大豆抗逆基因GmDREB3启动子的克隆及调控区段分析

Isolation and Regulative Region Analysis of Promoter of Stress-Related Gene GmDREB3 from Soybean

GmDREB3基因能提高转基因烟草和拟南芥的抗逆性。利用SiteFinding-PCR技术,从大豆品种铁丰8号基因组中分离到大豆抗逆基因GmDREB3启动子片段,长度1648bp。该片段富含A/T碱基,还含有TATA-box、低温响应元件MYC及其他顺式元件MYB、CAAT-box等。将该启动子分区段与GUS报告基因连接构建表达载体,利用基因枪法转化小麦愈伤组织,并进行干旱、高盐、低温等处理,通过组织化学染色和GUS荧光定量测定分析各区段调控元件的活性。结果表明,在干旱和低温的诱导下,该启动子能激活下游GUS基因的表达,在–285～–1117区域存在与低温和干旱应答有关的重要调控元件,在–1464～–1648区域内存在抑制启动子活性的调控元件。由此推断,在逆境条件下通过启动子区域正、负调控元件的共同作用,使GmDREB3基因的表达维持在一个恰当的水平。

In order to analyze transcriptional regulative mechanism of GmDREB3 gene, the GmDREB3 promoter （1 648 bp） was isolated from soybean genome using SiteFinding-PCR method. The sequences is abundant in A/T base and predicted to contain a lot of putative cis-element, such as low-temperature responsive element （MYC）, dehydration responsive element （MYB）, ABA responsive element （ABRE）, and so on, as well as TATA-box in biological database. To identify the key promoter regulative region controlling gene expression, GmDREB3 promoter was truncated according to the prediction of putative cis-element and inserted into the site upstream of GUS reporter gene. Then, vectors containing different length GmDREB3 promoters were transferred into wheat callus by particle bombardment, and after different stress treatments, function of these putative cis-elements was analyzed by identifying activation of GUS using histochemistry staining and GUS fluorescence intensity analysis. The results indicated that the promoters between -285 and -1 648 bp （relative to the translational start site） activated expression of GUS report gene under drought and low temperature stresses. But only the promoter region between -285 and -1 117 bp could activate expression of maximal GUS gene in wheat callus. Another promoter region between -1 117 and -1 464 bp weakened the ability for activating expression of GUS gene under drought and low-temperature stress conditions. Thus, it was suggested that there are some positive regulating motif between -285 and -1 117 bp and negative regulating motif between -1 117 and -1 464 bp of GmDREB3 promoter. As a result, the expression of GmDREB3 gene can be kept in an appropriate level response to various stresses through the regulation of both positive and negative regulating motifs.