Strawberry (Fragaria spp.) is a kind of herbaceous perennial plant that propagates vegetatively. The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of LTR retrotransposons were amplified from the cultivated strawberry (Fragaria x ananassa Duch.). Sequence analysis of clones demonstrated that 5 of 19 Ty1-copia group unique sequences and 2 of 10 Ty3-gypsy unique sequences in F. x ananassa genome possessed either stop codon or frameshift. Ty1-copia group sequences are highly heterogeneous (divergence ranged from 1 to 69.8%), but the Ty3-gypsy group sequences are less (divergence ranged from 1 to 10%). Southern dot blot hybridization result suggested that both of the LTR retrotransposons are present in the genome of cultivated strawberry with high copy number (Ty1-copia group 2,875 Ty3-gypsy group 348). RT-PCR amplification from total RNA, which was extracted from leaves of micropropagated strawberry plants, did not yield either of the RT fragments. This is the first report on the presence of RT sequences of Ty1-copia and Ty3-gypsy group retrotransposons in F. x ananassa genome.
The emergence of New Delhi metal beta‐lactamase (NDM‐1)‐producing bacteria and their worldwide spread pose great challenges for the treatment of drug‐resistant bacterial infections. These bacteria can hydrolyze most β‐lactam antibacterials. Unfortunately, there are no clinically useful NDM‐1 inhibitors. In the current work, we manually collected NDM‐1 inhibitors reported in the past decade and established the first NDM‐1 inhibitor database. Four machine‐learning models were constructed using the structural and property characteristics of the collected compounds as input training set to discover potential NDM‐1 inhibitors. In order to distinguish between high active inhibitors and putative positive drugs, a three‐classification strategy was introduced in our study. In detail, the commonly used positive and negative divisions are converted into strongly active, weakly active, and inactive. The accuracy of the best prediction model designed based on this strategy reached 90.5%, compared with 69.14% achieved by the traditional docking‐based virtual screening method. Consequently, the best model was used to virtually screen a natural product library. The safety of the selected compounds was analyzed by the ADMET prediction model based on machine learning. Seven novel NDM‐1 inhibitors were identified, which will provide valuable clues for the discovery of NDM‐1 inhibitors.
Long terminal repeat (LTR) retrotransposons are powerful tools for studying genetic biodiversity, genome evolution, gene mutation, gene cloning and gene expression. The scarcity of retrotransposon sequence information restricts the development of these studies in higher plants. In the present study, 31 reverse transcriptase (RT) genes of Ty1-copia-like retrotransposons were identified from the apple genome by amplifying the RT coding region using degenerate primers. Nineteen RT genes showed extreme heterogeneity in terms of fragment size, base pair composition and open reading frame integrality. Originating from one 266 bp cloned RT gene, a 1966 bp Ty1-copia-like retrotransposon (named Tcrm1), including RT-ribonuclease H-LTR domain sequences, was achieved by chromosome walking based on modified SiteFinding-polymerase chain reaction. The comparison between Tcrm1 and other LTR retrotransposons in gene structure and sequence homology shows that Tcrm1 is the first Ty1-copia-like retrotransposon including an LTR domain in the apple genome. Dot blot analysis revealed that Tcrm1 copy number in the apple was approximately 1 x 10(3) copies per haploid genome.
The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of long terminal repeat (LTR) retrotransposons were isolated from the Malus domestica genome using degenerate oligonucleotide primers. Sequence analysis showed that 45% of Ty1-copia and 63% of Ty3-gypsy RT sequences contained premature stop codons and/or indels disrupting the reading frame. High heterogeneity among RT sequences of both Ty1-copia and Ty3-gypsy group retrotransposons was observed, but Ty3-gypsy group retrotransposons in the apple genome are less heterogeneous than Ty1-copia elements. Retrotransposon copy number was estimated by dot blot hybridizations for Ty1-copia (approximately 5,000) and Ty3-gypsy (approximately 26,000). All elements of the two types of LTR retrotransposons comprise approximately 38% of the M. domestica genome, with the Ty3-gypsy group contribution being higher (33.5%) than the Ty1-copia one (4.6%). Transcription was not detected by reverse transcription-polymerase chain reaction for either Ty1-copia or Ty3-gypsy retrotransposons in the leaves of plants in vitro or in leaf explants cultured on medium supplemented with high concentration benzylaminopurine. This research reveals the differences in heterogeneity and copy number between Ty1-copia and Ty3-gypsy retrotransposons in the apple genome. Ty1-copia retrotransposon has higher heterogeneity than Ty3-gypsy retrotransposon, but the latter has a higher copy number, which implies that Ty3-gypsy retrotransposons may play a more important role in the apple genome evolution.
Retrotransposon-based molecular markers are a powerful tool for mapping and diversity studies. The scarcity of retrotransposon long terminal repeat (LTR) sequences limits the application of retrotransposon-based molecular marker systems. Here, we isolated two novel complete Ty1-copia retrotransposons (CTcrm1 and CTcrm2) in apple using a genome walking strategy. The CTcrm retrotransposons are nearly 5 kb long, and they have all the features of Ty1-copia retrotransposons. The differences in gene organization and nucleotide sequence length between the CTcrm retrotransposons and other reported complete retrotransposons in apple showed that CTcrm1 and CTcrm2 are the first two distinct complete Ty1-copia retrotransposons in the apple genome. To investigate the potential utility of the two retrotransposons as molecular markers, primers complementary to the CTcrm LTRs were designed to develop sequence-specific amplification polymorphism markers for discriminating bud sports of Fuji apple. Multiple polymorphisms corresponding to CTcrm1 and CTcrm2 were detected and could easily be used to discriminate bud sports from their Fuji progenitor, as well as from each other.
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