synthesis pathway. Transgenic tobacco plants overexpressing MdIPT3a have a typical cytokinin-specific phenotype which could be a potential selectable marker when transfered MdIPT3a in apple cisgenesis. To elucidate the regulation of MdIPT3a native promoter on gene expression, in this study, the sequence of MdIPT3a native promoter was isolated from apple cultivar Fuji. Gene fusion constructs of the GUS driven by MdIPT3a native promoters with different lengths and CaMV35S promoter as control were generated and transformed in Nicotiana tobacum Wisconsin 38, respectively. The qualitative and quantitative analysis of GUS expressions in transgenic plants were conducted to study the function of the MdIPT3a native promoter. The results showed that GUS activity was detected in all tested tissues and organs in transformed tobacco plants, and the levels of GUS expression dirven by different promoter lengths of MdIPT3a were significantly lower than by 35S promoter. The MdIPT3a promoter with different lengths were able to drive GUS expression, and a short promoter fragment of 446 bp was sufficent to confer the gene expression. This study provides technical support for the implementing the apple cisgenesis with MdIPT3a.