Abstract: 【Objective】The purpose of the present investigation was to construct the large-scale isolation and high-throughput identification systems for the Waxy proteins in rye seeds so as to provide more information about how to understand its bio-chemical features, functions and genetic diversity in rye cultivars. 【Method】Waxy gene was isolated by using a set of gene specific primers from the full-length cDNA library derived from the immature seeds of Austria rye, followed by prokaryotic expression in Escherichia coli system. The extracted product of the DuoFlow system from Austria rye seeds, together with the purified recombinant Waxy protein by Ni-NTA resin, were confirmed by SDS-PAGE and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and then followed by the alignment of protein sequences which deduced from the Waxy gene originated from Austria rye and common wheat. The above Waxy protein purified by DuoFlow workstation was then employed as the sample to construct the optimal capillary electrophoresis (CE) system for the waxy of rye seeds. Both the DuoFlow isolation system and CE identification system were verified based on the analysis results of rye cultivars. 【Result】 Sequence analysis revealed that the gene isolated from Austria cDNA (KF559182) encoded a 60 kD mature Waxy protein. The optimal isolation system for DuoFlow Workstation was the following: fixation Waxy proteins using 20 mM Tris-Hcl buffer (pH 9.5), and then elution the target components using 20 mmol•L-1 Tris + 1 mol•L-1 NaCl buffer (pH 9.5) from the UNO Q1 anion exchange column. All the above results, including SDS-PAGE, MS identified peptides and seqeunces semilarity of deduced amino acids based on the Waxy sequence, confirmed that the purified products by DuoFlow system in the present study was consistant with the characters of rye Waxy and therefore belongs to the Waxy protein itself. The CE was carried out using the purified protein by DuoFlow system under the conditions of 25℃, 20 kV and 0.5 psi × 5 s, together with the electrophoresis buffer 0.05 mM borax + 15% acetonitrile + 1% SDS solution (pH 9.5), and a single chromatographic peak was obtained aroud 11-12 min through the above CE system. The results also showed that the Waxy protein which extracted from the seeds could be used directly for the identification of rye Waxy in CE system. Meanwhile, the testing results of rye cultivars showed that the DuoFlow and CE system were suitable for the separation and identification of rye Waxy subunits, respectively. 【Conclusion】The present study successfully isolated the complete coding sequence of Waxy gene from cDNA library of Austria rye using the gene-specific primers, and constructed the DuoFlow large-scale isolation system and CE high-throughput identification system for Waxy subunits of rye grains.

Key words: Austria rye (Secale cereale L.) , waxy protein , capillary electrophoresis , BioLogic DuoFlow protein purification system , MALDI-TOF MS