Studies On The Separation And Purification, Identification, And CDNA Cloning Of Alcohol Dehydrogenase During Embryogenesis In Longan (Dimocarpus Longan Lour.)

A certain type of dehydrogenase from longan embryogenic calli (EC) was found and the preliminary studies indicated that the proteins were probably NAD⁺-dependent putative sugar alcohol dehydrogenases (SADHs). The activity of the enzymes decreased gradually with the development of embryoids, the zymogram pattern did not change in the embryogenic callus, globular embryo and cotyledonary embryo but the staining intensity decreased during somatic embryogenesis, which indicated that the enzymes located in embryogenic calli of longan were correlated to somatic embryogenesis. In order to identify the characteristics of these putative sugar alcohol dehydrogenases and reveal their involvement during somatic embryogenesis, further studies including separation, purification, identification and cDNA cloning of the enzymes were performed, and the main results showed as follows:1. Separation and purification of the putative SADHs. The putative SADHs were sensitive to 0.5mmol/L DTT and the stability of the enzymes increased when added with 10% glycerol. After separation experiments performed on different column media, a protocol for purification of the putative SADHs was developed. After homogenization and centrifugation, the crude enzyme extract was fractionated with 0～80% saturation of ammonium sulphate [(NH₄)₂SO₄]. The precipitate was redissolved and then desalted by gel filtration on Sephadex G-25. Two isozymes Lc.A and Lc.B of putative sugar alcohol dehydrogenase in longan embryogenic calli were purified when chromatographed on DEAE-Sepharose F.F., DEAE-52, Phenyl Sepharose HP and Superdex 200 subsequently. The Lc.A and Lc.B were homodimer and carried different isoelectric points when compared with the results of native linear gradient PAGE and SDS-PAGE, and the apparent molecular weight of subunit estimated was the same of 47.4kD.2. Identification of the putative SADHs by biology MS. Matrix-assisted laser desorption/ ionization time of flight-mass spectrometry (MALDI-TOF MS) was applied to the analysis of peptide mass fingerprinting (PMF) in Lc.A and Lc.B respectively. The PMF data of Lc.A and Lc.B were used in Mascot search, and the Lc.A and Lc.B were identified as alcohol dehydrogenase (ADH) preliminarily. Then, the technique of electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF2 MS) was used in Lc.A and Lc.B identification. Four tryptic digested peptide fragments in Lc.B, GTFFGNYKPR, KFGVTEFVNPK, AFEYMLGGDGR and FITHEVPF SEINK, were sequenced by ESI-MS/MS; another four fragments in Lc.A, FGVTEFVNPK, GTFFGN YKPR, FITHEVPFSEINK and DYDKPVQEVIAEMTDGGVDR (or DYDKPVKEVLAEMTDNVD R), were obtained by ESI-MS/MS sequencing. The results of database search and multiple alignments showed that Lc.A and Lc.B matched to the ADH protein significantly. Therefore, the putative sugar alcohol dehydrogenases Lc.A and Lc.B were identified as two isozymes of ADH from longan EC.3. Measurement of the activity and zymogram pattern of ADH during somatic embryogenesis in longan. The results showed that the activity of ADH were high in embryogenic calli and decreased in globular embryo and cotyledonary embryo; the zymogram pattern showed that the staining intensity...