关键词: 荔枝, 结合态过氧化物酶, 纯化, 质谱鉴定, 基因表达, 着色和成熟

Abstract:

【Objective】 In a previous study, the considerable activity of ionically bound peroxidase (BPox) was found in litchi pericarp. The BPox revealed a close relationship with the fruit maturation, but its role was unclear. This work was aimed to elucidate the biochemical properties and gene expression pattern of BPox for the further investigation of its involvement in the process of litchi coloration and maturation.【Method】The mature pericarp of litchi (Litchi chinensis Sonn. cv. Wuye) was used as material, and the BPox was extracted and purified through column chromatography of Streamline Phenyl, CM-52, Phenyl Sepharose and Superdex-200, respectively. The optimal pH and temperature, the substrate specificity and the inhibitors were measured, respectively. The K_m values of BPox for guaiacol and (-)-epicatechin and V_max values were determined by using double reciprocal plots, respectively. The purified Bpox protein was conducted to SDS-PAGE and in-gel digestion by trypsin, and the sequences of the peptide fragments were identified by using MALDI tandem TOF MS. Total RNA was isolated from litchi pericarp, and the cDNA encoding BPox was cloned. The fruits were harvested 58, 69, 76, 80 and 90 days after full blooming (DAFB), the determination of BPox activity changes in the pericarp and the analysis of BPox gene expression using real-time quantitative PCR, were performed, respectively.【Result】Two most major fractions of ionically bound cationic peroxidase, named BPox-2 and BPox-3, were purified from litchi pericarp, respectively. The apparent molecular weights of the two isoforms were the same, and were estimated to be 30 and 34 kD by gel filtration and SDS-PAGE, respectively. For the BPox-2 and the BPox-3, the optimal pH was 6.0, and the optimal temperature was 40℃ and 45℃, respectively. In the presence of H₂O₂, similar substrate affinity was revealed, while guaiacol and (-)-epicatechin (EC) were the favorable substrates for the two BPoxs. The metal ions test exhibited poor effect on the activity and the most effective inhibitors for litchi BPoxs were dithiothreitol, ascorbate and L-cysteine. The K_m values of BPox-2 and BPox-3 for guaiacol were 2.97 and 2.58 mmol∙L^-1, and the V_max values were 38.60×10⁶and 19.85×10⁶ U∙mg^-1, respectively. The K_m values of BPox-2 and BPox-3 for EC were 3.49 and 3.24 mmol∙L^-1, and the V_max values were 38.72×10⁶ and 23.06×10⁶ U∙mg^-1, respectively, illustrating that the catalytic efficiency (V_max/K_m) of BPox-2 was higher than that of BPox-3. The result of MALDI TOF MS demonstrated differences of peptide mass fingerprint (PMF) between the BPox-2 and the BPox-3; however, a common peptide fragment digested from the two peroxidases corresponding to the amino acid sequence of TASLSAANSDLPSPFADLATLIAR was identified by tandem MS and Mascot database search. The ORF of cDNA for litchi BPox2, containing 960 bp in length was cloned, encoding a polypeptide of 319 amino acid residue. The results of analysis revealed that, the polypeptide coded by the cDNA contained a putative 26-mer signal peptide and was absent of vacuolar sorting sequence on the C-terminus, and only one potential N-glycosylation site was found in the sequence. The molecular weight and the pI value of the mature polypeptide were predicted to be 31.35 kD and 7.71, respectively. The activity of BPox was very weak in the pericarp of young fruit. From the onset of pericarp coloration at 76 DAFB, the BPox activity increased remarkably, and then, it rose significantly coinciding with the subsequent process of fruit maturation until 90 DAFB. The qPCR results showed that the transcript level of BPox2 gene was low in the pericarp of young fruit at 58 DAFB and 69 DAFB. It increased dramatically, reached a peak at 76 DAFB, being 60.56-fold of that at 69 DAFB, and then declined. The transcript level increased significantly with the process of fruit maturation at 90 DAFB.【Conclusion】Characterization of the BPox illustrated that, its pH optimal, temperature optimal and substrate specificity etc., were similar to those of soluble peroxidases (SPox) in litchi pericarp and other plant peroxidases; however, the catalytic efficiency (V_max/K_m) of BPox for guaiacol and EC was much higher than that of litchi SPox. The results of MALDI MS/MS identification suggested that the BPox-2 and the BPox-3 were the two isoforms coded by the BPox2 gene and distinct due to different post-translational modification. The molecular weight of the predicted mature polypeptide coded by the BPox2 cDNA was near to that of the purified protein, suggesting its relative low degree of post-translational modification. Litchi BPox played a role in the pericarp maturation and was regulated at the transcriptional level.

Key words: litchi, ionically bound peroxidase, purification, MALDI MS/MS identification, gene expression, coloration and maturation