CDNA-AFLP Analysis On Dangui And Yingui Petals And Molecural Characteristics Of OfMYB1 Gene

Osmanthus fragrans is an evergreen broad-leaved tree of the Osmanthus genus. Native to southwest China, it is one of the ten most renowned traditional ornamental plants and widely used for urban greening. Adored for its upright posture, evergreen leaves, strong aroma, and resistance to various stresses, O. fragrans has been cultivated for more than 2,500 years. Centuries of natural evolution and artificial selection have resulted in numerous O. fragrans cultivars, which are categorized into 4 groups:Aurantiacus, Latifolius, Thunbergii, and Semperflorens. Using the cDNA-AFLP technique, we analyzed petals of blossoming Aurantiacus and Latifolius cultivars, isolated differentially expressed genes, and cloned the OfMYB1 gene. The main findings are as follows:1. Using 256 primer pairs, cDNA-AFLP analysis yielded 7,680 amplification fragments of 50bp to1000bp. There were 390 differentially expressed cDNA fragments. These were purified and sequenced, generating 102 gene fragments. Of these differentially expressed genes,57 were up-regulated in Aurantiacus,45 were up-regulated in Latifolius. According to the functions of the genes and their corresponding proteins, these differentially expressed genes were divided into 8 categories:signal transduction, photosynthesis, metabolism, transport, stimulus response, defense response, secondary metabolism, unknown.2. Based on sequencing results of cDNA-AFLP differential fragments, we obtained a DNA fragment that was homologous to the MYB transcription factor, then accordingly designed a primer and cloned the entire cDNA of the MYB gene using RACE technology. This cDNA had a length of 749bp. The coding region was 585bp in length, encoding a protein of 195 amino acids. Phylogenetic analysis of the amino acids revealed the MYB gene to be highly homologous with PsMYB26 and PhEoBI. A comparison of the amino acid sequences showed that this gene had 2 MYB domains, which were typical R2 and R3 MYB transcription factors.3. We constructed the recombinant vector 35S-OfMYB1-GFP-NOS, which was transformed into Arabidopsis thaliala leaf protoplast, incubated for 12 hours in the dark, then observed using fluorescence microscopy. The results showed that OfMYBl was located in the nucleus.