Abstract:

In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585 bp cDNA fragment was amplified and cloned. The 1 585 bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of diffe rent sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定

徐军望  魏晓丽  李旭刚  陈蕾  冯德江  朱祯^*

(中国科学院遗传和发育生物学研究所,北京 100101)

摘要：根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.

关键词 水稻EPSP合酶 cDNA序列 序列分析 拷贝数 表达

*通讯作者 Email: zzhu@genetics.ac.cn

Key words: rice EPSP synthase, cDNA sequence, sequence analysis, copy numbers, expression