大麦与油菜种皮特异启动子表达模式的比较

Comparison of the expression pattern of two seed coat-specific promoters from barley and seedrape

关键词:  种皮特异启动子  GUS组织化学染色  非生物胁迫  大麦  油菜

Key words:  seed coat-specific promoters  GUS histochemical staining  abiotic stress  Hordeum vulgare  Brassica napus

摘要:

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件; 转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高; 油菜Bntt种皮特异启动子表达较弱; 成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性; Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。

Abstract:

Promoter is a segment of DNA molecule which locates in the upstream of transcription start site and specifically binds to RNA polymerase. Efficient genetic modification of transgenic plant requires promoter as the regulatory sequence to drive the expression of foreign gene. Specific promoters with spatial and temporal control of ectopic gene expression are important for acquirement of the transgenic plant and its product with high quality. To understand the expression pattern of seed-coat specific promoters, based on the reported sequences, two seed-coat specific promoters-Gerb and Bntt were isolated from Brassica napus and Hordeum vulgare by homologous-based cloning method, analysis of the elements in both promoters, then Gerb::GUS and Bntt::GUS were constructed and introduced into Arabidopsis, and the expression patterns of Gerb and Bntt were observed by GUS histochemical staining. The results of bioinformatics showed that multi-copies of seed specific expression cis-acting elements and various stress response elements were identified in both promoters. Histochemical GUS staining of the transgenic plant indicated that in seedling stage, Gerb promoter showed strong blue GUS staining with the cotyledon and hypocotyl while relatively weaker of GUS activity with the true leaves and roots; Bntt promoter showed weak GUS staining in whole seedling; for adult plant, various tissues(leaf, stem, inflorescence, silique, etc.)with promoter Gerb presented visible GUS staining while with promoter Bntt a very weak staining was only observed on vascular bundle of silique and the leaf. Under various stresses, the expression pattern of transgenic line with Gerb::GUS showed no significant change, however, for Bntt::GUS, NaCl stress could cause strong GUS staining with silique and seeds, while no significant staining was detected in other tissues or under other stresses. These results indicated that Gerb and Bntt promoters displayed different temporal and spatial expression patterns, which may be helpful for screening seed-coat specific promoter in practice, however, the details of regulation mechanism needs further study to validate.