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Transcriptome sequencing of transgenic Nicotiana benthamiana producing moth pheromones [1]

ABSTRACT: Insect sex pheromones are an important resource in integrated pest management. Encouraging steps have been made towards their sustainable production in plant biofactories, but important obstacles remain to be resolved. We have used transcriptomic analysis to determine the molecular perturbations behing growth penalty associated with high pheromone production in transgenic Nicotiana benthamiana lines with constitutive expression of genes for biosynthesis of moth sex pheromones - SexyPlants v1.0. We analyzed lines with high production (homozygous lines) and strong growth penalty as well as lines with low production (heterozygous lines) and negligible growth penalty.

ORGANISM(S): Nicotiana benthamiana

PROVIDER: GSE191269 | GEO | 2022/09/26

REPOSITORIES: GEO

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Project description:Sex pheromones rapidly affect endocrine physiology and behaviour, but little is known about their effects on gene expression in the neuroendocrine tissues that mediate olfactory processing. In this study we exposed male goldfish for 6 h to waterborne 17,20betaP (4.3 nM) and PGF2α (3 nM), the main pre-ovulatory and post-ovulatory pheromones, respectively. Both treatments elevated milt volume (P = 0.001). Microarray hybridizations were used to male telencephalon samples following PGF2α treatment and identified 71 unique transcripts that were differentially expressed (q < 5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF2α pheromone exposure affects diverse biological processes including nervous system functions, energy metabolism, cholesterol/lipoprotein transport, translational regulation, transcription and chromatin remodelling, protein processing, cytoskeletal organization, and signalling. By using real-time RT-PCR, we further validated three candidate genes, ependymin-II, calmodulin-A and aldolase C, which exhibited 3 - 5 fold increase in expression following PGF2α exposure. Expression levels of some other genes that are thought to be important for reproduction were also determined using real-time RT-PCR. Expression of sGnRH was increased by PGF2α, but not 17,20betaP, whereas cGnRH expression was increased by 17,20betaP but not PGF2α. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and gamma-aminobutyric acid (GABAA gamma2) receptor subunit mRNAs. This gene expression data link milt release and rapid modulation of neuronal transcription as part of the response to female sex pheromones. A total of four microarray slides were hybridized to study the effects of PGF2α on gene expression in the telencephalon. Control samples (N = 4) were pooled to form our reference or technical replicates while treatment samples (N = 4), which formed our biological replicates, were ran separately. In addition, two dye swaps were performed for our biological samples for the treatments.

Project description:Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown at 20ËC constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14). Diamondback moth (DBM, Plutella xylostella) larvae were maintained on cabbage (Brassica oleracea) plants in a climate-controlled room at 25ÂºC, 12 hr photoperiod with 50%-60% relative humidity. Two days before exposing A. thaliana plants to herbivore treatment, plants were transferred to a climate-controlled room (22ÂºC, 50-60% humidity, 12 hr photoperiod). For insect treatment, seven Diamondback moth (DBM, Plutella xylostella) larvae (third to fifth instars) were placed on a group of four or five plants until time of harvest, for each time point separately. All rosette leaves were harvested at 1h, 4h, 12h, and 24h after onset of continuous herbivory and flash frozen in liquid nitrogen. Control plants were maintained under the same conditions and harvested in parallel. Keywords: time course, stress response, herbivory response For each time point (1h, 4h, 12h, and 24h) two biological replicates (DBM treatment) were performed. Control plants were harvested in parallel. Total RNA from each sample was used for two labelling reactions using dye-flips (Cy5 or Cy3) and labelled cDNA from treatment and control plants were co-hybridized. Thus, for each time-point four replicates were analyzed each comparing treatment with the corresponding control (rep1 and rep2 are dye-flips of biological replicate 1, and rep3 and rep4 are dye-flips of biological replicate 2). For background correction, we defined the mean of the lowest 10% of spot intensities from a particular subgrid as the background for that subgrid. This mean was subtracted from each spot in the subgrid. Signal intensities that did not exceed the background plus 3 standard deviations thereof were defined as not detectable and were excluded from further analyses. We normalized using loess curves thus generating log2 transformed expression ratios comparing DBM treatment with control. These ratios are given in the VALUE columns of each array.

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Transcriptome sequencing of transgenic Nicotiana benthamiana producing moth pheromones [1]

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