scholarly journals Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ruimin Zhang ◽  
Jingjing Chang ◽  
Jiayue Li ◽  
Guangpu Lan ◽  
Changqing Xuan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Male Sterility ◽  
Molecular Mechanisms ◽  
Citrullus Lanatus ◽  
Anther Development ◽  
Dose Effect ◽  
Luciferase Reporter ◽  
Bhlh Transcription Factor ◽  
Transcriptional Level ◽  
Hybrid Seed Production

AbstractAlthough male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

scholarly journals Knockout of SlMS10 Gene (Solyc02g079810) Encoding bHLH Transcription Factor Using CRISPR/Cas9 System Confers Male Sterility Phenotype in Tomato

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1189
Author(s):  
Yu Jin Jung ◽  
Dong Hyun Kim ◽  
Hyo Ju Lee ◽  
Ki Hong Nam ◽  
Sangsu Bae ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Male Sterility ◽  
Mutant Line ◽  
Pcr Analysis ◽  
Bhlh Transcription Factor ◽  
Hybrid Seed Production ◽  
Sequencing Analysis ◽  
Alternative Approach ◽  
Deep Sequencing Analysis ◽  
Male Sterility Phenotype

The utilization of male sterility into hybrid seed production reduces its cost and ensures high purity of tomato varieties because it does not produce pollen and has exserted stigmas. Here, we report on the generation of gene edited lines into male sterility phenotype by knockout of SlMS10 gene (Solyc02g079810) encoding the bHLH transcription factor that regulates meiosis and cell death of the tapetum during microsporogenesis in the tomato. Twenty-eight gene edited lines out of 60 transgenic plants were selected. Of these, eleven different mutation types at the target site of the SlMS10 gene were selected through deep sequencing analysis. These mutations were confirmed to be transmitted to subsequent generations. The null lines without the transferred DNA (T-DNA) were obtained by segregation in the T1 and T2 generations. In addition, we showed that the cr-ms10-1-4 mutant line exhibited dysfunctional meiosis and abnormal tapetum during flower development, resulting in no pollen production. RT-PCR analysis showed that the most genes associated with pollen and tapetum development in tomatoes had lower expression in the cr-ms10-1-4 mutant line compared to wild type. We demonstrate that modification of the SlMS10 gene via CRISPR/Cas9-mediated genome editing results in male sterility of tomato plants. Our results suggest an alternative approach to generating male sterility in crops.

A Single Nucleotide Polymorphism in an R2R3 MYB Transcription Factor Triggers ms6 (Ames1) Male Sterility in Soybean

2021 ◽  
Author(s):  
Junping Yu ◽  
Guolong Zhao ◽  
Wei Li ◽  
Ying Zhang ◽  
Peng Wang ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Transcription Factor ◽  
Male Sterility ◽  
Anther Development ◽  
Hybrid Breeding ◽  
Myb Transcription Factor ◽  
Hybrid Seed Production ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
R2r3 Myb

Abstract Soybean [Glycine max (L.) Merr.] is an important crop providing vegetable oils and proteins. Increasing demand on soy products heightens the urgency of soybean yield improvement. Hybrid breeding with male sterility system is an effective method to improve crop production. Cloning of genic male sterile (GMS) gene combined with biotechnology method can contribute to constructing GMS-based hybrid Seed Production Technology (SPT) to promote soybean performance and yield. In this research, we identified a soybean GMS locus, GmMS6, by combining bulked segregant analysis (BSA)-sequencing and map-based cloning technology. GmMS6 encodes an R2R3 MYB transcription factor, whose mutant allele in ms6 (Ames1) harbors a single nucleotide polymorphism (SNP) substitution, leading to the 76th Leucine to Histidine change in the DNA binding domain. Phylogenetic analysis demonstrates GmMS6 is a homolog of Tapetal Development and Function 1 (TDF1)/MYB35 that is an anther development key factor co-evolved with angiosperm. It has a recently duplicated homolog GmMS6LIKE (GmMS6L), both of which can rescue the male fertility of Arabidopsis homologous mutant attdf1 while GmMS6L76H cannot, denoting that both proteins are functional and L76 is a critical residue for TDF1’s function. However, compared to anther specific expressed GmMS6, GmMS6L is constitutively expressed at a very low level, explaining deficiency of GmMS6 alone causes pollen abortion. Moreover, the expression levels of major regulatory and structural genes for anther development are significantly decreased in ms6, unveiling that GmMS6 is a core transcription factor regulating soybean anther development.

