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1.
J Zhejiang Univ Sci B ; 21(6): 460-473, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32478492

RESUMO

Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.

2.
J Vis Exp ; (151)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31524866

RESUMO

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

3.
Genomics ; 111(2): 142-158, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29476784

RESUMO

Tobacco (Nicotiana tabacum) serve as the top leading commercial, non-food, and model crop worldwide. Cyclic nucleotide-gated channels (CNGCs) are ligand-gated, calcium-permeable, divalent, cation-selective channels, involved in important biological functions. Here, we systematically characterized thirty-five CNGC genes in the genome of Nicotiana tabacum, and classified into four phylogenetic groups. Evolutionary analysis showed that NtabCNGC family of N. tabacum originated from the parental genome of N. sylvestris and N. tomentosiformis, and further expanded via tandem and segmental duplication events. Tissue-specific expression analysis showed that twenty-three NtabCNGC genes are involved in the development of various tobacco tissues. Subsequent RT-qPCR analyses indicated that these genes are sensitive towards external abiotic and biotic stresses. Notable performances were exhibited by group-I and IV CNGC genes against black shank, Cucumber mosaic virus, Potato virus Y, cold, drought, and cadmium stresses. Our analyses also suggested that NtabCNGCs can be regulated by phosphorylation and miRNAs, and multiple light, temperature, and pathogen-responsive cis-acting regulatory elements present in promotors. These results will be useful for elaborating the biological roles of NtabCNGCs in tobacco growth and development.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Evolução Molecular , Proteínas de Plantas/genética , Tabaco/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Genoma de Planta , Proteínas de Plantas/metabolismo
4.
J Zhejiang Univ Sci B ; 19(8): 620-629, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30070085

RESUMO

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.


Assuntos
Mutação INDEL , Mutagênese , Oryza/genética , Produtos Agrícolas/genética , Produtos Agrícolas/efeitos da radiação , Raios gama , Técnicas Genéticas , Genoma de Planta , Homozigoto , Oryza/efeitos da radiação , Melhoramento Vegetal , Reação em Cadeia da Polimerase , Sementes , Análise de Sequência de DNA , Deleção de Sequência
5.
Nat Plants ; 4(6): 338-344, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29735983

RESUMO

Rice is one of the world's most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.


Assuntos
Oryza/metabolismo , Serotonina/metabolismo , Animais , Regulação da Expressão Gênica de Plantas , Hemípteros , Herbivoria , Mariposas , Oryza/fisiologia , Reguladores de Crescimento de Planta/metabolismo , Ácido Salicílico/metabolismo
6.
BMC Genomics ; 18(1): 869, 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-29132315

RESUMO

BACKGROUND: The cyclic nucleotide-gated ion channel (CNGC) family affects the uptake of cations, growth, pathogen defence, and thermotolerance in plants. However, the systematic identification, origin and function of this gene family has not been performed in Brassica oleracea, an important vegetable crop and genomic model organism. RESULTS: In present study, we identified 26 CNGC genes in B. oleracea genome, which are non-randomly localized on eight chromosomes, and classified into four major (I-IV) and two sub-groups (i.e., IV-a and IV-b). The BoCNGC family is asymmetrically fractioned into the following three sub-genomes: least fractionated (14 genes), most fractionated-I (10), and most fractionated-II (2). The syntenic map of BoCNGC genes exhibited strong relationships with the model Arabidopsis thaliana and B. rapa CNGC genes and provided markers for defining the regions of conserved synteny among the three genomes. Both whole-genome triplication along with segmental and tandem duplications contributed to the expansion of this gene family. We predicted the characteristics of BoCNGCs regarding exon-intron organisations, motif compositions and post-translational modifications, which diversified their structures and functions. Using orthologous Arabidopsis CNGCs as a reference, we found that most CNGCs were associated with various protein-protein interaction networks involving CNGCs and other signalling and stress related proteins. We revealed that five microRNAs (i.e., bol-miR5021, bol-miR838d, bol-miR414b, bol-miR4234, and bol-miR_new2) have target sites in nine BoCNGC genes. The BoCNGC genes were differentially expressed in seven B. oleracea tissues including leaf, stem, callus, silique, bud, root and flower. The transcript abundance levels quantified by qRT-PCR assays revealed that BoCNGC genes from phylogenetic Groups I and IV were particularly sensitive to cold stress and infections with bacterial pathogen Xanthomonas campestris pv. campestris, suggesting their importance in abiotic and biotic stress responses. CONCLUSION: Our comprehensive genome-wide analysis represents a rich data resource for studying new plant gene families. Our data may also be useful for breeding new B. oleracea cultivars with improved productivity, quality, and stress resistance.


