scholarly journals Targeted Deletion of the First Intron of the Wxb Allele via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice

Rice ◽  
2022 ◽  
Vol 15 (1) ◽  
Author(s):  
Xingdan Liu ◽  
Qi Ding ◽  
Wenshu Wang ◽  
Yanling Pan ◽  
Chao Tan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cooking Quality ◽  
Amylose Content ◽  
Inbred Lines ◽  
Transcriptional Level ◽  
Wild Type ◽  
Targeted Deletion ◽  
First Intron ◽  
Eating And Cooking Quality ◽  
Mutant Lines

Abstract Background The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5′splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often harbours intermediate to high amylose contents, whereas Wxb rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content. Results CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wxb), X32 (Wxb), X35 (Wxa) and X55 (Wxlv). Deletion of the 1st intron occurred in 8.6–11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wxb allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wxa and Wxlv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed. Conclusions This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wxb allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.

scholarly journals Targeted Deletion of the Wx Gene First Intron via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice With a Wxb Allele, but Not in Rice With a Wxa Allele

2021 ◽  
Author(s):  
Xingdan Liu ◽  
Qi Ding ◽  
Wenshu Wang ◽  
Yanling Pan ◽  
Chao Tan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cooking Quality ◽  
Amylose Content ◽  
Mrna Level ◽  
Foreign Gene ◽  
Rice Grain ◽  
Wild Type ◽  
Targeted Deletion ◽  
First Intron ◽  
Eating And Cooking Quality

Abstract Background: Rice Waxy (Wx) gene plays a major role in seed amylose synthesis, and consequently controls grain amylose content. The expression of Wx gene is highly regulated at both transcriptional and post-transcriptional levels. Particularly, the GT/TT poplymorphism at the 5` splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often has intermediate to high amylose content, whereas Wxb rice has low to intermediate amylose content. A previous study indicates that rice Wx 1st intron significantly enhances gene expression when it is inserted into the 5` UTR (untranslated region) of a foreign gene. By deleting Wx 1st intron with the CRISPR/Cas9 technology, we intended to create a totally noval Wx allele, and further to investigate how the intron removal affects Wx gene expression and rice grain amylose content.Results: CRISPR/Cas9-mediated targeted deletion of Wx 1st intron was performed on 4 rice inbreds, KY131(Wxb), X32(Wxb), X35(Wxa) and X55(Wxlv). Complete deletion of the 1st intron occurred in 8.6%-11.8% of the primary transformants of these 4 inbreds. Transgene-free, homozygous mutants were obtained. Their grain amylose content and Wx gene expression were analyzed. Compared to the amylose content of wild type plants, mutants’ amylose content was significantly increased from 13.0% to about 24% in KY131 and X32 which both carried the Wxb allele. However, no significant differenece in aylose content was observed between wild type plants and mutants of X35 and X55 which carried the Wxa and Wxlv allele, respectively. Results of Wx gene expression analysis on wild type plants and mutants showed a high consistence with their amylose content results. Mutants of KY131 and X32 accumulated much more steady mRNA transcripts than their wild type plants, while steady mRNA level remained somehow unchanged between wild type plants and mutants of X35 and X55. Grain quality including appearance quality and ECQ(eating and cooking quality) that are tightly linked to amylose content was also evalued on wild type plants and mutants, and data were presented and analyzed. Conclusions:This study presents a novel and fast strategy to increase amylose content for rice inbreds carrying a Wxb allele. Our data strongly suggest that rice Wx 1st intron regulates Wx gene expression mainly at the post-transcriptinal level, not as previously thought that it influences Wx gene transcription as well. In addition, removal of the first intron creates a completely noval Wx allele. Further studies on this new Wx allele would provide invaluable insights into the regulation of Wx gene expression, which will help researchers to engineer more new alleles that leads to the breeding of rice cultivars with better eating and cooking quality.

