Objective The reverse transcriptase (RT) gene sequences of Ty3-gypsy-like retrotransposons were isolated from the sugarcane genome, and their characteristics, differences, and phylogenetic relationships analyzed to study the transcriptional activities and regulatory functions associated with the retrotransposons.
Method Degenerate primers were designed according to the conserved region of the RT gene sequences of Ty3-gypsy-like retrotransposons. The genomic DNA of sugarcane variety, ROC22, was amplified by PCR. The recovered, cloned, and sequenced target band was analyzed by bioinformatics.
Result Thirty-six RT gene sequences were successfully isolated. Only one sequence of the target bands was 430 bp in length, while the remainders were 432 bp. The proportions of AT in the sequence ranged from 56.71% to 64.81%, and the ratio of AT to GC from 1.31 to 1.84. A high heterogeneity existed among the nucleotide sequences, as the similarity was shown to be 46.2% to 99.3%. The cluster analysis divided the 36 RT gene sequences into 5 families. Family I and Family IV contained most of the sequences. After translated into amino acids, 6 sequences displayed nonsense mutations. The similarity between amino acid sequences ranged from 10.1% to 100% exhibiting a high degree of heterogeneity. The conserved motifs of 34 of the sequences were identical, indicating a highly conservative property. The overall configuration of the tertiary structure of the representative proteins in each family was basically similar, however, there were significant differences in the number of hydrogen bonds and turns, suggesting a certain degree of heterogeneity and polymorphism. The phylogenetic tree showed 7 distinct groups among the RT gene sequences. Of which, 18 in Group I were highly homology with BAB40828.1 of Arabidopsis thaliana, and SoRT3-26 in Group VII was genetically closest to the BAB40834.1 of soybean, BAB40833.1 of spinach, and BAB40824.1 of japonica rice. It indicated that a horizontal transfer of Ty3-gypsy-like retrotransposons might have occurred in evolution between sugarcane and those plant species. By comparing the sugarcane EST database, 10 Ty3-gypsy-like retrotransposons were found with transcriptional activity in ROC22.
Conclusion Thirty-six RT gene sequences of Ty3-gypsy-like retrotransposons in sugarcane were obtained with 10 of them showing a transcriptional activity. The results provided the basic information for the isolation of full-length Ty3-gypsy-like retrotransposons, further understanding of the transcriptional, transposon activity and functions of the retrotransposons, and the development of sugarcane molecular markers based on LTR retrotransposons.
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