Assuntos
Brassica/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Genômica , Proteínas de Plantas/genética , Sintenia , Sequência de Aminoácidos , Brassica/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Evolução Molecular , Duplicação Gênica , Ontologia Genética , Filogenia , Proteínas de Plantas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
8.
Front Plant Sci ; 8: 901, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28620402

RESUMO

The xantha marker trait, which is controlled by a down-regulating epi-mutation of OsGUN4, has been applied to the production of hybrid rice. However, the molecular basis for the ability of xantha mutants to attain high photosynthetic capacity even with decreased chlorophyll contents has not been characterized. In the present study, we observed that the total chlorophyll content of the xantha mutant was only 27.2% of that of the wild-type (WT) plants. However, the xantha mutant still accumulated 59.9% of the WT δ-aminolevulinic acid content, 72.8% of the WT Mg-protoporphyrin IX content, and 63.0% of the WT protochlorophyllide a content. Additionally, the protoporphyrin IX and heme contents in the mutant increased to 155.0 and 160.0%, respectively, of the WT levels. A search for homologs resulted in the identification of 124 rice genes involved in tetrapyrrole biosynthesis and photosynthesis. With the exception of OsGUN4, OsHO-1, and OsHO-2, the expression levels of the genes involved in tetrapyrrole biosynthesis were significantly higher in the xantha mutant than in the WT plants, as were all 72 photosynthesis-associated nuclear genes. In contrast, there were no differences between the xantha mutant and WT plants regarding the expression of all 22 photosynthesis-associated chloroplast genes. Furthermore, the abundance of 1O2 and the expression levels of 1O2-related genes were lower in the xantha mutant than in the WT plants, indicating 1O2-mediated retrograde signaling was repressed in the mutant plants. These results suggested that the abundance of protoporphyrin IX used for chlorophyll synthesis decreased in the mutant, which ultimately decreased the amount of chlorophyll in the xantha mutant. Additionally, the up-regulated expression of photosynthesis-associated nuclear genes enabled the mutant to attain a high photosynthetic capacity. Our findings confirm that OsGUN4 plays an important role in tetrapyrrole biosynthesis and photosynthesis in rice. GUN4, chlorophyll synthesis pathways, and photosynthetic activities are highly conserved in plants and hence, novel traits (e.g., xantha marker trait) may be generated in other cereal crops by modifying the GUN4 gene.

9.
J Zhejiang Univ Sci B ; 17(12): 905-915, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27921396

RESUMO

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10-6-9.8×10-6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.


Assuntos
Raios gama , Mutação , Oryza/genética , Cruzamento , Produtos Agrícolas/genética , Variações do Número de Cópias de DNA , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala
10.
J Zhejiang Univ Sci B ; 17(12): 992-996, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27921404

RESUMO

Agrobacterium-mediated transformation has been widely used in producing transgenic plants, and was recently used to generate "transgene-clean" targeted genomic modifications coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Although tremendous variation in morphological and agronomic traits, such as plant height, seed fertility, and grain size, was observed in transgenic plants, the underlying mechanisms are not yet well understood, and the types and frequency of genetic variation in transformed plants have not been fully disclosed. To reveal the genome-wide variation in transformed plants, we sequenced the genomes of five independent T0 rice plants using next-generation sequencing (NGS) techniques. Bioinformatics analyses followed by experimental validation revealed the following: (1) in addition to transfer-DNA (T-DNA) insertions, three transformed plants carried heritable plasmid backbone DNA of variable sizes (855-5216 bp) and in different configurations with the T-DNA insertions (linked or apart); (2) each transgenic plant contained an estimated 338-1774 independent genetic variations (single nucleotide variations (SNVs) or small insertion/deletions); and (3) 2-6 new Tos17 insertions were detected in each transformed plant, but no other transposable elements or bacterial genomic DNA.


Assuntos
Agrobacterium/genética , Oryza/genética , Biologia Computacional , Elementos de DNA Transponíveis , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Plantas Geneticamente Modificadas , Transformação Bacteriana
11.
Breed Sci ; 66(4): 552-559, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27795680

RESUMO

Doubled haploid (DH) populations, particularly those from subspecies crosses possessing the wide compatible gene S5n , are important germplasm resources for rice genetic studies and breeding, but their feature and potential have not been fully assessed and explored. In the present study, we produced a DH population from the hybrid of japonica 668B and wide compatible indica T23. Genotyping of the S5 locus with allele-specific markers for ORF3, ORF4 and ORF5 revealed a potential recombination hot spot in the ORF3-ORF4 region. Haplotyping analysis revealed that 21/34 subspecies specific Indel markers segregated in distortion in the DH population, with a few lines having indica alleles either extremely low (1.7%) or high (98.3%), with little effect of the S5 allele. While DH lines with the S5n allele had higher frequency of indica alleles, no effect of the S5n allele was observed on all agronomic traits but flowering time. Taken together, the present study advanced understanding of the genetics of wide crosses in general, and DH production in particular between the two rice subspecies, and the new DH population generated will become a useful resource for rice genetic study and breeding in the future.