Abstract 531: Role of Rna Splicing In Mediating Lineage-specific Expression of the Von Willebrand Factor Gene in the Endothelium

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Lei Yuan ◽  
Lauren Janes ◽  
David Beeler ◽  
Katherine C Spokes ◽  
Joshua Smith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Rna Splicing ◽  
Coagulation Factor ◽  
Transcriptional Level ◽  
Specific Expression ◽  
First Intron ◽  
Von Willebrand ◽  
Willebrand Factor

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we employed transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human beta-globin, mouse DSCR-1 or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells, but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a post-transcriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

scholarly journals Adaptation of Sucrose Metabolism in the Escherichia coli Wild-Type Strain EC3132†

2002 ◽  
Vol 184 (19) ◽  
pp. 5307-5316 ◽  
Author(s):  
Knut Jahreis ◽  
Lars Bentler ◽  
Jürgen Bockmann ◽  
Stephan Hans ◽  
Astrid Meyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Hot Spot ◽  
Chromosomal Rearrangements ◽  
Enteric Bacteria ◽  
Transcriptional Level ◽  
Wild Type ◽  
New Host ◽  
E Coli ◽  
K 12

ABSTRACT Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.

scholarly journals Effect of indica pedigree on eating and cooking quality in rice backcross inbred lines of indica and japonica crosses

2017 ◽  
Vol 67 (5) ◽  
pp. 450-458 ◽  
Author(s):  
Mingyu Fan ◽  
Xiaojing Wang ◽  
Jian Sun ◽  
Qun Zhang ◽  
Zhengjin Xu ◽  
...  
Keyword(s):  
Cooking Quality ◽  
Inbred Lines ◽  
Backcross Inbred Lines ◽  
Eating And Cooking Quality

scholarly journals Subtle perturbations of the maize methylome reveal genes and transposons silenced by DNA methylation

2017 ◽  
Author(s):  
Sarah N. Anderson ◽  
Greg Zynda ◽  
Jawon Song ◽  
Zhaoxue Han ◽  
Matthew Vaughn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Zea Mays ◽  
Natural Variation ◽  
Chromatin Modification ◽  
Inbred Lines ◽  
Specific Sequence ◽  
Number Of Genes ◽  
Context Specific ◽  
Mutant Lines

ABSTRACTDNA methylation is a chromatin modification that can provide epigenetic regulation of gene and transposon expression. Plants utilize several pathways to establish and maintain DNA methylation in specific sequence contexts. The chromomethylase (CMT) genes maintain CHG (where H = A, C or T) methylation. The RNA-directed DNA methylation (RdDM) pathway is important for CHH methylation. Transcriptome analysis was performed in a collection of Zea mays lines carrying mutant alleles for CMT or RdDM-associated genes. While the majority of the transcriptome was not affected, we identified sets of genes and transposon families sensitive to context-specific decreases in DNA methylation in mutant lines. Many of the genes that are up-regulated in CMT mutant lines have high levels of CHG methylation, while genes that are differentially expressed in RdDM mutants are enriched for having nearby mCHH islands, providing evidence that context-specific DNA methylation directly regulates expression of a small number of genes. The analysis of a diverse set of inbred lines revealed that many genes regulated by CMTs exhibit natural variation for DNA methylation and gene expression. Transposon families with differential expression in the mutant genotypes show few defining features, though several families up-regulated in RdDM mutants show enriched expression in endosperm, highlighting the importance for this pathway during reproduction. Taken together, our findings suggest that while the number of genes and transposon families whose expression is reproducibly affected by mild perturbations in context-specific methylation is small, there are distinct patterns for loci impacted by RdDM and CMT mutants.

scholarly journals LacI-Family Transcriptional Regulator DagR Acts as a Repressor of the Agarolytic Pathway Genes in Streptomyces coelicolor A3(2)

2021 ◽  
Vol 12 ◽  
Author(s):  
Maral Tsevelkhoroloo ◽  
So Heon Shim ◽  
Chang-Ro Lee ◽  
Soon-Kwang Hong ◽  
Young-Soo Hong
Keyword(s):  
Gene Expression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Degradation Products ◽  
Mrna Level ◽  
Regulation Of Gene Expression ◽  
Hydrolytic Enzymes ◽  
Upstream Region ◽  
Transcriptional Level ◽  
Wild Type

