Dataset Information

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

ABSTRACT: In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.

SUBMITTER: Nawaz G

PROVIDER: S-EPMC7504442 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

Nawaz Gul G Usman Babar B Zhao Neng N Han Yue Y Li Zhihua Z Wang Xin X Liu Yaoguang Y Li Rongbai R

International journal of molecular sciences 20200826 17

In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated t ...[more]

PMID: 32859098

Similar Datasets

Project description:BackgroundRice plant growth is comprised of distinct phases, such as vegetative, reproductive, grain filling and maturity phases. In these phases synthesis and availability of primary and secondary metabolites including volatile organic compounds (VOC's) is highly variable. In scented rice, aroma volatiles are synthesized in aerial plant parts and deposited in mature grains. There are more than 100 VOCs reported to be responsible for flavor in basmati rice. It will be interesting to keep track of aroma volatiles across the developmental stages in scented rice. Therefore, the aroma volatiles contributing in aroma with special reference to the major compound 2 acetyl-1-pyrroline (2AP) were screened at seven developmental stages in scented rice cultivars Basmati-370 and Ambemohar-157 along with non-scented rice cultivar IR-64 as a control following HS-SPME-GC-MS method. In addition, the expression levels of key genes and precursor levels involved in 2AP biosynthesis were studied.ResultsThe study indicated that volatilome of scented rice cultivars is more complex than non-scented rice cultivar. N-heterocyclic class was the major distinguishing class between scented from non-scented rice. A total of 14 compounds including, 2AP were detected specifically in scented rice cultivars. Maximum number of compounds were synthesized at seedling stage and decreased gradually at reproductive and maturity. The seedling stage is an active phase of development where maximum number green leaf volatiles were synthesized which are known to act as defense molecules for protection of young plant parts. Among the 14 odor active compounds (OACs), 10 OACs were accumulated at higher concentrations significantly in scented rice cultivars and contribute in the aroma. 2AP content was highest in mature grains followed by at booting stage. Gene expression analysis revealed that reduced expression of betaine aldehyde dehydrogenase 2 (badh2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elevated level of triose phosphate isomerase (TPI) and Δ1-Pyrolline-5-carboxylic acid synthetase (P5CS) transcript enhances 2AP accumulation.ConclusionsMost diverse compounds were synthesized at seedling stage and OACs were accumulated more at flowering followed by seedling stage. Distinct accumulation pattern exists for 2AP and other aroma volatiles at various developmental stages. The study revealed the mechanism of 2AP accumulation such that 2AP in mature grains might be transported from leaves and stem sheath and accumulation takes place in grains.

Project description:Basmati is considered a unique varietal group of rice (Oryza sativa L.) because of its aroma and superior grain quality. Previous genetic analyses of rice showed that most of the Basmati varieties are classified into the aromatic group. Despite various efforts, genomic relationship of Basmati rice with other varietal groups and genomic variation in Basmati rice are yet to be understood. In the present study, we resequenced the whole genome of three traditional Basmati varieties at a coverage of more than 25X using Illumina HiSeq2500 and mapped the obtained sequences to the reference genome sequences of Nipponbare (japonica rice), Kasalath (aus rice), and Zhenshan 97 (indica rice). Comparison of these sequences revealed common single nucleotide polymorphisms (SNPs) in the genic regions of three Basmati varieties. Analysis of these SNPs revealed that Basmati varieties showed fewer sequence variations compared with the aus group than with the japonica and indica groups. Gene ontology (GO) enrichment analysis indicated that SNPs were present in genes with various biological, molecular, and cellular functions. Additionally, functional annotation of the Basmati mutated gene cluster shared by Nipponbare, Kasalath, and Zhenshan 97 was found to be associated with the metabolic process involved in the cellular aromatic compound, suggesting that aroma is an important specific genomic feature of Basmati varieties. Furthermore, 30 traditional Basmati varieties were classified into three different groups, aromatic (22 varieties), aus (four varieties), and indica (four varieties), based on genome-wide SNPs. All 22 aromatic Basmati varieties harbored the fragrant-inducing Badh2 allele. We also performed comparative analysis of 13 key agronomic and grain quality traits of Basmati rice and other rice varieties. Three traits including length-to-width ratio of grain (L/W ratio), panicle length (PL), and amylose content (AC) showed significant (P < 0.05 and P < 0.01) differences between the aromatic and indica/aus groups. Comparative analysis of genome structure, based on genome sequence variation and GO analysis, revealed that the Basmati genome was derived mostly from the aus and japonica groups. Overall, whole-genome sequence data and genetic diversity information obtained in this study will serve as an important resource for molecular breeding and genetic analysis of Basmati varieties.

Project description:Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

Dataset Information

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

Publications

CRISPR/Cas9 Directed Mutagenesis of <i>OsGA20ox2</i> in High Yielding Basmati Rice (<i>Oryza sativa</i> L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets