荸荠球茎膨大相关基因COL5的克隆及表达分析

Cloning and Expression Analysis of Corm Enlargement Related Gene COL5 of Chinese Water-chestnut

荸荠(Eleocharis dulcis)的膨大球茎是其主要的可食部位,而参与膨大过程调控的相关基因尚不清楚。COL5(CONSTANS-Like 5)基因是COL基因家族成员之一,可能在植物储存器官发育和膨大过程中发挥重要作用。为了研究荸荠COL5在球茎膨大过程中的表达模式及可能的作用,从荸荠转录组数据库中筛选与其它植物COL基因同源性较高的转录本片段并克隆其cDNA序列以及DNA全长,进而对其进行生物信息学分析和时空表达模式分析。结果表明:克隆得到的荸荠膨大相关COL基因ORF长度为1017 bp,可编码338个氨基酸,DNA序列长1275 bp,包含1个长度为257 bp的内含子,将其命名为CwCOL5(ON934922)。生物信息学分析显示,CwCOL5蛋白分子式为C_(1592)H_(2515)N_(477)O_(508)S_(17),预测蛋白质相对分子量为37010.39 Da,理论等电点为5.89,总平均亲水值(gravy)为-0.462,不稳定系数为45.63,属亲水性不稳定蛋白质,有31个磷酸位点,无跨膜结构,无信号肽,亚细胞预测分析显示其定位于细胞核。CwCOL5含有2个B-box Zinc finger和1个高度保守CCT结构域。系统进化分析表明,CwCOL5与莲藕(Nelumbo nucifera)、拟南芥(Arabidopsis)的COL5蛋白亲缘关系较近。荧光定量PCR结果显示,CwCOL5在叶片与荸荠皮中有较高的表达量,在球茎膨大前期表达快速上升。荸荠COL5属于典型的锌指蛋白转录因子家族,推测该基因可能参与荸荠球茎膨大过程。本研究在荸荠球茎中克隆COL5基因并分析其表达模式,为植物球茎膨大的分子机制研究提供基础。

The enlarged corm of Chinese water chestnut(Eleocharis dulcis)is the main edible part,and the related genes involved in the regulation of the corm enlargement process remain unclear.COL5(CONSTANS-Like 5)gene is a member of COL gene family,which may play an important role in the development of plant storage organs.The transcript frag-ment with high homology to COL gene in other plants was screened from the Chinese water-chestnut transcriptome da-tabases and cloned its cDNA sequence and full-length DNA,and then the bioinformatics analysis and spatiotemporal expression pattern analysis were analyzed to study the expression pattern and possible role of Chinese water-chestnut COL5 during corm enlargement.The results showed that the cloned COL5 gene related to Chinese water-chestnut enlargement had an ORF of 1017 bp,encoding 338 amino acids,and the DNA sequence was 1275 bp,containing an intron of 257 bp,which was named CwCOL5(ON934922).Bioinformatics analysis showed that the CwCOL5 protein with the formula C_(1592)H_(2515)N_(477)O_(508)S_(17),the relative molecular weight of the predicted protein was 37010.39 Da,and a theoretical isoelectric point of 5.89.The total average hydrophilic value(gravy)was-0.462,the instability coefficient was 45.63,which was a hydrophilic unstable protein.There were 31 phosphosites,no transmembrane structure,and no signal peptide,and additionally subcellular prediction analysis showed that it was located in the nucleus.CwCOL5 contained two B-box Zinc fingers and a highly conserved CCT domain.Phylogenetic analysis showed that CwCOL5 was closely related to the COL protein of Nelumbo nucifera and Arabidopsis.The results of PCR showed that CwCOL5 had a high expression in the leaves and water-chestnut peel,and the expression of CwCOL5 increased rapidly in the early stage of corm enlargement.The CwCOL5 gene belongs to a typical transcription factor family of zinc finger protein,and the gene may be involved in the process of corm enlargement of Chinese water-chestnut.In this study,COL5 gene was cloned from the corm of Chinese water-chestnut and its expression pattern was analyzed,which would provide a theoretical basis for molecular mechanism of plant corm enlargement.