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.

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.

Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1376-1376
Author(s):  
Soumyadeep Dey ◽  
David J. Curtis ◽  
Stephen Jane ◽  
Stephen J. Brandt
Keyword(s):  
Transcription Factor ◽  
Real Time ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Critical Role ◽  
Pcr Analysis ◽  
Bhlh Transcription Factor ◽  
Lacz Gene ◽  
Macrophage Differentiation ◽  
In Cells

Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.

scholarly journals Overexpression of ThMYC4E Enhances Anthocyanin Biosynthesis in Common Wheat

2019 ◽  
Vol 21 (1) ◽  
pp. 137 ◽  
Author(s):  
Shuo Zhao ◽  
Xingyuan Xi ◽  
Yuan Zong ◽  
Shiming Li ◽  
Yun Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Common Wheat ◽  
Anthocyanin Biosynthesis ◽  
Pcr Analysis ◽  
Bhlh Transcription Factor ◽  
Anthocyanin Content ◽  
Metabolome Analysis ◽  
Helix Loop Helix ◽  
Transgenic Lines ◽  
Ethanol Extracts

The basic helix-loop helix (bHLH) transcription factor has been inferred to play an important role in blue and purple grain traits in common wheat, but to date, its overexpression has not been reported. In this study, the bHLH transcription factor ThMYC4E, the candidate gene controlling the blue grain trait from Th. Ponticum, was transferred to the common wheat JW1. The positive transgenic lines displayed higher levels of purple anthocyanin pigments in their grains, leaves and glumes. Stripping the glumes (light treatment) caused white grains to become purple in transgenic lines. RNA-Seq and qRT-PCR analysis demonstrated that the transcript levels of structural genes associated with anthocyanin biosynthesis were higher in transgenic wheat than the wild-type (WT), which indicated that ThMYC4E activated anthocyanin biosynthesis in the transgenic lines. Correspondingly, the anthocyanin contents in grains, roots, stems, leaves and glumes of transgenic lines were higher than those in the WT. Metabolome analysis demonstrated that the anthocyanins were composed of cyanidin and delphinidin in the grains of the transgenic lines. Moreover, the transgenic lines showed higher antioxidant activity, in terms of scavenging DPPH radicals, in the ethanol extracts of their grains. The overexpression of ThMYC4E sheds light on the traits related to anthocyanin biosynthesis in common wheat and provide a new way to improve anthocyanin content.

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.

scholarly journals iTRAQ-Based Proteomic Analysis of Ogura-CMS Cabbage and Its Maintainer Line

2018 ◽  
Vol 19 (10) ◽  
pp. 3180 ◽  
Author(s):  
Fengqing Han ◽  
Xiaoli Zhang ◽  
Limei Yang ◽  
Mu Zhuang ◽  
Yangyong Zhang ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Male Sterility ◽  
Molecular Mechanisms ◽  
Transcriptome Profiling ◽  
Pcr Analysis ◽  
Hybrid Seed Production ◽  
Protein Species ◽  
Absolute Quantitation ◽  
Maintainer Line ◽  
Cms Line

Ogura cytoplasmic male sterility (CMS) contributes considerably to hybrid seed production in Brassica crops. To detect the key protein species and pathways involved in Ogura-CMS, we analysed the proteome of the cabbage Ogura-CMS line CMS01-20 and its corresponding maintainer line F01-20 using the isobaric tags for the relative and absolute quantitation (iTRAQ) approach. In total, 162 differential abundance protein species (DAPs) were identified between the two lines, of which 92 were down-accumulated and 70 were up-accumulated in CMS01-20. For energy metabolism in the mitochondrion, eight DAPs involved in oxidative phosphorylation were down-accumulated in CMS01-20, whereas in the tricarboxylic acid (TCA) cycle, five DAPs were up-accumulated, which may compensate for the decreased respiration capacity and may be associated with the elevated O2 consumption rate in Ogura-CMS plants. Other key protein species and pathways involved in pollen wall assembly and programmed cell death (PCD) were also identified as being male-sterility related. Transcriptome profiling revealed 3247 differentially expressed genes between the CMS line and the fertile line. In a conjoint analysis of the proteome and transcriptome data, 30 and 9 protein species/genes showed the same and opposite accumulation patterns, respectively. Nine noteworthy genes involved in sporopollenin synthesis, callose wall degeneration, and oxidative phosphorylation were presumably associated with the processes leading to male sterility, and their expression levels were validated by qRT-PCR analysis. This study will improve our understanding of the protein species involved in pollen development and the molecular mechanisms underlying Ogura-CMS.

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 Conserved and non-conserved functions of the rice homologs of the Arabidopsis trichome initiation-regulating MBW complex proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kaijie Zheng ◽  
Xutong Wang ◽  
Yating Wang ◽  
Shucai Wang
Keyword(s):  
Transcription Factor ◽  
Anthocyanin Biosynthesis ◽  
Transcriptional Activator ◽  
Myb Transcription Factor ◽  
Bhlh Transcription Factor ◽  
Seed Color ◽  
Transcription Activator ◽  
Root Hair Formation ◽  
Protoplast Transfection ◽  
Mbw Complex

Abstract Background Trichome initiation in Arabidopsis is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex formed by the R2R3 MYB transcription factor GLABRA1 (GL1), MYB23 or MYB82, the bHLH transcription factor GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). However, the functions of the rice homologs of the MBW complex proteins remained uncharacterized. Results Based on amino acid sequence identity and similarity, and protein interaction prediction, we identified OsGL1s, OsGL3s and OsTTG1s as rice homologs of the MBW complex proteins. By using protoplast transfection, we show that OsGL1D, OsGL1E, OsGL3B and OsTTG1A were predominantly localized in the nucleus, OsGL3B functions as a transcriptional activator and is able to interact with GL1 and TTG1. By using yeast two-hybrid and protoplast transfection assays, we show that OsGL3B is able to interact with OsGL1E and OsTTG1A, and OsGL1E and OsTTG1A are also able to interact with GL3. On the other hand, we found that OsGL1D functions as a transcription activator, and it can interact with GL3 but not OsGL3B. Furthermore, our results show that expression of OsTTG1A in the ttg1 mutant restored the phenotypes including alternations in trichome and root hair formation, seed color, mucilage production and anthocyanin biosynthesis, indicating that OsTTG1A and TTG1 may have similar functions. Conclusion These results suggest that the rice homologs of the Arabidopsis MBW complex proteins are able to form MBW complexes, but may have conserved and non-conserved functions.

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