[In silico cloning of glucose-6-phosphate dehydrogenase cDNA from rice (Oryza sativa L.)].
In silico cloning was a new strategy of gene cloning developed with the development of genome, EST projects and bioinformatics. Using wheat glucose-6-phosphate dehydrogenase cDNA (clone: Tagpdl) sequence as a querying probe, one highly homologous BAC clone sequence was obtained from rice sequence database of GenBank and the putative cDNA sequence of rice glucose-6-phosphate dehydrogenase was assembled according to the wheat clone. Furthermore, the full-length cDNA of rice glucose-6-phosphate dehydrogenase was cloned by RT-PCR with two primers designed based on this assembled cDNA sequence. Since this fragment contained a complete ORF of 1515 bp with a stop codon in its upstream and poly(A) signal in its downstream, it could be concluded that a full-length gene (GenBank accession number AY078072), which was named as OsG6PDH. Homology analysis of OsG6PDH showed a 88% identity with wheat and the deduced amino acid showed 89%, 79% and 80% homology with G6PDH from wheat, tomato and tobacco respectively. OsG6PDH was expressed in inflorescence, embryo, root and leaf of rice, with a slightly higher in inflorescence and root. It was also discussed in this paper that the application of in silico cloning in the isolation of functional genes from rice.
Huang
Ji
J
National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China.
Wang
Jian-Fei
JF
Zhang
Hong-Sheng
HS
Cao
Ya-Jun
YJ
Lin
Chang-Fa
CF
Wang
Dong
D
Yang
Jin-Shui
JS
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D005954:Glucosephosphate Dehydrogenase