菠萝COL基因的克隆及响应乙烯的表达特性分析

Cloning of Pineapple COL Genes and Expression Analysis of the Response to Ethephon

COL(CONSTANS Like)基因不仅响应光周期调控,还参与植物器官的发育及非生物胁迫的响应。本研究克隆了4个菠萝COL(AcCOL)同源基因,预测其蛋白的结构理化性质和启动子顺式作用元件,并探究AcCOL基因在乙烯利处理后的时空表达模式。结果表明:AcCOL1、AcCOL2基因分别编码387、243个氨基酸,都含有CCT结构域,属于第Ⅳ类COL基因;AcCOL3、AcCOL4基因分别编码394、411个氨基酸,均含有1个B-box和1个CCT结构域,属于第Ⅲ类COL基因。启动子顺式作用元件预测表明4个AcCOL的启动子中均含有光响应元件、昼夜节律表达相关元件。AcCOL1和AcCOL2启动子均含有生长素响应元件和乙烯响应元件;AcCOL3和AcCOL4启动子均含有细胞分裂素响应元件。实时荧光定量PCR分析结果显示,AcCOL基因主要在叶中表达。乙烯利处理后,4个AcCOL同源基因均有不同程度上调。与各自时期的对照相比,AcCOL1和AcCOL4在茎中表达上调较多;AcCOL2和AcCOL3在叶中表达上调较多。另外,AcCOL2基因在3个组织部位中与各自时期的对照相比均有显著上调。研究表明,4个AcCOL同源基因在乙烯利处理后,表达水平均有上调;其中AcCOL2更为显著,在3个组织部位中,与另外3个基因相比表达上调更多。说明AcCOL2基因对外施的乙烯利信号更加敏感,可能在乙烯作用途径中发挥作用。

COL(CONSTANS Like)gene not only responds to photoperiod regulation,but also participates in the development of plant organs and abiotic stress response.In this study,four homologous genes of AcCOL were cloned,then bioinformatics was used to analyze the amino acid sequence and the cis acting elements of AcCOL,finally the spatiotemporal expression pattern of the genes after ethephon treatment was explored.The results showed that the genes of AcCOL1 and AcCOL2 encoded 387 and 243 amino acids respectively,both of which contained CCT domains and belonged to the group IV COL genes;the genes of AcCOL3 and AcCOL4 encoded 394 and 411 amino acids respectively,both of which contained a b-box and a CCT domain and beloned to the class III COL genes.The prediction of promoter cis acting elements showed that the four promoters of AcCOL contained light response elements and circadian rhythm expression related elements.AcCOL1 and AcCOL2 promoters both contained auxin responsive elements and ethylene responsive elements,and AcCOL3 and AcCOL4 promoters both contained cytokinin responsive elements.The results of real-time PCR showed that the expression of AcCOL genes were mainly in leaves.After ethephon treatment,the four homologous genes of AcCOL were up-regulated in varying degrees.AcCOL1 and AcCOL4 were up-regulated in the stem,and AcCOL2 and AcCOL3 were up-regulated in the leaves.In addition,the AcCOL2 gene was significantly up-regulated in the three tissue sites compared with the control in each period.The results showed that the expression level of the four AcCOL homologous genes was up-regulated after ethephon treatment,of which AcCOL2 was more significant,and in three tissue sites,the expression was up-regulated more than the other three genes.It is suggested that AcCOL2 gene was more sensitive to ethylene signal and may play an important role in ethylene pathway.