Cloning and analysis of reverse transcriptase of Ty3-gypsy retrotransposon in Hylocereus undatus

Abstract: 【Objective】In order to provide a fundamental clue for genetic evolution and variation mecha⁃nisms in Hylocereus undatus, reverse transcriptase (RT) sequences of Ty3-gypsy retrotransposon werecloned and analyzed in the genome of pitaya.【Methods】Using degenerate oligonucleotide primers basedon the conserved domains of the Ty3-gypsy LTR retrotransposon RT sequence, 430 bp fragment was amplified through a polymerase chain reaction (PCR) from the genomic DNA of red-flesh pitaya‘( Xinhonglong’), white-flesh pitaya‘( Baiyulong’) plants and their mutants. The amplicons were cloned, then se⁃quenced and analyzed. Sequence homology queries for nucleotide and deduced amino acid sequences ofTy3-gypsy LTR retrotransposon RT sequences were performed using BLASTn and BLASTx (https://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively. Multiple sequence alignments were conducted using the ClustalW program. Phylogenetic trees were generated using MEGA 6.06 software by p-distance and neighborjoiningmethods by 1 000 bootstrap replications. Special primers were designed at the basis of all RT se⁃quences of pitaya using Primer Premier 5.0 software, and were employed to detect their transcriptional ac⁃tivities from the plants grown normally in the field and the tissue culture seedlings obtained by reversetranscription PCR (RT-PCR). RT-PCR was carried out with the corresponding cDNA, which was reversetranscribed from the total RNA of the test samples‘( Zihonglong’). The PCR products were electropho⁃resed in 1% agarose gel and observed under UV light after staining with Goldview.【Results】The PCR am⁃plification with degenerate primers yielded an expected 430 bp fragment. The fragment within this bandwas recovered and sequenced. Totally, 31 RT sequences from the red-flesh type and 51 from the whitefleshtype were obtained, which were named HURT. Sequences ranged in length from 431 bp to 444 bp,most of which were AT-rich, with AT content of 48.6%-63.0%. When the nucleotide sequences of the RTsequences were compared, the identities among the red-flesh type‘Xinhonglong’and its bud variety‘Zi⁃honglong’ranged from 36.75% to 98.39%. And the similarity of the RT nucleotide sequences among thewhite-flesh type‘Baiyulong’and its mutants ranged from 33.63% to 99.31%. All RT sequences werehighly heterogeneous in pitaya, and were highly homologous with Ty3-gypsy retrotransposons from otherspecies. These RT sequences were submitted to GenBank with the accession numbers of KU977005-KU977006, KU977008- KU977029, KU977031 - KU977050, KU977052- KU977063, KU977065-KU977089 and KX090146. Of the amino acid sequences, 55 (67%) presented premature stop codons, andfive sequences demonstrated frameshift mutation, indicating a high heterogeneity among the Ty3-gypsyRT sequences of pitaya. Alignment of putative amino acid sequences showed that the RT sequences isolat⁃ed from pitaya genome contained motifs MCVDY at their 5’ends. However, the RT sequences of the Ty3-gypsy retrotransposons of the white-flesh type pitaya genome were shown to be less conserved than those ofthe red-flesh type. For multiple sequence alignment purposes, 13 Ty3-gypsy LTR retrotransposons RTamino acid sequences of other organisms from the GenBank database were chosen for comparison with theTy3-gypsy retrotransposons RT sequences of pitaya. The RT sequences of the Ty3-gypsy group retrotrans⁃posons were divided into ten groups, and the genetic distances between them ranged from 0 to 0.953. GroupⅠwas the largest one, including 55 sequences obtained from all test pitaya samples，indicating that thered-flesh type and white-flesh type of pitaya had a closer relationship in their evolutionary history. Theother 27 RT sequences from pitaya were unequally assigned to group Ⅱ, group Ⅲ and group Ⅸ. Six ofTy3-gypsy retrotransposons RT sequences from pitaya were associated with AAL79340 isolated from Ory⁃za sativa, which were clustered in the group Ⅴ. AAD19758 from Arabidopsis thaliana involved in group Ⅷcontained three sequences from pitaya. This suggests that the RT sequences had considerable homologywith those from O. sativa and A. thaliana. Based on these results, we concluded that a vertical and horizon⁃tal transmission had occurred during the evolution of the LTR retrotransposons in pitaya. To investigate thetranscriptional activity of the 82 RT sequences, RT- PCR was employed. Only three RT sequences(HURT1, HURT56 and HURT70) were transcriptionally activated in the in vitro shoots.【Conclusion】TheRT nucleotide sequences of Ty3-gypsy retrotransposon demonstrated high heterogeneity in pitaya, whichwere characterized in deletion, frameshift or stop codon mutation. Phylogenetic analysis indicated that theymight share a common origin, and a horizontal transmission of retrotransposons had occurred among theplant species in their evolutionary history. Three Ty3-gypsy retrotransposons and RT sequences in pitayacould be activated for transcription as subjected to tissue culture conditions.