Cloning And Activation Analysis Of Reverse Transcriptase Of Ty3-gypsy Retrotransposon In Pitaya

Retrotransposons are a kind of mobile genetic factor prevalent in eukaryotic genome.They have many properties such as multiple copies,insertion polymorphism and high heterogeneity.Reverse transcriptase(RT)is a part of the long terminal repeat retrotransposons with transcriptional activity.Cloning and analysis of RT sequence are of great value in the further study of retrotransposons in plant genome evolution,genetic variation and related trait formation.Pitaya has a lot of bud sport resources,from which the main cultivars are obtained.The cause of bud sport are complicated,among which contributed to the activation of retrotransposon.Currently,pitaya variety ‘Xinhonglong’(red peel red flesh)and its budding variety ‘Zihonglong’,red peel white flesh pitaya ‘Baiyulong’ and its in vitro mutants were taken as the test materials.The conserved domains of Ty3-gypsy LTR retrotransposon RT sequences were amplified from the genomic DNA of this germplasms by polymerase chain reaction(PCR)using degenerate oligonucleotide primers.Reverse transcription PCR(RT-PCR)were employed to detect RT sequences transcriptional activity among the field-grown plants and in vitro plants.Then,semi-quantitative RT-PCR was carried out to quantify the relative expression level of RT sequences with transcriptional activity upon exposure to the abiotic stress treatments as well as exogenous uses of plant growth regulators.The main results were as follows:1)PCR amplification with degenerate primers yielded an expected 430 bp fragment,which was cloned,then sequenced and analyzed.Sequence homology queries for nucleotide and deduced amino acid sequences of Ty3-gypsy LTR retrotransposon RT sequences were performed using BLASTn and BLASTx(https://blast.ncbi.nlm.nih.gov/Blast.cgi),respectively.After removing duplicate and too short sequences,a total of 82 fragments of Ty3-gypsy reverse transcriptase were obtained,which were named as HURT1~HURT86.Among them,31 RT sequences were derived from ‘Xinhonglong’ and its budding type ‘Zihonglong’,51 from white-flesh type ‘Baiyulong’ and its mutants.These RT sequences were submitted to Gen Bank with the accession numbers of KU977005~KU977006,KU977008~ KU977029,KU977031~KU977050,KU977052~KU977063,KU977065~KU977089 and KX090146.2)Bioinformatics analysis showed that the similarity of RT nucleotide sequences among red-flesh type and white-flesh type ranged from 36.75% to 98.39% and 33.63% to 99.31%,respectively.Most of the RT sequences were AT-rich,with AT content of 48.6%~63.0%.Of the amino acid sequences,55(67%)presented premature stop codons,and five demonstrated frameshift mutation.Phylogenetic analysis suggested that the RT sequences of pitaya had high homology with that of Oryza sativa and Arabidopsis thaliana.3)To investigate the transcriptional activity of 82 RT sequences and unravel their relative expression as exposure to stress,semi-quantitative RT-PCR were employed.Only three RT sequences(HURT1,HURT56 and HURT70)were transcriptionally activated in vitro shoots,and diversely expressed among the abiotic stress treatments as well as exogenous uses of plant growth regulators.4)To further explore the structure and function of pitaya Ty3-gypsy retrotransponnsons,the 3’ end sequence RT-RNase H,which named REHu1,was successfully obtained by two-step nested PCR amplification using chromosome walking method.Sequence analysis results showed that the length of REHu1 was 1,293 bp.