Diversity analysis of Ty3-gypsy-like retrotransposons' RT gene sequences in fruit sugarcane

【Objective】To analyze the characteristics,diversity,phylogenetic evolution and transcriptional activity of Ty3-gypsy-like retrotransposons in fruit sugarcane so as to provide a theoretical reference for the in-depth study of the fulllength sequence,transcriptional transposition activity and biological function of Ty3-gypsy-like retrotransposons(RTs) in sugarcane.【Method】Using the fruit sugarcane variety Badila as the test material,degenerate primers were designed corresponding to the conserved region of Ty3-gypsy-like RT for PCR amplification. The amplified targets were recovered,purified and ligated into pMD18-T vectors. After transformation into Escherichia coli competent cells DH5α,positive clones were picked for sequencing. Ty3-gypsy-like retrotransposon sequences were analyzed by bioinformatics.【Result】Fiftyone sequences(SoRT4-1-SoRT4-51) were amplified from fruit sugarcane Badila. After removing repetitive sequences and non-RT sequences,a total of forty-four RT sequences were obtained,with lengths of 423-433 bp, an AT content of 56.84%-64.97%, and an AT:GC ratio of 1.32-1.85. The similarity between the nucleotide sequences was 44.4%-99.3%, whereas the similarity between the encoded amino acid sequences was 10.8%-100.0%,indicating that the amino acid sequences showed higher heterogeneity than nucleotide sequences. The forty-four RT gene sequences were divided into four families, of which I and III were the main families. Nonsense mutations occurred in twenty of the forty-four RT genes,indicating a high mutation rate. There were five conserved motifs in the forty-four RT protein sequences,of which twentynine sequences contained motifs 1-4 while the conserved motifs of the remaining fifteen sequences varied greatly. The protein tertiary structures of representative RT gene sequences in four families differ in the number of hydrogen bonds and corner. A phylogenetic tree analysis showed that the forty-four RT gene sequences were divided into seven classes. The sugarcane RT gene sequences in class I and II accounted for 40.91% and 27.27% of the total sequences, respectively. Some sugarcane RT gene sequences in class II and VI were closely related to those of BAB40828.1 of Arabidopsis thaliana, BAB40824.1 of Japonica rice, BAB40833.1 of spinach and BAB40834.1 of soybean. It is speculated that the lateral transmission of Ty3-gypsy-like retrotransposons occurred in the evolution of these species. One Ty3-gypsy-like retrotransposon (SoRT4-40) was initially found to have transcriptional activity.【Conclusion】Forty-four Ty3-gypsy-like retrotransposons' RT gene sequences in fruit sugarcane were obtained,of which only one sequence had transcriptional activity. Molecular markers based on these Ty3-gypsy-like retrotransposons can be used in future analyses of sugarcane genetics.