三褶虾脊兰CtARF57和CtIAA12基因克隆及功能分析

Cloning and Functional Analysis of CtARF57 and CtIAA12 From Calanthe triplicata

以三褶虾脊兰(Calanthe triplicata)不同发育时期的花瓣距为试材,基于转录组测序数据,通过克隆获得生长素信号转导途径中CtARF57和CtIAA12基因全长序列,并进行生物信息学分析,探究园林花卉三褶虾脊兰生长素相关响应因子(ARF和Aux/IAA)的表达特性及功能,以期揭示三褶虾脊兰花瓣距的发育机理。结果表明:cDNA全长为3 054 bp和1 124 bp,分别编码911、177个氨基酸。2个基因氨基酸序列与铁皮石斛同源基因的相似性较高,均超过84%。保守结构域分析表明CtARF57蛋白具有B3结构域、Auxin_resp结构域和Aux/IAA结构域;CtIAA12具有Aux/IAA蛋白的DomainⅠ、Ⅱ、Ⅲ和Ⅳ特异性结构域。实时荧光定量PCR结果显示,其转录水平受到生长素的诱导,不同时期基因表达水平差异明显,2个基因均在三褶虾脊兰花瓣距发育最后一个时期表达量最高,前2个时期表达量较低,与转录组FPKM值趋势一致。综上所述,初步推测2个响应因子基因参与生长素信号传导途径并调控园林花卉三褶虾脊兰花瓣距的形态建成和生长代谢过程。

The petal spur of Calanthe triplicata at different development stages was used as the test materials.Two genes that expressed differentially during the auxin signal transduction pathway, namely CtARF57 and CtIAA12,were cloned from petal spur of Calanthe triplicata based on transcriptome data and analyzed by bioinformatics, in order to investigate the expression characteristics and functions of auxin related response factors(ARF and Aux/IAA) in Calanthe triplicata,and to reveal the development mechanism of petal spur.The results showed that the full length of cDNA were 3 054 bp and 1 124 bp, encoding 911 and 177 amino acids, respectively.Homology analysis showed that the amino acid sequences of the two genes had high homology similarity with Dendrobium catenatum,and the similarity was more than 84%.Conserved domain analysis showed that CtARF57 protein had B3 domain, Auxin_resp domain and Aux/IAA domain;CtIAA12 contained the typical Aux/IAA protein DomainⅠ,Ⅱ,Ⅲ and Ⅳ.The results of real-time fluorescence quantitative PCR showed that the transcriptional level was induced by auxin, and the expressions of the two genes were significantly different at different stages.The expression levels of the genes were the highest at the last stage of petal spur development, and were lower in the first and second periods, which was consistent with the trend of FPKM value of the transcriptomic.The above results preliminarily suggested that two response factor genes were involved in the auxin signal transduction pathway to regulate the morphogenesis, growth and metabolism of Calanthe triplicata in petal spur of the garden flower.