Cloning And Characterization Of MADS-box Genes In Equisetum Arvense

MADS-box genes are a group of sequence specific homologous alien genesexisting widely in plants, which encode MADS proteins as transcription factors,mainly involved in the development of angiosperm floral organs. Understanding theorigin and evolution of the floral development specific genes is the base to understandthe origin and evolution of the flower, and researches on MADS-box genes have animportant significance to resolve the above questions. Ferns, as an importanttransition group in plant kingdom, is an indispensable part for the study of geneevolution. However, the studies on MADS-box genes are focused on seed plants, lessstudies of MADS-box genes are on algae, moss and ferns. This study selectedEquisetum arvense, a member of Equisetum, the only remaining genus of theEquisetaceae, Equisetales, Subdivision Sphenophytina, which has a special position inthe evolution of ferns system, as material. We successfully isolated two MADS-boxgenes from Equisetum arvense, named EaMADS1and EaMADS2, using RACEmethod. The results of this experiment provide more data for complementing theevolutionary spectrum of MADS-box genes in plants. In the meantime, it alsoaccumulate data for the functional studies of Equisetum arvense MADS-box genes.The total RNA was extracted using Equisetum arvense reproductive branches,and used for the cDNA synthesis. Two pairs of degenerate primers were designedbased on sequence homology alignment results of plants MADS-box genes inGenbank. Two MADS-box gene core fragments,148bp and150bp respectively, wereisolated from Equisetum arvense. Through the RACE method, a503bp of5’ end and1589bp of3’ end, a300bp of5’ end and1116bp3’ end, respectively were obtained. Finally, two pairs of specific primers were designed according to the splicing resultsof the core, the5’ and3’, and PCR was performed to isolate two complete full-lengthcDNA sequences of MADS-box genes. The results showed that the two MADS-boxgenes complete sequences obtained from Equisetum arvense were2264bp and1584bp,with330and287amino acids encoded, respectively.Using bioinformatics analysis, we compared two MADS-box genes obtainedfrom Equisetum arvense with MADS-box gene sequences of other plants in Genbank.Results show that: EaMADS1shares a high similarity with Dryopteris fragrans, andEaMADS2shares a high similarity with Triticum aestivum in the core sequences.Amino acid sequences alignment results show that: EaMADS1and EaMADS2geneprotein sequences both have a MADS region and a K region, which are the relativelyconserved structural domains peculiar to typical MIKC-type MADS-box genes, so wecan confirm that Equisetum arvense EaMADS1and EaMADS2genes are belonged tothe MADS-box gene family. Among these, EaMADS1shares a relatively highsimilarity with MADS-box genes from Lycopodium annotinum, Selaginellamoellendorffii and Cucumis sativus, while EaMADS2shares a relatively highsimilarity with MADS-box genes from Arabidopsis thaliana, Lycopodium annotinum,Ricinus communis, Solanum tuberosum, Citrus aurantium and Glycine max.Sequence comparison analysis reveals that N terminal amino acid sequence ofEaMADS1and EaMADS2MADS region have no extension, both have distinctMADS region and K region, and no obvious extension in I region, so Equisetumarvense EaMADS1and EaMADS2genes are both belonged to MIKCc-typeMADS-box genes. Sequence alignment analysis results also show that MADS regionand K region of EaMADS1and EaMADS2are relatively conserved and I region andC-terminal region have significant changes compared with MADS-box genes fromother plants.Phylogenetic analysis shows that, EaMADS1and EaMADS2share a highsimilarity with Physcomitrella patens, Lycopodium annotinum and Selaginellaremotifolia, while have a certain genetic distance with MADS-box genes from algaeand other ferns, such as Ceratopteris and Dryopteris fragrant. The phylogenetic tree also shows that Equisetum arvense EaMADS1and EaMADS2are not belonged to anybranch of the seed plants, so the branch of EaMADS1and EaMADS2may be anindependent branch in the MADS-box gene family.