Actinobacteria utilize various polysaccharides in the soil as carbon source by degrading them via extracellular hydrolytic enzymes. Agarose, a marine algal polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG), is one of the carbon sources used by S. coelicolor A3(2). However, little is known about agar hydrolysis in S. coelicolor A3(2), except that the regulation of agar hydrolysis metabolism is strongly inhibited by glucose as in the catabolic pathways of other polysaccharides. In this study, we elucidated the role of DagR in regulating the expression of three agarase genes (dagA, dagB, and dagC) in S. coelicolor A3(2) by developing a dagR-deletion mutant (Δsco3485). We observed that the Δsco3485 mutant had increased mRNA level of the agarolytic pathway genes and 1.3-folds higher agarase production than the wild type strain, indicating that the dagR gene encodes a cluster-suited repressor. Electrophoretic mobility shift assay revealed that DagR bound to the upstream regions of the three agarase genes. DNase 1 footprinting analysis demonstrated that a palindromic sequence present in the upstream region of the three agarase genes was essential for DagR-binding. Uniquely, the DNA-binding activity of DagR was inhibited by AHG, one of the final degradation products of agarose. AHG-induced agarase production was not observed in the Δsco3485 mutant, as opposed to that in the wild type strain. Therefore, DagR acts as a repressor that binds to the promoter region of the agarase genes, inhibits gene expression at the transcriptional level, and is derepressed by AHG. This is the first report on the regulation of gene expression regarding agar metabolism in S. coelicolor A3(2).

scholarly journals Rac2, a Hematopoiesis-Specific Rho GTPase, Specifically Regulates Mast Cell Protease Gene Expression in Bone Marrow-Derived Mast Cells

2002 ◽  
Vol 22 (21) ◽  
pp. 7645-7657 ◽  
Author(s):  
Yi Gu ◽  
Michael C. Byrne ◽  
Nivanka C. Paranavitana ◽  
Bruce Aronow ◽  
Jamie E. Siefring ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Gene Transcription ◽  
Rho Gtpase ◽  
Regulation Of Gene Expression ◽  
Transcriptional Level ◽  
Wild Type ◽  
Mast Cell Protease

ABSTRACT Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-κB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2−/− bone marrow-derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2−/− mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2-deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-κB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2−/− mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases.

CARP Plays a Key Role in Cellular Growth Under Hypertrophic Stimuli in Neonatal Rat Ventricular Myocytes

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Ventricular Myocytes ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Dual Nature ◽  
Hypertrophic Growth ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes ◽  
Over Time

Cardiac hypertrophy is a growth process that occurs in response to stress stimuli or injury, and leads to the induction of several pathways to alter gene expression. Under hypertrophic stimuli, sarcomeric structure is disrupted, both as a consequence of gene expression and local changes in sarcomeric proteins. Cardiac-restricted ankyrin repeat protein (CARP) is one such protein that function both in cardiac sarcomeres and at the transcriptional level. We postulate that due to this dual nature, CARP plays a key role in maintaining the cardiac sarcomere. GATA4 is another protein detected in cardiomyocytes as important in hypertrophy, as it is activated by hypertrophic stimuli, and directly binds to DNA to alter gene expression. Results of GATA4 activation over time were inconclusive; however, the role of CARP in mediating hypertrophic growth in cardiomyocytes was clearly demonstrated. In this study, Neonatal Rat Ventricular Myocytes were used as a model to detect changes over time in CARP and GATA4 under hypertrophic stimulation by phenylephrine and high serum media. Results were detected by analysis of immunoblotting. The specific role that CARP plays in mediating cellular growth under hypertrophic stimuli was studied through immunofluorescence, which demonstrated that cardiomyocyte growth with hypertrophic stimulation was significantly blunted when NRVMs were co-treated with CARP siRNA. These data suggest that CARP plays an important role in the hypertrophic response in cardiomyocytes.

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