Transcriptome analysis implicates involvement of long noncoding RNAs in cytoplasmic male sterility and fertility restoration in cotton

2019 ◽  
Author(s):  
Bingbing Zhang ◽  
Xuexian Zhang ◽  
Meng Zhang ◽  
Liping Guo ◽  
Tingxiang Qi ◽  
...  
Keyword(s):  
Cytoplasmic Male Sterility ◽  
Male Sterility ◽  
Molecular Mechanisms ◽  
Fertility Restoration ◽  
Noncoding Rnas ◽  
Anther Development ◽  
Long Noncoding Rnas ◽  
Pcr Analysis ◽  
Hybrid Seed Production ◽  
Restorer Of Fertility

Abstract Background Hybrid cotton has greatly contributed to global increase in cotton productivity. The cytoplasmic male sterility (CMS)/restorer-of-fertility system is an important tool to exploit heterosis because it is convenient for commercial hybrid seed production. The importance of long noncoding RNAs (lncRNAs) in plant development is recognized, few analyses of lncRNAs during anther development of three-line hybrid cotton (CMS-D2 line A, maintainer line B, restorer-of-fertility line R) have been reported. Results Here, we performed transcriptome sequencing during anther development in Upland cotton carrying cytoplasmic male sterile Gossypium harknessii (D2) cytoplasm. Totally 80,695 lncRNAs were identified, in which 43,347 and 44,739 lncRNAs were differentially expressed in A–B and A–R comparisons, respectively. These lncRNAs represent functional candidates involved in CMS and fertility restoration. Gene ontology enrichment analysis indicated that cellular hormone metabolic processes and oxidation–reduction reaction processes might be involved in CMS, and cellular component morphogenesis and small molecular biosynthetic processes might participate in fertility restoration. Analysis of the putative relationship between lncRNAs and miRNAs revealed that 63 lncRNAs were identified as putative precursors of 35 miRNAs, and qRT-PCR analysis showed a similar expression pattern to that of RNA-sequencing data. Furthermore, construction of lncRNA regulatory networks indicated that several miRNA–lncRNA–mRNA networks might be involved in CMS and fertility restoration. Conclusion Our findings provide systematic identification of lncRNAs during anther development and lays a solid foundation for future investigation of the regulatory molecular mechanisms and utilization in ­­breeding of hybrid cotton.

scholarly journals A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1)

2021 ◽  
Author(s):  
Junping Yu ◽  
Guolong Zhao ◽  
Wei Li ◽  
Ying Zhang ◽  
Peng Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Glycine Max ◽  
Male Sterility ◽  
Bulked Segregant Analysis ◽  
Soybean Protein ◽  
Anther Development ◽  
Hybrid Breeding ◽  
Myb Transcription Factor ◽  
Male Sterile ◽  
R2r3 Myb

Abstract Key message Identification and functional analysis of the male sterile gene MS6 in Glycine max. Abstract Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

scholarly journals Hsa_circ_0026628 promotes the development of colorectal cancer by targeting SP1 to activate the Wnt/β-catenin pathway

2021 ◽  
Vol 12 (9) ◽  
Author(s):  
Xuexiu Zhang ◽  
Jianning Yao ◽  
Haoling Shi ◽  
Bing Gao ◽  
Haining Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcription Factor ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Transcriptional Level ◽  
Sp1 Transcription Factor ◽  
Reporter Assays ◽  
Sphere Formation

