CDNA Cloning And Tissue Epreession Analysis Of Bovine Liver NADP(H)-dependent Retinol Dehydrogenase/Reductase

ObjectiveRetinoids play important roles in various physiological processes, and many enzymes involved in retinoic acid synthesis have been reported as follows. In 1997,Huang, D. Y. etc purified and identified a novel NADP( H) dependent retinol dehydrogenase/reductase( NRDR) from rabbit liver cytosol. NRDR has much higher substrate specificity and afiBnity than reported cytosolic alcohol dehydrogenase( ADH) and microsomal short - chain dehydrogenase/reductase(SDR) ,and metabolize the first step in the all - trans - retinoic acid biosynthesis. Now the full - length cDNA of human, mouse and rabbit NRDR have been cloned and submitted to GenBank ([ AB045133 ] [ AB045132 ] [ AB045131 ] ). But no paper is reported. Here According to the conserved sequences of human, mouse and rabbit NRDR cDNA, we designed specific primers and cloned the full - length of bovine NRDR cDNA (AF487454) by using rapidly amplify cDNA ends (RACE) methods and the sequence was analyzed . It provided a reliable foundation to investigate the biological function of this protein and retinoic acid biosynthesis.MethodsTotal RNA isolation and cDNA was obtained: The total RNA was isolated from bovine liver using Trizol reagent and quantitated on the basis of the absorbance at 260/280. The cDNA library was obtained using oligo dT primer by Reverse Transcriptase XL(AMV) Kit .The biosynthesis of primers and the amplification of NRDR cDNA fragment: According to the conserved sequences of human, mouse and rabbit NRDR cDNA, we designed a pair of primer using Jellyfish program to amplify a 294 bp bovine liver NRDR DNA fragment.The 3 ' RACE method: 3 ' RACE reactions were performed u-sing the TaKaRa 3 ' - full RACE core set following the manufacturer ' s instruction. The 3 ' RACE product was amplified using oligo dT- 3 sites adaptor primer and F primer which is designed with the obtained 294bp fragment as template. The RACE products were sub-cloned into PGEM - T vectors and sequenced.The 5 ' RACE methods:5 ' RACE reactions were performed u-sing the TaKaRa 5 ' - full RACE core set following the manufacturer ' s instruction. First, we designed one 5 ' - end - phosphorylated RT primer and two pairs of nested primers ( SI and Al, S2 and A2) u-sing the obtained 294bp fragment as template. 1st strand cDNA is synthesized by reverse transcription from target RNA using 5 ' - end- phosphorylated RT primer which is specific to the target RNA. Second , degraded the hybrid DNA - RNA to separate RNA with RNase H. Third, carried out the circularization of single - stranded cDNA or formation of concatemers by RNA ligase. Finally , nested 5 RACE reactions were performed according to the manufacturer's in-structions; 1st PCR reaction with primers SI and Al , and then a nest PCR reaction with primers S2 and A2. Hie RACE products were sub-cloned into PGEM - T vectors and sequenced.Sequences analysis: DNA sequence analysis was performed using online the National Center for Biotechnology Information (NCBI) blast nucleotide and local software Jellyfish. The sequence homology search was conducted at the protein level using the BLAST network service of NCBI. The sequence alignment among proteins was performed using the program Clustalw. Protein subsequence motifs were identified u-sing the network service SMART ( information for bovine NRDR was analysis using the ( Hofman et al,1999).ResultsThe results of RACE: The total RNA was lOOug, the 260/280 value is 1.84 -2. 0. We designed 3 ' RACE and 5 ' RACE using Jellyfish program with the obtained 294bp fragment as template. The 3 ' RACE products were subcloned into PGEM - T vectors and obtained 1200bp. The 5 ' RACE products were subcloned into PGEM -T vectors and obtained 400bp and 200bp fragments, the sequenced result revealed the 200bp fragment was the target product.Identification and sequence analysis of the full - length bovine NRDR cDNA : The 200bp fragment of 5 ' nested RACE and 1200bp fragment of 3 ' RACE products inserted into PGEM - T vectors were sequenced with primers T7and SP6 an...