Isolation, expression, and characterization of a hydroperoxide lyase gene from cucumber

• Published in: International journal of molecular sciences Vol. 14; no. 11; pp. 22082 - 22101
• Main Authors: Wan, Xu-Hua, Chen, Shu-Xia, Wang, Cong-Ying, Zhang, Ran-Ran, Cheng, Si-Qiong, Meng, Huan-Wen, Shen, Xiao-Qing
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 07.11.2013; Molecular Diversity Preservation International (MDPI)
• Subjects: Alcohol; Aldehyde-Lyases - biosynthesis; Aldehyde-Lyases - chemistry; Aldehyde-Lyases - genetics; Aldehyde-Lyases - isolation & purification; Aldehydes; Amino Acid Sequence; C6 and C9 aldehyde content; Chromatography; Cloning; Cloning, Molecular; Cucumis sativus; Cucumis sativus (cucumber); Cucumis sativus - enzymology; Cytochrome P-450 Enzyme System - biosynthesis; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Dehydrogenases; DNA, Complementary - genetics; Enzymes; expression mode; Fruits; gene cloning; Gene expression; Gene Expression Regulation, Plant; Hydrogen Peroxide - chemistry; Hydrogen Peroxide - metabolism; hydroperoxide lyase; hydroperoxide lyase activity; Linoleic Acids - chemistry; Lipid Peroxides - chemistry; Mass spectrometry; Scientific imaging; sequence analysis; China
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms141122082

A full-length cDNA coding for hydroperoxide lyase (CsHPL) was isolated from cucumber fruits of No. 26 (Southern China type) and No.14-1 (Northern China type), which differed significantly in fruit flavor. The deduced amino acid sequences of CsHPL from both lines show the same and significant similarity to known plant HPLs and contain typical conserved domains of HPLs. The recombinant CsHPL was confirmed to have 9/13-HPL enzymatic activity. Gene expression levels of CsHPL were measured in different organs, especially in fruits of different development stages of both lines. The HPL activities of fruit were identified basing on the catalytic action of crude enzyme extracts incubating with 13-HPOD (13-hydroperoxy-(9Z,12E)-octadecadienoic acid) and 13-HPOD + 9-HPOD (9-hydroperoxy-(10E,12Z)-octadecadienoic acid), and volatile reaction products were analyzed by GC-MS (gas chromatography-mass spectrometry). CsHPL gene expression in No. 26 fruit occurred earlier than that of total HPL enzyme activity and 13-HPL enzyme activity, and that in No. 14-1 fruit was consistent with total HPL enzyme activity and 9-HPL enzyme activity. 13-HPL enzyme activities decreased significantly and the 9-HPL enzyme activities increased significantly with fruit ripening in both lines, which accounted for the higher content of C6 aldehydes at 0-6 day post-anthesis (dpa) and higher content of C9 aldehydes at 9-12 dpa.