Cloning, Expression, and Mapping of GDP-D-mannose Pyrophosphorylase cDNA from Tomato (Lycopersicon esculentum) |
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Authors: | Li-Ping ZOU Bo OUYANG Zhi-Biao YE |
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Institution: | a National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China b College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, China |
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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 homologous 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 sequence. 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. |
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Keywords: | tomato GDP-D-mannose pyrophosphorylase molecular cloning gene expression bin mapping |
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