AbstractCircular RNAs (circRNAs) have been reported to play crucial roles in the progression of various cancers, including colorectal cancer (CRC). SP1 (Sp1 transcription factor) is a well-recognized oncogene in CRC and is deemed to trigger the Wnt/β-catenin pathway. The present study was designed to investigate the role of circRNAs which shared the same pre-mRNA with SP1 in CRC cells. We identified that hsa_circ_0026628 (circ_0026628), a circular RNA that originated from SP1 pre-mRNA, was upregulated in CRC cells. Sanger sequencing and agarose gel electrophoresis verified the circular characteristic of circ_0026628. Functional assays including CCK-8, colony formation, transwell, immunofluorescence staining, and sphere formation assay revealed the function of circ_0026628. RNA pull-down and mass spectrometry disclosed the proteins interacting with circ_0026628. Mechanistic assays including RIP, RNA pull-down, CoIP, ChIP, and luciferase reporter assays demonstrated the interplays between molecules. The results depicted that circ_0026628 functioned as a contributor to CRC cell proliferation, migration, EMT, and stemness. Mechanistically, circ_0026628 served as the endogenous sponge of miR-346 and FUS to elevate SP1 expression at the post-transcriptional level, thus strengthening the interaction between SP1 and β-catenin to activate the Wnt/β-catenin pathway. In turn, the downstream gene of Wnt/β-catenin signaling, SOX2 (SRY-box transcription factor 2), transcriptionally activated SP1 and therefore boosted circ_0026628 level. On the whole, SOX2-induced circ_0026628 sponged miR-346 and recruited FUS protein to augment SP1, triggering the downstream Wnt/β-catenin pathway to facilitate CRC progression.

scholarly journals Ablation of the MiR-17-92 MicroRNA Cluster in Germ Cells Causes Subfertility in Female Mice

2018 ◽  
Vol 45 (2) ◽  
pp. 491-504 ◽  
Author(s):  
Jian Wang ◽  
Bo Xu ◽  
Geng G. Tian ◽  
Tao Sun ◽  
Ji Wu
Keyword(s):  
Germ Cells ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Conditional Knockout ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Follicular Atresia ◽  
Apoptotic Pathway ◽  
Female Mice ◽  
Important Regulator

Background/Aims: Oogenesis is a highly complex process that is intricately regulated by interactions of multiple genes and signaling molecules. However, the underlying molecular mechanisms are poorly understood. There is emerging evidence that microRNAs contribute to oogenesis. Here, we aimed to investigate the role of miR-17-92 cluster in regulating oogenesis. Methods: The miR-17-92 cluster was genetically ablated in germ cells of female mice by applying the Cre-loxp system for conditional gene knockout. Mating experiment, superovulation and histological analysis were used to assess the fertility of the model female mice. TUNEL assay was used to identify apoptotic cells in ovaries. The expression level of apoptosis- and follicular atresia- related genes was evaluated by qRT-PCR. Western blotting was performed to detect protein expression. Bioinformatics software and dual luciferase reporter assay were applied to predict and verify the target of miR-17-92 cluster. Results: Deletion of miR-17-92 cluster in germ cells of female mice caused increased oocyte degradation and follicular atresia, perturbed oogenesis, and ultimately led to subfertility. Genes involved in follicular atresia and the mitochondrial apoptotic pathway were obviously up-regulated. Furthermore, we verified that miR-19a regulated oogenesis at the post-transcriptional level by targeting Bmf in the ovaries of miR-17-92 cluster conditional knockout female mice. Conclusion: The miR-17-92 cluster is an important regulator of oogenesis. These findings will assist in better understanding the etiology of disorders in oogenesis and in developing new therapeutic targets for female infertility.

scholarly journals Sp1 and Smad transcription factors co-operate to mediate TGF-β-dependent activation of amyloid-β precursor protein gene transcription

2004 ◽  
Vol 383 (2) ◽  
pp. 393-399 ◽  
Author(s):  
Fabian DOCAGNE ◽  
Cecilia GABRIEL ◽  
Nathalie LEBEURRIER ◽  
Sylvain LESNÉ ◽  
Yannick HOMMET ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Amyloid Β ◽  
Transforming Growth Factor Β ◽  
Precursor Protein ◽  
Transcriptional Level ◽  
Amyloid Β Peptide ◽  
Aβ Amyloid

