Transcriptome-wide characterization of miRNA-directed and non-miRNA-directed endonucleolytic cleavage using Degradome analysis under low ambient temperature in Phalaenopsis aphrodite subsp. formosana.

Plant microRNAs (miRNAs) regulate gene expression through post-transcriptional gene silencing. Phalaenopsis aphrodite subsp. formosana is an orchid species native to Taiwan, which has high economic value and a high frequency of floral polymorphism. To date, few studies have focused on the regulatory roles of miRNAs and functional small RNAs (sRNAs) in orchids although understanding the regulation of flower development and flowering time is potentially important. Here, we combined analyses of the transcriptome, sRNAs and the degradome to identify sRNA-directed transcript cleavages in Phalaenopsis. Degradome analysis provided large-scale evidence of conserved and novel miRNA-directed cleavage of target transcripts, and 46 abundant sRNA groups and their target transcripts were identified. Low temperature-responsive sRNAs were validated with normalized reads from an sRNA library and quantitative stem-loop reverse transcription-PCR (RT-PCR) analysis. According to gene ontology (GO) categorization, target transcripts of the novel miRNAs and sRNAs are functionally involved in metabolic processes or responses to stress. One particular homologous gene, Allcontig28452, which encodes digalactosyldiacylglycerol synthase 2 (DGD2), was found to be targeted by natural antisense transcripts (NATs) unique to Phalaenopsis. In summary, comprehensive analyses of the transcriptome, sRNAs and degradome using deep sequencing technology provided a useful platform for investigating miRNA-directed and non-miRNA-directed endonucleolytic cleavage in a non-model plant, the orchid Phalaenopsis.

Sequencing-based approaches reveal low ambient temperature-responsive and tissue-specific microRNAs in phalaenopsis orchid.

Plant small RNAs (smRNAs) are short, non-coding RNA molecules that mediate RNA silencing and regulate a group of genes involved in plant development and responses to environmental stimuli. Low temperature is necessary to initiate stalk development in the orchid Phalaenopsis aphrodite subsp. formosana. To identify smRNAs in Phalaenopsis responding to low temperatures, a smRNA profiling analysis using high-throughput sequencing technology was performed. Subsequent bioinformatic analysis was applied to categorize the miRNAs identified. A total of 37,533,509 smRNA reads yielded 11,129 independent orchid miRNA sequences, representing 329 known miRNA families identified in other plant species. Because the genomic resources available for Phalaenopsis are limited, a transcriptomic database was established using deep sequencing data sets to identify miRNAs precursors and their target transcripts. Comparing small RNAs and the transcriptomic database, 14 putative miRNA precursors of 10 miRNA families were identified, as were hundreds of putative targets. Comparing sequencing data and smRNA northern hybridization results identified miR156, miR162, miR528 and miR535 as low temperature-induced miRNAs. In addition, tissue-specific expression of these miRNAs was investigated. It was concluded that miR156 and miR172 may be components of a regulatory pathway mediating transition from the vegetative to the reproductive phase in Phalaenopsis. The smRNA and transcriptomic databases could be the foundations for further research aimed at elucidating the control of the flowering time in orchids.

Molecular characterization of two novel deltamethrin-inducible P450 genes from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Two novel P450 genes, CYP6CE1 and CYP6CE2 (GenBank accession number: EF421245 and EF421246), were cloned and characterized from psocid, Liposcelis bostrychophila. CYP6CE1 and CYP6CE2 contain open reading frames of 1,581 and 1,563 nucleotides that encode 527 and 521 amino acid residues, respectively. The putative proteins of CYP6CE1 and CYP6CE2 show predicted molecular weights of 60.76 and 59.83 kDa with a theoretical pI of 8.58 and 8.78, respectively. CYP6CE1 and CYP6CE2 share 74% identity with each other, and the deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Both CYP6CE1 and CYP6CE2 share the closest identities with Hodotermopsis sjoestedti CYP6AM1 at 48% among the published sequences. Phylogenetic analysis showed a closer relationship of CYP6CE1 and CYP6CE2 with CYP6 members of other insects than with those from other families. Quantitative real-time RT-PCR showed that both CYP6CE1 and CYP6CE2 are expressed at all developmental stages tested. Interestingly, CYP6CE2 transcripts decreased from the highest in 1st nymph to the lowest in adults, which seemed to suggest developmental regulation. However, neither CYP6CE1 nor CYP6CE2 were stage specific. The CYP6CE1 and CYP6CE2 transcripts in adults increased significantly after deltamethrin exposure. Recombinant protein expression studies are needed to determine the real functions of these proteins.

Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae).

Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lbalpha1 and Lbalpha8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full-length cDNAs of Lbalpha1 (GenBank accession number: EU871527) and Lbalpha8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608 bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lbalpha1 had the closest phylogenetic relationship to Agamalpha1 from Anopheles gambiae and Amelalpha1 from Apis mellifera, and Lbalpha8 shares the highest identity with Agamalpha8 from An. gambiae and Amelalpha8 from A. mellifera. Quantitative real-time PCR analysis showed that Lbalpha1 was expressed 2.03-6.54-fold higher than Lbalpha8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lbalpha1 and Lbalpha8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lbalpha1, whereas there was a descending expression pattern for Lbalpha8 in the 1st through the 4th nymphal stadia.

Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters.

This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4). Glycans were conjugated using an ester or ether bond as the linker between 2'-paclitaxel and the 2'-glucose or glucuronic acid moiety. These prodrugs showed good water solubility and selective cytotoxicity against cancer cell lines, but showed reduced toxicity toward normal cell lines and cancer cell lines with low expression levels of GLUTs. The ester conjugated prodrug 1 showed the most cytotoxicity among the prodrugs examined and could be transported into cells via GLUTs. Fluorescent and confocal microscopy demonstrated that targeted cells exhibited morphological changes in tubulin and chromosomal alterations that were similar to those observed with paclitaxel treatment. Therefore, these glycan-based prodrugs may be good drug candidates for cancer therapy, and the glycan conjugation approach is an alternative method to enhance the targeted delivery of other drugs to cancer cells that overexpress GLUTs.