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  • MDPI AG  (9)
  • Cho, Yong-Gu  (9)
  • 1
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    Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene
    MDPI AG ; 2022
    In:  Plants Vol. 12, No. 1 ( 2022-12-22), p. 56-
    Details
    In: Plants, MDPI AG, Vol. 12, No. 1 ( 2022-12-22), p. 56-
    Abstract: We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.
    Type of Medium: Online Resource
    ISSN: 2223-7747
    URL: Article
    DOI: 10.3390/plants12010056
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2704341-1
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  • 2
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    Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.)
    MDPI AG ; 2021
    In:  Genes Vol. 12, No. 12 ( 2021-11-26), p. 1891-
    Details
    In: Genes, MDPI AG, Vol. 12, No. 12 ( 2021-11-26), p. 1891-
    Abstract: In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.
    Type of Medium: Online Resource
    ISSN: 2073-4425
    URL: Article
    DOI: 10.3390/genes12121891
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2527218-4
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  • 3
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    Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice
    MDPI AG ; 2022
    In:  International Journal of Molecular Sciences Vol. 23, No. 18 ( 2022-09-08), p. 10383-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 23, No. 18 ( 2022-09-08), p. 10383-
    Abstract: Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms231810383
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 4
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    Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System
    MDPI AG ; 2022
    In:  International Journal of Molecular Sciences Vol. 24, No. 1 ( 2022-12-21), p. 109-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 24, No. 1 ( 2022-12-21), p. 109-
    Abstract: Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography–tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change 〉 2 and adjusted p-value 〈 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms24010109
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2019364-6
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  • 5
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    Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice
    MDPI AG ; 2020
    In:  International Journal of Molecular Sciences Vol. 21, No. 15 ( 2020-07-31), p. 5492-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 21, No. 15 ( 2020-07-31), p. 5492-
    Abstract: The rice SLR1 gene encodes the DELLA protein (protein with DELLA amino acid motif), and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms21155492
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2019364-6
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  • 6
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    Knockout of SlMS10 Gene (Solyc02g079810) Encoding bHLH Transcription Factor Using CRISPR/Cas9 System Confers Male Sterility Phenotype in Tomato
    MDPI AG ; 2020
    In:  Plants Vol. 9, No. 9 ( 2020-09-11), p. 1189-
    Details
    In: Plants, MDPI AG, Vol. 9, No. 9 ( 2020-09-11), p. 1189-
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 2223-7747
    URL: Article
    DOI: 10.3390/plants9091189
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
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  • 7
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    Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice
    MDPI AG ; 2023
    In:  International Journal of Molecular Sciences Vol. 24, No. 6 ( 2023-03-10), p. 5338-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 24, No. 6 ( 2023-03-10), p. 5338-
    Abstract: Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2 OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein–protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms24065338
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2019364-6
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  • 8
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    Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity
    MDPI AG ; 2022
    In:  Agronomy Vol. 12, No. 1 ( 2022-01-10), p. 164-
    Details
    In: Agronomy, MDPI AG, Vol. 12, No. 1 ( 2022-01-10), p. 164-
    Abstract: Nowadays, rice production faces significant challenges due to population pressure, global climate change, and outbreak of various pests and diseases. Breeding techniques used to improve rice traits include mutant breeding, cross breeding, heterogeneity, transformation, molecular markers, genome-wide association study (GWAS), and so on. Since the recently developed CRISPR/Cas9 technology can directly target a specific part of a desired gene to induce mutation, it can be used as a powerful means to expand genetic diversity of crops and develop new varieties. So far, CRISPR/Cas9 technology has been used for improving rice characteristics such as high yield, good quality, abundant nutrition, pest and disease resistance, herbicide resistance, and biotic and abiotic stress resistance. This review highlights the mechanisms and optimization of the CRISPR system and its application to rice crop, including resistance to biotic and abiotic stresses, and improved rice quality and yield.
    Type of Medium: Online Resource
    ISSN: 2073-4395
    URL: Article
    DOI: 10.3390/agronomy12010164
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2607043-1
    SSG: 23
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  • 9
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    Development of SNP Markers from GWAS for Selecting Seed Coat and Aleurone Layers in Brown Rice (Oryza sativa L.)
    MDPI AG ; 2022
    In:  Genes Vol. 13, No. 10 ( 2022-10-06), p. 1805-
    Details
    In: Genes, MDPI AG, Vol. 13, No. 10 ( 2022-10-06), p. 1805-
    Abstract: Ninety-five percent of the general nutrients in rice are concentrated in the rice bran and germ, and many nutrients such as vitamins, minerals, dietary fiber, and essential fatty acids, as well as antioxidants such as tocopherol, are lost during milling. In this study, we investigated the thickness of seed coat and aleurone layers using a 294 rice core collection, and found candidate genes related to thickness of seed coat and aleurone layers, by performing a genome wide association study (GWAS) analysis using whole genome resequencing data. Two primer pairs that can be used as high-resolution melting (HRM) markers were developed. As a result of genotyping BC2F2 individuals derived from a cross between “Samgwang” and “Seolgaeng”, and using corresponding HRM markers, it was possible to finally develop HRM markers for selecting seed coat and aleurone layer thickness. This is expected to be used as basic data for the application of gene editing using CRISPR/Cas9 technology and for establishing a breeding strategy for high eating quality rice using molecular genetic technology.
    Type of Medium: Online Resource
    ISSN: 2073-4425
    URL: Article
    DOI: 10.3390/genes13101805
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2527218-4
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