Cloning Of GLDH Gene Full-Length CDNA From Non-Heading Chinese Cabbage

L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3,GLDH) was a key enzyme that catalyzed the final step in the L-ascorbic acid synthetic pathway of plants. This gene was cloned from non-heading Chinese cabbage which could be used to increase ascorbate production, the nutritional value and stress tolerance through gene engineering technique.1. Suzhouqing and Wutacai were treated with light and continuous darkness. The result indicated that the enzyme activity and L-ascorbic acid content exhibited a diurnal change. The contents of L-ascorbic acid and total L-ascorbic acid were in a low level in the morning and increased during the day. However, the levels of L-ascorbic acid and total L-ascorbic acid treated with continuous darkness changed differently. Similar diurnal change was also observed in GLDH activity, suggesting that the enzyme activity was induced by light.2. The PCR primers were designed on the basis of L-galactono-1,4-lactone dehydrogenase gene cDNA sequences from cauliflower. A 1779bp fragment of GLDH cDNA from Suzhouqing was obtained using RT-PCR technique. Based on the known fragment of GLDH, primers were designed for the 5' and 3' extensions by RACE techniques.The resulting 453bp and 622bp from the 5' and 3' ends were sequenced and the combined information of the cloned cDNA fragments of non-heading Chinese cabbage GLDH resulted in a total of 2034bp. The accession no. of this gene in GenBank was AY899298. The complete sequence of GLDH gene cDNA revealed an open reading frame of 1806bp encoding 601 amino acids. There were a A (position 3) and a TGA(position 13-15) terminator codon included in the same frame upstream from the ATG initiator codon.A hexanucleotide AATAAA consensus signal for polyadenylation was found 25 nucleotides before the poly(A)+ tract. The presence of these elements showed that the obtained cDNA corresponding to the character of full-length cDNA. It contained a 24bp 5' untranslated region and a 203bp 3' untranslated region; the encoded protein was 67, 921 Da with isolectric point 8.66. The amino acid sequence comparison with Brassica oleracea GLDH gene and Arabidopsis thaliana GLDH gene showed that identity was 97% and 89%,...