Abnormal deposition of Aβ (amyloid-β peptide) is one of the hallmarks of AD (Alzheimer's disease). This peptide results from the processing and cleavage of its precursor protein, APP (amyloid-β precursor protein). We have demonstrated previously that TGF-β (transforming growth factor-β), which is overexpressed in AD patients, is capable of enhancing the synthesis of APP by astrocytes by a transcriptional mechanism leading to the accumulation of Aβ. In the present study, we aimed at further characterization of the molecular mechanisms sustaining this TGF-β-dependent transcriptional activity. We report the following findings: first, TGF-β is capable of inducing the transcriptional activity of a reporter gene construct corresponding to the +54/+74 region of the APP promoter, named APPTRE (APP TGF-β-responsive element); secondly, although this effect is mediated by a transduction pathway involving Smad3 (signalling mother against decapentaplegic peptide 3) and Smad4, Smad2 or other Smads failed to induce the activity of APPTRE. We also observed that the APPTRE sequence not only responds to the Smad3 transcription factor, but also the Sp1 (signal protein 1) transcription factor co-operates with Smads to potentiate the TGF-β-dependent activation of APP. TGF-β signalling induces the formation of nuclear complexes composed of Sp1, Smad3 and Smad4. Overall, the present study gives new insights for a better understanding of the fine molecular mechanisms occurring at the transcriptional level and regulating TGF-β-dependent transcription. In the context of AD, our results provide additional evidence for a key role for TGF-β in the regulation of Aβ production.

scholarly journals Longitudinal noninvasive monitoring of transcription factor activation in cardiovascular regulatory nuclei using bioluminescence imaging

2008 ◽  
Vol 33 (2) ◽  
pp. 292-299 ◽  
Author(s):  
Jeffrey R. Peterson ◽  
David W. Infanger ◽  
Valdir A. Braga ◽  
Yulong Zhang ◽  
Ram V. Sharma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Gene Transfer ◽  
Molecular Mechanisms ◽  
Bioluminescence Imaging ◽  
Brain Regions ◽  
Luciferase Reporter ◽  
Viral Gene ◽  
Adenoviral Delivery ◽  
Wide Range

The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-κB-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for 1 mo. Importantly, systemic endotoxin, a stimulator of NF-κB activity, induced dramatic and dose-dependent increases in NF-κB-dependent bioluminescence in the SFO up to 30 days after gene transfer. Cotreatment with a dominant-negative IκBα mutant significantly prevented endotoxin-dependent NF-κB activation, confirming the specificity of the bioluminescence signal. NF-κB-dependent luminescence signals were also stable and inducible 1 mo following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-κB reporters. These results demonstrate that BLI, in combination with virally mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.

Novel nuclear target for thrombin: activation of the Elk1 transcription factor leads to chemokine gene expression

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3696-3706 ◽  
Author(s):  
Qi-Jing Li ◽  
Sucheta Vaingankar ◽  
Frances M. Sladek ◽  
Manuela Martins-Green
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Gene Activation ◽  
Tumor Development ◽  
Growth Factor Receptor ◽  
Early Response ◽  
Transcriptional Level ◽  
Binding Domains ◽  
Thrombin Activation

Thrombin is primarily known for its role in homeostasis and thrombosis. However, this enzyme also plays important roles in wound healing and pathologic situations such as inflammation and tumorigenesis. Among the molecules stimulated by thrombin in these latter processes are the stress response proteins, chemokines. Chemokines are also known for their roles in inflammatory responses and tumor development. These correlative observations strongly suggest that chemokines may be mediators of some of thrombin's functions in these processes. Elucidation of the molecular mechanisms of stimulation of chemokines by thrombin may help to unravel the ways in which their expression can be modulated. Up-regulation of the chemokine 9E3/cCAF by thrombin occurs via its proteolytically activated receptor with subsequent transactivation of the epidermal growth factor receptor tyrosine kinase. This study shows that stimulation by thrombin very rapidly activates this chemokine at the transcriptional level, that 2 Elk1 binding elements located between −534 and −483 bp of the promoter are major thrombin response elements, that activation occurs via the Elk1 transcription factor, and that the latter is directly activated by MEK1/ERK2. The common occurrence of Elk1 binding domains in the promoters of immediate early response genes suggests that it may be characteristically involved in gene activation by stress-inducing agents.

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