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园艺学报 ›› 2016, Vol. 43 ›› Issue (10): 2039-2049.doi: 10.16420/j.issn.0513-353x.2016-0406

• 研究报告 • 上一篇    下一篇

丝瓜过氧化氢酶基因CAT1的克隆及表达分析

温庆放*,刘建汀,朱海生,陈敏氡,王 彬,张前荣   

  1. (福建省农业科学院作物研究所,福建省农业科学院蔬菜研究中心,福建省蔬菜工程技术研究中心,福州350013)
  • 出版日期:2016-10-25 发布日期:2016-10-25

Cloning and Expression Analysis of Catalase CAT1 Gene from Luffa cylindrical

WEN Qing-fang*,LIU Jian-ting,ZHU Hai-sheng,CHEN Min-dong,WANG-Bin,and ZHANG Qian-rong   

  1. (Crops Research Institute,Fujian Academy of Agricultural Sciences;Vegetable Research Center,Fujian Academy of Agricultural Sciences;Fujian Engineering Research Center for Vegetables,Fuzhou 350013,China)
  • Online:2016-10-25 Published:2016-10-25

摘要:

采用RT-PCR和RACE技术从普通丝瓜果肉中分离到1条长达1 755 bp的cDNA序列,并对其进行序列分析。结果表明,该序列包含1个1 479 bp的开放读码框(ORF);预测编码492个氨基酸,理论分子量为56.98 kD,等电点为7.126;编码的蛋白与黄瓜(Cucumis sativus)、拟南芥(Arabidopsis thaliana)和萝卜(Raphanus sativus)同源蛋白的相似性均在92%以上,显示其高度的保守性,将基因命名为LcCAT1,GenBank登录号为:KP222260。Wolf Psort预测其亚细胞定位于过氧化物酶体(Peroxisomal)内;Motif Scan分析显示,蛋白氨基酸序列344 ~ 352位和54 ~ 70位分别为过氧化氢酶近端血红素—配体及血红素活性位点特征序列,推测其属于典型过氧化氢酶类(typical catalase,tCAT)。荧光定量PCR分析显示,LcCAT1具有组织表达特异性,在普通丝瓜品种‘福丝3号’叶片中表达量最高,将近为花、果实、根和茎中表达量的5倍。LcCAT1在所选的6个丝瓜品种的果肉中呈差异表达,普通丝瓜中的表达量均高于有棱丝瓜;在普通丝瓜品种‘福丝3号’鲜切及采后储藏褐变过程中随时间增加而呈现上调表达趋势,且与其酶活性的变化趋势基本一致。LcCAT1可能在丝瓜果肉褐变产生过程中发挥着一定的调控作用。

关键词: 丝瓜, 褐变, CAT1, 基因克隆, 实时荧光定量PCR

Abstract:

A length of 1 755 bp cDNA was isolated from Luffa cylindrical by using RACE and RT-PCR techniques,which contained a 1 479 bp open reading frame(ORF)that encoded 492 amino acids,with a predicted molecular weight of 56.98 kD and a hypothetical isoelectric point of 7.126. It shared over 92% identity with the homologous proteins from Cucumis sativusArabidopsis thaliana and Raphanus sativus,proving that it was highly conservative. This gene was named LcCAT1 and the GenBank accession was KP222260. Wolf Psort protection indicated that LcCAT1 protein was located in the peroxisome,and Motif Scan analysis showed that LcCAT1 protein had the characteristic amino acid sequences of catalase proximal heme-ligand and heme active sites in the position of 344–352 and 54–70 sites,respectively,which  suggested that LcCAT1 protein was a typical hydrogen peroxide enzyme(tCAT).The Real time PCR revealed that LcCAT1 exhibited a tissue specific expression,and the expression level in Luffa cylindrical‘Fusi 3leaves was the highest,which was almost five times than that in flowers,fruits,roots and stems. The levels of LcCAT1 were different among six luffa varieties,and the expression in Luffa cylindrical was higher than that in Luffa acutangula Roxb. What is more,the level of LcCAT1 in‘Fusi 3was up-regulated during fresh-cut and post-harvest storage browning processes,which was consistent with the changes observed in peroxidase activity,suggesting that LcCAT1 gene may play a regulation role in luffa browning process.

Key words: Luffa cylindrical, browning, CAT1, gene cloning, real time PCR

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