关键词: 枸杞, 木糖异构酶, 原核表达, 多克隆抗体, Western blot检测

Abstract: In this study, the full-length sequence of the xylose isomerase gene (LbxylA) was obtained from Chinese wolfberry fruit from the cultivar ‘Ningqi 1’ (Lycium barbarum Linn.) by using reverse transcription-polymerase chain reaction (RTPCR). The open reading frame (ORF) of LbxylA was 1 446 bp in length encoding a polypeptide of 481 amino acids. The relative molecular mass was 53.54 kDa and the theoretical isoelectric point (pI) was 5.37. The deduced amino acid sequence of LbxylA shared a high similarity (95%) to those of the tobacco and potato xylose isomerase genes as revealed by bioinformatics analysis. The recombinant plasmid pGEX4T-1-LbxylA was constructed and transformed into E. coli Rosetta (DE3) for prokaryotic expression induced by isopropyl-β-D-thiogalactoside (IPTG), and the optimum expression conditions were determined as induction at 18 ℃ for 10 h with 100 μmol/L IPTG. The fused protein, partially soluble, was purified and used as an antigen to immunize Japanese big-ear white rabbits for the preparation of polyclonal antibody against the LbxylA protein. As a result, the serum antibody titer was higher than 1:81 000 as determined by indirect ELISA. The relative expression level of LbxylA in the fruit reached its maximum at 9 d after full bloom, and then decreased gradually with fruit development. The changes of LbxylA protein level as detected by Western Blot were basically consistent with the changes of LbxylA relative expression. The protein expression level of LbxylA was lower at 9 d after full bloom than at 15 d, and then decreased gradually from the 15th day onwards. Both the gene and protein expression levels reached the lowest values at the end of fruit ripening. This study indicated that the protein expression of LbxylA lags behind the gene transcription possibly owning to the post-translation processing at the early stage of fruit development. The results of this study are helpful to gain further insights into LbxylA function and its effect on sugar accumulation during fruit development.

Key words: Lycium barbarum Linn., xylose isomerase gene, prokaryotic expression, polyclonal antibody, Western blot