遗 传 学 报 Acta Genetica Sinica, August 2006, 33 (8)：757–764 ISSN 0379-4172 Cloning, Expression, and Mapping of GDP-D-mannose Py- rophosphorylase cDNA from Tomato (Lycopersicon esculen- tum) ZOU Li-Ping 1 , LI Han-Xia 2 , OUYANG Bo 1 , ZHANG Jun-Hong 1 , YE Zhi-Biao 1,① 1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; 2. College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, China Abstract: GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly ho- mologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA se- quence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1 086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii. Key words: tomato; GDP-D-mannose pyrophosphorylase; molecular cloning; gene expression; bin mapping Received: 2005-08-12; Accepted: 2005-09-16 This work was supported by the National Natural Science Foundation of China (No. 30370980). ① Corresponding author. E-mail: zbye@mail.hzau.edu.cn L-ascorbic acid (AsA, vitamin C) is essential for cardiovascular function, immune cell development, connective tissue, and iron utilization. Although plants and most animals can synthesize ascorbic acid, humans lack L-gulono-1,4-lactone oxidoreductase, which is required for the final step in AsA synthesis. Because AsA cannot be stored in human bodies, the vitamin must be acquired regularly from dietary sources, primarily from plants rich in AsA. As many plant foods are not rich in AsA, the ability to increase the level of this vitamin in more plant foods would increase their nutritive value. AsA plays an important role as an antioxidant and protects the plant from oxidative damage by scavenging free radicals and active oxygen species (AOS) that are generated during photosynthesis, oxi- dative metabolism and various stresses including ex- cess light, soil water deficit, water logging, UV-B radiation, and ozone [1-5] . Recent studies indicate that ascorbate is involved in the process and regulation of both cell division and expansion [3,6,7] . Moreover, the AsA redox state, controlled by ascorbate oxidase (AO) activity levels, is mainly responsible for the apoplast capability of transmitting signals related to environ- mental changes or defence processes [5,8] . In the case of animals, the biosynthetic pathway of AsA has been completely worked out. AsA is synthesized in the liver or the kidney from D-glucose, which in turn is derived from D-glucuronate and L-gulono-1, 4-lactone (L-GulL) by an inversion in the carbon skeleton of the precursor [3] . On the other hand, in plants, it was not until 1998 that Wheeler et al. [9] proposed a pathway which