丝瓜过氧化氢酶CAT2基因的分离及表达分析

Isolation and Expression of Catalase CAT2 Gene from Luffa cylindrical

该研究对普通丝瓜品种‘WL-3’的褐变果肉开展转录组测序,对高质量片段进行拼接和组装后获得58 073条unigenes序列,其中,27 301条unigenes在褐变前后表达水平发生变化。从差异表达的基因中筛选得到1条功能注释为过氧化氢酶(catalase,CAT)的基因全长序列(unigene0033876)。分析发现,该序列全长1 690 bp,包含1个1 479 bp的开放读码框(open reading frame,ORF),预测编码492个氨基酸,理论分子量(molecular weight,Mw)为56.94 k Da,等电点(isoelectric point,p I)为7.31,编码的蛋白质与葫芦科作物南瓜、西葫芦和黄瓜中同源蛋白质的相似性均在97%以上,显示其高度的保守性,基因命名为Lc CAT2,Gen Bank登录号为KR184674。荧光定量PCR分析结果表明,Lc CAT2基因具有组织表达特异性,在丝瓜叶片中表达量最高,花中表达量最低。Lc CAT2基因在所选的6个丝瓜品种中的表达量存在一定的差异,普通丝瓜中的表达量均高于有棱丝瓜。Lc CAT2基因在普通丝瓜品种‘WL-3’采后储藏条件下的表达量随时间增加而呈上调表达趋势。初步推测,Lc CAT2基因在丝瓜果肉褐变过程中起着一定的调控作用。该研究旨在从转录组测序结果中挖掘与丝瓜果肉褐变相关的基因,为今后丝瓜品种的选育及进一步揭示其酶促褐变产生的分子机制奠定理论基础。

The RNA-seq technique was used to analyze the changes of transcriptomic occurring in the browning of fruits from Luffa cultivar ‘WL3＇. A total of 58 073 unigenes were assembled from high-quality reads, and 27 301 unigenes were differentially expressed during the browning process of Luffa fruits. In this study, full length of agene sequence（i.e., unigene0033876） annotated as catalase CAT gene was filtrated from the differentially expressed genes. The sequence analysis showed that unigene0033876 reached a length of 1 690 bp and contained a 1 479 bp open reading frame（ORF） that encoded 492 amino acids, with a predicted molecular weight of 56.94 k Da and a hypothetical isoelectric point of 7.31. It shared over 97% identity with the homologous proteins from Cucurbita moschata, Cucurbita pepo and Cucumis sativus, revealing that it was highly conservative, and the Gen Bank accession was KR184674. The results of q PCR（Real-time quantitative PCR） revealed that Lc CAT2 exhibited a tissue specific expression, the expression level in Luffa cultivar ‘WL-3＇ leaves was the highest, and minimally expressed in flowers on the contrary. The levels of Lc CAT2 were different among six luffa varieties, and the expression in Luffa cylindrical was higher than that in Luffa acutangula Roxb. Furthermore, the level of Lc CAT2 in ‘WL3＇ was up-regulated during post-harvest storage, suggesting that Lc CAT2 gene may play a regulatory role in luffa browning process. The survey aims to excavate the genes associated with Luffa browning from transcriptome sequencing results, and make contributions to breeding of the Luffa varieties and ulteriorly revealing the molecular mechanism of Luffa enzymatic browning process for the future.