摘要
为进一步研究脱水可诱导启动子的结构和功能,并为开展禾谷类植物转基因研究选择启动子提供依据,用大麦品种撒哈拉幼苗提取的总DNA为模板,利用设计合成的引物对进行PCR定向扩增,获得了HVA1s、Dhn4s和Dhn8s启动子片段。通过分离、纯化、酶切和连接,把启动子片段分别插入到带有GFP和GUS报告基因的表达载体质粒中。经测序确认,目标启动子片段与原报道的同源启动子序列一致性达95.3%以上。HVA1s、Dhn4s和Dhn8s启动子在构建的瞬间表达载体pHVA1sGFPG、pDhn4sGFPG、Dhn8sGFPG和pHVA1sGUSR、pDhn4sGUSR、pDhn8sGUSR中,都具有启动表达报告基因的能力,通过基因枪转化大麦幼苗可有效启动gfp和gus基因瞬间表达。
The objective of this experiment was to isolate dehydration-inducible promoters and construct their expression vectors for the further analyses of the structure and function, so as to provide with evidences for promoter selection in cereal plant transgenic researches. The fragments of HVAls, Dhn4s and Dhn8s promoters have been amplified through PCR in the experiment by using the designed primers and the total DNA from barley variety Sahara seedlings as template. Through isolation, purification, restriction digestion and ligation, the amplified fragments were inserted into expressional vectors with GFP or GUS reporter gene,respectively. By sequencing identification, the identity between the cloned promoters and the earlier reported ones reached greater than 95. 3% in DNA sequences. Promoters HVA1s, Dhn4s and DhnSs were all able to initiate the expression of reporter genes in the constructed vector pHVAlsGFPG, pDhn4sGFPG, pDhnSsGFPG, pHVAlsGUSR, pDhn4sGUSR, pDhn8sGUSR, which had been proved by the effectively transient expression of gfp and gus in barley seedlings transformed by genegun bombardment.
出处
《麦类作物学报》
CAS
CSCD
北大核心
2006年第3期1-7,共7页
Journal of Triticeae Crops
基金
云南省自然科学基金项目(2002C0042M)。
关键词
大麦
脱水
启动子
序列
表达载体
Barley
Promoter
Sequence, Dehydration
Expression vector