中国农业科学2017，Vol.50Issue(17)：3391-3404,14.DOI:10.3864/j.issn.0578-1752.2017.17.013
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
Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase Cu/Zn-SOD Gene Family from Luffa cylindrical
摘要
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 1011 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-SOD gene family was 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.关键词
丝瓜/褐变/Cu/Zn-SOD/表达分析/SOD活性Key words
Luffa cylindrical/browning/Cu/Zn-SOD/expression analysis/activity of SOD引用本文复制引用
朱海生,刘建汀,陈敏氡,李永平,王彬,张前荣,叶新如,林珲,温庆放..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[J].中国农业科学,2017,50(17):3391-3404,14.基金项目
福建省自然科学基金(2015J01118)、福建省属公益类科研院所基本科研专项(2017R1026-6)、福建省农业科学院创新团队 PI 项目(2016PI-40)、福建省农业科学院创新项目(2015QC-6、PC2017-7) （2015J01118）
