Cloning and analysis of reverse transcriptase of LTR retrotransposons in Eriobotrya japonica

Abstract: 【Objective】Loquat（Eriobotrya japonica）is a fruit tree with a long history of planting, and the degree of variation is high. The activation of retrotransposon is one of the reasons for plant variation, and reverse transcriptase is the key regulatory factor of retrotransposon. Therefore, two kinds of reverse transcriptase sequences, Ty1-copia and Ty3-gypsy, were cloned from the Loquat genome using degenerate primers. The possible relationship between retrotransposon and genetic variation of loquat are explored by studying the characteristics of retrotransposons.【Methods】Using the wild loquat leaves as materials, two kinds of reverse transcriptase sequences, Ty1- copia and Ty3- gypsy, were amplified from the Loquat genome using degenerate primers. After purification, the amplified products were linked to pMDTM19- T vector and transformed into E. coli DH5α competent cells. After culturing at 37 ℃ for 12-16 hours, blue and white spots were screened. Single colonies were picked out and cultured overnight in LB liquid medium containing 100 mg · L^-1 AMP. The positive clones identified by PCR were sequenced. RT amino acid sequence was obtained by deriving and comparing with blast pro-gram on NCBI, and multiple alignment of amino acid sequence was performed by ClustalW software. The phylogenetic tree was constructed by adjacency method using MEGA7 software. DNAsp5 was used to calculate the synonymous mutation rate and non- synonymous mutation rate.【Results】After PCR amplification, Ty1-copia and Ty3-gypsy like RT sequences were obtained in the tested materials, they were about 260 bp and 430 bp and were consistent with the length of Ty1-copia and Ty3-gypsy type RT sequences reported in other plants. After the characteristic fragments were recovered and sequenced, 57 Ty1-copia like RT sequences and 24 Ty3-gypsy RT sequences were obtained after removing the short sequences. The 57 Ty1-copia like RT sequences were compared with those of loquat accessed on NCBI. 19 sequences with more than 95% similarity with the registered sequences were removed. Finally, 38 Ty1-copia like RT sequences and 24 Ty3-gypsy RT sequences were obtained. The
RT sequences of Ty1-copia type retrotransposons were 257-266 bp in length, and the sequence similarity ranged from 22.6% to 97.6%, and the average sequence similarity was 42.1%. The cluster analysis showed that the Ty1-copia like RT nucleotide sequences had three branches: Maxium, TAR and Angela, and Maxium were the largest branch. In the Ty1-copia like RT sequences of loquat, Bianca, Ivana and Ale were not found. The phylogenetic tree was constructed from 38 Ty1-copia RT nucleotide sequences of loquat, and these sequences were divided into 4 groups according to the degree of branching. After being translated into amino acids, there were 4 frame shift mutations and 12 stop codon mutations. The phylogenetic tree was constructed with the same type sequences of other species. The results showed that the Ty1-copia RT sequences of loquat were close to those of apple and plum. The RT sequences of Ty3- gypsy retrotransposons ranged from 429 bp to 438 bp, and the sequence similarity ranged from 48.1% to 99.3%, and the average sequence similarity was 66.9%. The cluster analysis showed that there were two branches, Tat and Reina, and Reina contained the most sequences. In the Ty3-gypsy like RT sequences of loquat, Athina, CRM, Galadriel and Tekay were not found. The Phylogenetic tree was constructed from 24 Ty3-gypsy like RT nucleotide sequences of loquat, and these sequences were divided into 5 groups according to the degree of branching. After being translated into amino acids, there were three frame shift mutations and seven stop codon mutations. The phylogenetic tree was constructed with the same type sequences of other species. The results showed that the RT sequences of Ty3-gypsy in loquat had high homology and conservation. The 38 Ty1-copia RT sequences and 24 Ty3-gypsy RT sequences obtained in this study were analyzed by phylogenetic analysis. Ty1-copia type RT sequences could be divided into five types. The first type had a close genetic distance with those of apple and plum, among which EJRT38 was likely to be homologous with that of plum; the fifth type was EJRT23 and pepper. Ty3-gypsy type RT sequences could be divided into four types. The fourth type had a close genetic distance with that of plum, which might be caused by the lateral transmission of retrotransposons. The results of synonymous mutation rate and non-synonymous mutation rate showed that dN/dS of Ty1-copia and Ty3-gypsy RT sequences of loquat were all less than 1, indicating that the sequences were affected by purify selection in the evolution process【Conclusion】The RT nucleotide sequences of Ty1- copia and Ty3- gypsy retrotransposon were highly heterogeneous, and they were affected by the role of purification choices during evolution. The phylogenetic analysis demonstrated that loquat showed a common origin with other species, and a horizontal transmission of retrotransposons occurred among different species in the process of evolution.