Cloning, Expression Analysis Of FLD,FVE And HD1Homologous Genes And DNA Methylation In Aechmea Fasciata

Under natural conditions, Bromeliaceae plant flowering, for instance pineapples, is uncertain due their blooming time and rate significantly depend on environment factors, which lead to varied harvest period and great loss to producers. Ethylene and its derivatives are widely used to regulate the flowering phase and control harvest time. Although the research of ethylene inducing flowering have been reported a lot, the ethylene inducing molecular mechanism is still unclear. Aechmeas fasciata has a close evolution relationship with pineapple with shorter vegetative period and more sensitive reaction with ethylene than Pineapple. Due to these characters, Aechmeas fasciata was selected as great research materials to studying the ethylene inducing mechanism for Bromeliaceae. In this study,we used Aechmea fasciata as material and used RACE (rapid amplification of cDNA ends) homology-based cloning technique to analyze the flowering regulation pathway related genes FLD, FVE and HD1.The expression patterns of AfFLD, AfFVE, AfHDl gene during the processing of ethylene were investigated by quantitative real-time (qRT-PCR). Meanwhile, we used methylation sensitive amplified polymorphism (MSAP) technique to study the DNA stability, methylation degree and pattern change, expecting to understand the ethylene inducing pineapple flowering mechanism in epigenetic level. The results of the study are as follows:1. The FLD-like gene named AfFLD was cloned from Aechmea fasciata. The cDNA sequence of AfFLD is2878bp in length. By alignment analysis, the deduced amino acid sequence contained two domains:SWIRM domain and amine oxidase domain, both of which were highly conserved in LSD1-LIKE protein family. The deduced protein of AfFLD was72%and73%identical to homologs encode AtFLD in Arabidopsis thaliana and ZmFLD in Zea mays, respectively. Results showed that the expression of AfFLD reached the highest level on Id after ethylene processing, which the highest amount of expression2.5times than Oh.2. The FVE-like gene named AfFVE was cloned from Aechmea fasciata. The cDNA sequence of AfFVE is1672bp in length. By alignment analysis, the deduced amino acid sequence contained two domains:CAF-1domain and WD40domain. The deduced protein of AfFVE was86%and85%identical to homologs encode DhFVE in Doritaenopsis hybrid and DnMSI4in Dendrobium nobile, respectively. Results showed that the expression of AfFVE reached the highest level on1d after ethylene processing, which the highest amount of expression3.5times than Oh.3. The HD1-like gene named AfHD1was cloned from Aechmea fasciata. The cDNA sequence of AfHD1is1755bp in length. By alignment analysis, the deduced amino acid sequence contained one domains:HDAC domain. The deduced protein of AfHD1was87%and85%identical to homologs encode OsHD1in Oryza saliva and BdHDl in Brachypodium distachyon, respectively. Results showed that the expression of AfHD1reached the highest level on1d after ethylene processing, which the highest amount of expression5times than Oh.4.After analyzing the methylation change of genomic DNA with ethylene treatment and building Aechmea fasciata MSAP technology system, the total average methylation rate is36.69%, especially the total methylation as the main impact method rate is27.8%. Analyzed DNA methylation pattern change of Aechmea fasciata with1hour,6hours,1day and2days ethylene treatment and time corresponding deionized water control treatment, DNA methylation ration reached the peak after1hour ethylene treatment and then gradually decreased. Reversely demethylation ration had gradual increase tendency and overwhelmed methylation ration in1day treatment, reaching peak in2days treatment. Moreover, analysis methylation differences in MSAP fragment recycling sequencing and bioinformatics, the8of14different fragments found in Genbank successful matching with the of gene sequence, among them, there are2respectively with arabidopsis (Arabidopsis thaliana) DnaJ heat shock protein mRNA sequence and sitka (Picea sitchensis) late embryogenesis abundant protein is highly homologous mRNA sequence. The differences with these two functional genes homologous of MSAP fragments in the methylation status of variation between control and processing, suggests that it may control associated with the gene expression of ethylene induced response, dynamic stress or direct expression in ethylene stress mechanism, reduce the deleterious effects, and promote plant growth and development process.