脐橙CsNAC基因的克隆及其在果实贮藏过程中的表达分析

Cloning of CsNAC Gene from Navel Orange and Its Expression Analysis During the Storage of Fruits

从脐橙(Citrussinensis Osbeck)果皮褐变相关基因的cDNA抑制差减文库中,筛选了一个与NAC基因家族同源的EST序列,通过RACE技术成功克隆了CsNAC基因全长cDNA序列共1203bp,并对其进行了序列分析。结果表明,CsNAC包含一个918bp的开放阅读框(ORF),编码305个氨基酸,预测的蛋白质分子量为35.2kD,等电点为6.72;CsNAC蛋白N端具有一个高度保守的NAC结构域;进化树分析表明CsNAC属于NAC蛋白家族的ATAF亚家族,可能参与胁迫反应。荧光定量PCR表达分析结果表明,CsNAC在果实贮藏过程中,随着果皮褐变的发生,与对照相比褐变果皮中的表达水平明显增强。说明CsNAC基因与脐橙果实的果皮褐变具有密切关系。

Based on the cDNA subtraction library which had been constructed to identify differentially expressed genes in peel pitting of citrus fruit, the full-length cDNA sequence of CsNAC homologous to NAC gene family was isolated from navel orange （ Citrus sinensis Osbeck） by RACE （ rapid amplification of cDNA ends）. CsNAC is of 1 203 nucleotides encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2 kD and theoretical isoelectric point was 6.72. Sequence analysis showed that the CsNAC protein had a strikingly conserved region at the N-terminus, which is considered as the characteristic of NAC protein family. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to stress stimuli. The expression of CsNAC was enhanced in the pitting peel compared to healthy peel during the storage by real-time quantitative PCR, indicating that CsNAC is involved in the development of the peel pitting of navel orange fruits.