摘要
以日本晴水稻幼苗叶的基因组总DNA为模板,采用PCR方法扩增获得约1 300 bp大小的DNA片段,回收该片段并与pMD18-T载体连接,转化大肠杆菌感受态,并提取质粒DNA。用凝胶电泳检测和酶切鉴定,选取阳性克隆进行测序分析。测序结果显示,该片段含1 376个核苷酸对;应用NCB1网站的BLAST软件对本试验中克隆的序列与GenBank(NC_008401)公布的水稻硝酸还原酶基因启动子序列进行比对,有3个核苷酸差异,同源性为99%,证实本试验中克隆的DNA序列为水稻硝酸诱导基因启动子。该序列含有多个与转录调控有关的保守序列(如TATA-box、CAAT-box和GAGA-box)。此序列的成功克隆,为今后开展水稻等重要农作物转基因研究奠定基础。
About 1 300 bp DNA fragment was amplified by using PCR technique from seedings foliar genomic DNA of rice japonica eultivar ＂Nipponbare＂. Recovered and linked with pMD18 T vector, the DNA fragment was transferred to the competence E. coll. After using agarose gel electrophoresis and double enzymes digestion analyses, the recombinant was chosen and the sequence of 5r-flanking region of nitrate reductase promoter was identified. Comparing rice 5＇-flanking region of nitrate reductase promoter sequence cloned in this experiment with that reported in GenBank NC_008401 by using BLAST software of NCBI, we found that the DNA fragment composed of 1 376 bp nucleotide with homology 99% and they only have three nucleotides diversity. Based on tifs such as TATA box, CAAT-box and GAGA-box, which are related to regulation of transcription. The successfully cloning rice nitrate reductase promoter sets up bases for the prospective transgenic studies on important crops such as rice.
出处
《华中师范大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第2期284-288,共5页
Journal of Central China Normal University：Natural Sciences
基金
湖北省自然科学基金项目(2004ABA123)
关键词
日本晴水稻
硝酸还原酶基因
5’上游序列
基因克隆
Rice（Oryza sativa L. japonica, cv. Nipponbare）
nitrate reductase
5＇-flanking region
gene cloned
