墨兰CsAP1-A基因克隆及表达分析

Analysis on Cloning and Expression of CsAP1-A Gene in Cymbidium sinense

【目的】AP1基因在植物的花器官发育和开花调控中发挥重要作用,对墨兰AP1基因进行克隆与表达分析可为研究其在墨兰花发育和开花调控中的作用提供前期基础。【方法】以墨兰品种‘小香’为材料,克隆到1个AP1基因,命名为CsAP1-A。通过生物信息学分析其基因结构、蛋白结构域和进化关系,利用RTqPCR方法分别检测CsAP1-A在墨兰不同器官、不同花发育阶段和不同花组织部位的表达情况;通过转录组测序分析CsAP1-A在5个不同花型墨兰品种花组织部位的表达情况;通过蛋白互作预测软件分析CsAP1-A与其他蛋白的互作关系。【结果】CsAP1-A基因编码区为744 bp,编码248个氨基酸,含有高度保守的MADS-box和K-box结构域,符合MADS-box转录因子家族特征。CsAP1-A与其他兰科植物AP1蛋白相似性较高,其中与春兰AP1/FUL3(APY18447.1)和蕙兰MADS1(AGE15496.1)的同源性最高。RT-qPCR分析结果表明,CsAP1-A在墨兰不同器官中均有表达,在花中表达量最高,且集中在花芽分化初期(S1)高表达。在不同花型的墨兰品种中,CsAP1-A在WT(普通型)、MPV(重瓣花型)、LaPV(花瓣唇瓣化花型)和NLV(唇瓣萼片化花型)4种花型的合蕊柱中表达量均最高,而在萼片中表达量最低;在合蕊柱异常发育的MPV中,CsAP1-A在合蕊柱的表达量显著提高,而在花瓣蕊柱化的梅瓣花型(GPV)中整体表达量最高,且表达区域从合蕊柱扩展到花瓣。蛋白互作预测CsAP1-A蛋白可与MADS1、MADS6、MADS47、MADS8等10个蛋白存在互作关系。【结论】墨兰CsAP1-A的基因结构、进化关系、时空表达情况和蛋白互作预测可为墨兰花发育的研究提供理论依据,对进一步揭示CsAP1-A基因在墨兰成花过程中的作用奠定基础。

【Objective】AP1 gene plays an important role in the development of floral organs and regulation of flowering in plants.The analysis on cloning and expression of the AP1 gene of Cymbidium sinense can provide a preliminary foundation for the study of its role in the development of C.sinense flowers and the regulation of flowering.【Method】An AP1 gene,named CsAP1-A,was cloned from the C.sinense variety‘Xiao Xiang’,and its gene structure,protein structural domains and evolutionary relationship were analyzed by bioinformatics.RT-qPCR was used to detect the expression of CsAP1-A in different organs, flower development stages and flower tissue parts, respectively, and the expression of CsAP1-A in flower tissues of five different flower types of C. sinense was analyzed by RNA-seq. The interactions between CsAP1-A and other proteins were analyzed by the Protein Interaction Prediction Software.【Result】The coding region of CsAP1-A gene was 744 bp, encoding 248 amino acids, with highly conserved MADS-box and K-box structural domains, which is in line with the characteristics of the MADS-box family of transcription factors. The protein sequence comparison revealed that the similarity between CsAP1-A and the AP1 proteins of other orchid plants was relatively high, among which the highest homology was found with the AP1/FUL3(APY18447.1) of C. goeringii and MADS1 (AGE15496.1) of C. faberi. RT-qPCR showed CsAP1-A was expressed in different organs of C. sinense, the highest expression was in flowers, and concentrated in stage of early flower development (S1). In the five varieties with different floral patterns, the expression of CsAP1-A in common type variety (WT), multi-perianth variety (MPV), labellum-like petal variety (LaPV) and null-labellum variety (NLV) were the highest in gynostegiums and the lowest in sepals. CsAP1-A was significantly up-regulated expression in MPV with abnormal development of gynostegium. The overall expression was the highest in gynostegium-like petal variety (GPV), and the expression area expanded from gynostegium to petal. Protein interactions predicted that CsAP1-A could interact with 10 proteins, including MADS1, MADS6, MADS47 and MADS8 etc. 【Conclusion】The gene structure, evolutionary relationship, spatio-temporal expression and protein interaction prediction of CsAP1-A can provide theoretical basis for the study of flower development in C. sinense, and lay a foundation for further revealing the role of CsAP1-A gene in flower formation of C. sinense.