丝瓜铜锌超氧化物歧化酶Cu/Zn-SOD基因家族的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2017.17.013

Abstract

Abstract: 【Objective】The aim of this study was to clone the Cu/Zn-SOD gene family from Luffa cylindrical, investigate their sequence characteristics and analyze their expression in luffa browning. These findings will provide a scientific basis for further revealing the mechanism of luffa browning and lay a practical foundation for the genetic improvement of luffa. 【Method】 The cDNA sequences of Cu/Zn-SOD gene family were obtained by transcriptome sequencing and RT-PCR. The bioinformatics methods were used to analyze the putative amino acid sequence, and quantitative real-time PCR (qRT-PCR) method was used to study the expression of Cu/Zn-SOD gene family in different tissues and browning conditions. The superoxide dismutase enzyme activity was measured by NBT deoxidization method. The total phenols was measured by folin-cioncaleuc method. 【Result】Three cDNAs of Cu/Zn-SOD were cloned from luffa fruit, in turn being named LcCu/Zn-SOD1, LcCu/Zn-SOD2 and LcCu/Zn-SOD3. The cDNA sequence of LcCu/Zn-SOD1 was 758 bp in length, containing a 456 bp opening reading frame(ORF), encoded a polypeptide of 152 amino acids. The cDNA sequence of LcCu/Zn-SOD2 was 799 bp in length, containing a 471 bp ORF, encoded a polypeptide of 157 amino acids. The cDNA sequence of LcCu/Zn-SOD3 was 1 011 bp in length, containing a 663 bp ORF, encoded a polypeptide of 221 amino acids. They shared over 90% identity with the homologous proteins from Cucumis melo, Cucurbita pepo and Cucumis sativus. The bioinformatics analysis showed that three proteins were hydrophilic protein without signal-peptide and transmembrane region, and the Wolf Psort protection indicated that they were located in the cytoplasm. The expression of LcCu/Zn-SODgene familywas the highest in root and the lowest in flower. During post-harvest storage, the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 was up-regulated in the early, and then decreased. The expression levels of three genes were overall down-regulated in fresh-cut luffa fruit. Correlation analysis showed that the expression level of LcCu/Zn-SOD1 showed a extremely significant positive correlation with SOD activity, and the expression level of LcCu/Zn-SOD3 showed a significant positive correlation with SOD activity during fresh-cut and post-harvest storage. SOD activity was significantly and negatively correlated with total phenols content during fresh-cut conditions. LcCu/Zn-SOD1 and LcCu/Zn-SOD3 play an important role in regulating the activity of SOD, and the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may influence the activity of SOD and the process of luffa browning.【Conclusion】Three cDNAs of Cu/Zn-SOD were firstly obtained and characterized from luffa fruit, LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may play an important role in luffa browning process.

Key words: Luffa cylindrical, browning, Cu/Zn-SOD, expression analysis, activity of SOD

ZHU HaiSheng, LIU JianTing, CHEN MinDong, LI YongPing, WANG Bin, ZHANG QianRong, YE XinRu, LIN Hui, WEN QingFang. Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase Cu/Zn-SOD Gene Family from Luffa cylindrical[J].Scientia Agricultura Sinica, 2017, 50(17): 3386-3399.

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Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase Cu/Zn-SOD Gene Family from Luffa cylindrical

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