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Background: Iron (Fe) deficiency affects 30-50% of the world's population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5' leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF)., Results: We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5' LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression., Conclusions: Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance., (© 2024. The Author(s).)

The Bardet-Biedl Syndrome 7 ( Bbs7 ) gene was identified as the most likely candidate gene causing juvenile obesity in the Berlin Fat Mouse Inbred (BFMI) line. Bbs7 expression is significantly lower in the brain, adipose tissue, and liver of BFMI mice compared to lean C57BL/6NCrl (B6N) mice. A DNA sequence comparison between BFMI and B6N revealed 16 sequence variants in the Bbs7 promoter region. Here, we tested if these mutations contribute to the observed differential expression of Bbs7 . In a cell-based dual-luciferase assay, we compared the effects of the BFMI and the B6N haplotypes of different regions of the Bbs7 promotor on the reporter gene expression. A single-nucleotide polymorphism (SNP) was identified causing a significant reduction in the reporter gene expression. This SNP (rs29947545) is located in the 5' UTR of Bbs7 at Chr3:36.613.350. The SNP is not unique to BFMI mice but also occurs in several other mouse strains, where the BFMI allele is not associated with lower Bbs7 transcript amounts. Thus, we suggest a compensatory mutation in the other mouse strains that keeps Bbs7 expression at the normal level. This compensatory mechanism is missing in BFMI mice and the cell lines tested.

Santalum album L., a semi-parasitic evergreen tree, contains economically important essential oil, rich in sesquiterpenoids, such as ( Z ) α- and ( Z ) β-santalol. However, their transcriptional regulations are not clear. Several studies of other plants have shown that basic-helix-loop-helix (bHLH) transcription factors (TFs) were involved in participating in the biosynthesis of sesquiterpene synthase genes. Herein, bHLH TF genes with similar expression patterns and high expression levels were screened by co-expression analysis, and their full-length ORFs were obtained. These bHLH TFs were named SaMYC1 , SaMYC3 , SaMYC4 , SaMYC5 , SabHLH1 , SabHLH2 , SabHLH3 , and SabHLH4 . All eight TFs had highly conserved bHLH domains and SaMYC1 , SaMYC3 , SaMYC4 , and SaMYC5 , also had highly conserved MYC domains. It was indicated that the eight genes belonged to six subfamilies of the bHLH TF family. Among them, SaMYC1 was found in both the nucleus and the cytoplasm, while SaMYC4 was only localized in the cytoplasm and the remaining six TFs were localized in nucleus. In a yeast one-hybrid experiment, we constructed decoy vectors pAbAi-SSy1G-box, pAbAi-CYP2G-box, pAbAi-CYP3G-box, and pAbAi-CYP4G-box, which had been transformed into yeast. We also constructed pGADT7- SaMYC1 and pGADT7- SabHLH1 capture vectors and transformed them into bait strains. Our results showed that SaMYC1 could bind to the G-box of SaSSy, and the SaCYP736A167 promoter, which SaSSy proved has acted as a key enzyme in the synthesis of santalol sesquiterpenes and SaCYP450 catalyzed the ligation of santalol sesquiterpenes into terpene. We have also constructed pGreenII 62-SK- SaMYC 1, pGreenII 0800-LUC- SaSSy and pGreenII 0800- LUC - SaCYP736A167 via dual-luciferase fusion expression vectors and transformed them into Nicotiana benthamiana using an Agrobacterium -mediated method. The results showed that SaMYC 1 was successfully combined with SaSSy or SaCYP736A167 promoter and the LUC/REN value was 1.85- or 1.55-fold higher, respectively, than that of the control group. Therefore, we inferred that SaMYC1 could activate both SaSSy and SaCYP736A167 promoters.

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo ( Citrus grandis ). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043 , that is involved in the regulation of lignin biosynthesis in Citrus grandis , which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of 'San hong' as the material, the staining for lignin with HCl-phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46 , which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043 , was transformed into the mesocarp of 'San hong' by Agrobacterium -mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

Transcription factor 4 (TCF4) gene plays an important role in nervous system development and it always associated with the risk of schizophrenia. Since miRNAs regulate targetgenes by binding to 3'UTRs of target mRNAs, the functional variants located in 3'UTR of TCF4 are highly suggested to affect the gene expressions in schizophrenia. To test the hypothesis regarding the effects of the variants located in 3'UTR of TCF4, we conducted an in silico analysis to identify the functional variants and their predicted functions. In this study, we sequenced the 3'UTR of TCF4 in 13 multiplex schizophrenia families and 14 control families. We found two functional variants carried by three unrelated patients. We determined that the C allele of rs1272363 and the TC insert of rs373174214 might suppress post- transcriptional expression. Secondly, we cloned the region that flanked these two variants into a dual luciferase reporter system and compared the luciferase activities between the pmirGLO-TCF4 (control), pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263. Both pmirGLO-TCF4-rs373174214 and pmirGLO-TCF4-rs1273263 caused lower reporter gene activities, as compared to the control. However, only the C allele of rs1272363 reduced the luciferase activity significantly (p = 0.0231). Our results suggested that rs1273263 is a potential regulator of TCF4 expression, and might be associated with schizophrenia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug., (Copyright © 2017 Elsevier B.V. All rights reserved.)