水稻26kDa球蛋白基因启动子克隆及序列分析

以水稻品种日本晴基因组总DNA为模板,采用PCR扩增获得约900bp大小的DNA片段,回收该片段并与pUCm-T载体连接,转化感受态大肠杆菌.进行PCR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904个核苷酸对;采用vector NTI软件将本试验中克隆的序列与Genbank（AY427575）公布的日本晴球蛋白基因启动子序列比对,有9个核苷酸差异,同源性为99%,证实本试验中克隆的DNA序列为水稻球蛋白基因启动子;在重要功能区段上,两者核苷酸序列完全一致.本研究认为造成长期不在一个环境中种植的同一水稻品种球蛋白基因启动子序列差异的原因之一,可能是核苷酸中性突变.水稻球蛋白基因启动子的成功克隆,为今后开展水稻等重要农作物转基因研究奠定基础.

Cloning and Nucleotide Sequence Analysis of 26kDa Aalpha-Globulin Promoter in Rice (Oryza sativa L.)

About 900bp DNA fragment was amplified by using PCR technique from genomic DNA of rice japonica cultivar"Nipponbare". Recovered and linked with pUCm-T vector, the DNA fragment was transferred to the competence E.coli. After using double enzymes digestion analyses, the recombinant was chosen and the sequence of alpha-globulin promoter was identified. Comparing rice globulin promoter sequence cloned in this experiment with that reported in GenBank AY427575 by using vector NTI software, we found that the DNA fragment composed of 904bp nucleotide with homology 99%. There were no differences between the cloned promoter sequence and the sequence published in GenBank in dominant functional regions, based on which we suggested that the nucleotide neutral mutation would result in the main difference between the two sequences of rice globulin promoter of"Nipponbare". The successfully cloning rice globulin promoter sets up bases for the prospective transgenic studies on important crops such as rice.