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Vol. 45 No. 3 SCIENCE IN CHINA (Series C)June 2002Cloning of genomic DNA of rice 5-enolpyruvylshikimate3-phosphate synthase gene and chromosomal localization ofthe geneXU Junwang (徐军望), FENG Dejiang (冯德江), LI Xugang (李旭刚),CHANG Tuanjie (常团结) & ZHU Zhen (朱 祯)Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, ChinaCorrespondence should be addressed to Zhu Zhen (email: zzhen@genetics.ac.cn)Received July 16, 2001Abstract The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs)is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for ricegenomic library screening. In a screen of approximately 8.0library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone,E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequencewas obtained. Sequence analysis and homologous comparison revealed that epsps gene is com-posed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with theprobe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with anindica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSPsynthase from monocotyledons.104 clones from the rice genomicKeywords: 5-enolpyruvylshikimate 3-phosphate synthase, gene isolation, DNA sequence, Chromosomal locating.The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC2.5.1.19) is a critical enzyme in the shikimate pathway, which cata-lyzes the addition of the enolpyruvyl moiety of phosphoenolpyruvate to shikimate-3-phosphate[1].Interest in the characterization of plant EPSPS has been increased significantly since the enzymewas identified as the primary target of the broad-spectrum, nonselective herbicide glyphosate (N-phosphonomethyl- glycine)[2]. It has been shown that glyphosate can inhibit EPSPS activity in acompetitive manner with regard to shikimate-3-phosphate (S-3-P). EPSP synthase has been usedto engineer glyphosate tolerance in transgenic plants either by the overproduction of the wild-typeEPSPS or by the expression of a mutant gene (aroA) encoding glyphosate-resistant EPSPs[3,4].Shah et al. (1986) have obtained high-level expression of petunia EPSPs in transgenic plants. Thepetunia cDNA clone was linked to a Cauliflower Mosaic Virus 35S promoter. Expression of thewild-type gene from this promoter resulted in a 20-fold increase in EPSPs activity in transgenicpetunia plants. The plants tolerated applications of glyphosate approximately four times greaterthan that needed to kill nontransformed plants[5].EPSPs has been detected and studied for a number of organisms including higher plants.万方数据