12.
J Zhejiang Univ Sci B ; 17(2): 100-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26834011

RESUMO

The OsLpa1 gene (LOC_Os57400) was identified to be involved in phytic acid (PA) metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter::GUS transgenic plants (GUS: ß-glucuronidase); GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts (OsLpa1.1, OsLpa1.2, OsLpa1.3) are produced due to alternative splicing; quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa all tissues. OsLpa1.2 is predominant in germinating seeds (about 5 times that of OsLpa1.1), but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF (day after flowering) seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein (GFP) in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.


Assuntos
Processamento Alternativo/fisiologia , Oryza/fisiologia , Ácido Fítico/metabolismo , Proteínas de Plantas/fisiologia , Frações Subcelulares/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/ultraestrutura , Plantas Geneticamente Modificadas/fisiologia , Análise Espaço-Temporal , Frações Subcelulares/ultraestrutura , Distribuição Tecidual
13.
J Agric Food Chem ; 63(36): 8008-16, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26317332

RESUMO

An association mapping of quantitative trait loci (QTLs) regulating the concentrations of eight elements in brown rice (Oryza sativa L.) was performed using USDA mini-core subset cultivated in two different environments. In addition, correlation between the grain elemental concentrations was also studied. A total of 60 marker loci associated with 8 grain elemental concentrations were identified, and these loci were clustered into 37 genomic regions. Twenty new QTLs were found to be associated with important elements such as Zn, Fe, and P, along with others. Fe concentration was associated with the greatest number of markers in two environments. In addition, several important elemental/metal transporter genes were identified in a few mapped regions. Positive correlation was observed within all grain elemental concentrations. In summary, the results provide insight into the genetic basis of rice grain element accumulation and may help in the identification of genes associated with the accumulation of Zn, Fe, and other essential elements in rice.


Assuntos
Genoma de Planta , Oryza/genética , Locos de Características Quantitativas , Mapeamento Cromossômico , Cromossomos de Plantas , Estudo de Associação Genômica Ampla , Oryza/química , Sementes/química , Sementes/genética
14.
J Zhejiang Univ Sci B ; 16(2): 113-22, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25644466

RESUMO

The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Herbicidas/farmacologia , Oryza/enzimologia , Frações Subcelulares/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Distribuição Tecidual
15.
BMC Genomics ; 15: 853, 2014 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-25280591

RESUMO

BACKGROUND: Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable cation transport channels, which are present in both animal and plant systems. They have been implicated in the uptake of both essential and toxic cations, Ca2+ signaling, pathogen defense, and thermotolerance in plants. To date there has not been a genome-wide overview of the CNGC gene family in any economically important crop, including rice (Oryza sativa L.). There is an urgent need for a thorough genome-wide analysis and experimental verification of this gene family in rice. RESULTS: In this study, a total of 16 full length rice CNGC genes distributed on chromosomes 1-6, 9 and 12, were identified by employing comprehensive bioinformatics analyses. Based on phylogeny, the family of OsCNGCs was classified into four major groups (I-IV) and two sub-groups (IV-A and IV- B). Likewise, the CNGCs from all plant lineages clustered into four groups (I-IV), where group II was conserved in all land plants. Gene duplication analysis revealed that both chromosomal segmentation (OsCNGC1 and 2, 10 and 11, 15 and 16) and tandem duplications (OsCNGC1 and 2) significantly contributed to the expansion of this gene family. Motif composition and protein sequence analysis revealed that the CNGC specific domain "cyclic nucleotide-binding domain (CNBD)" comprises a "phosphate binding cassette" (PBC) and a "hinge" region that is highly conserved among the OsCNGCs. In addition, OsCNGC proteins also contain various other functional motifs and post-translational modification sites. We successively built a stringent motif: (LI-X(2)-[GS]-X-[FV]-X-G-[1]-ELL-X-W-X(12,22)-SA-X(2)-T-X(7)-[EQ]-AF-X-L) that recognizes the rice CNGCs specifically. Prediction of cis-acting regulatory elements in 5' upstream sequences and expression analyses through quantitative qPCR demonstrated that OsCNGC genes were highly responsive to multiple stimuli including hormonal (abscisic acid, indoleacetic acid, kinetin and ethylene), biotic (Pseudomonas fuscovaginae and Xanthomonas oryzae pv. oryzae) and abiotic (cold) stress. CONCLUSIONS: There are 16 CNGC genes in rice, which were probably expanded through chromosomal segmentation and tandem duplications and comprise a PBC and a "hinge" region in the CNBD domain, featured by a stringent motif. The various cis-acting regulatory elements in the upstream sequences may be responsible for responding to multiple stimuli, including hormonal, biotic and abiotic stresses.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Oryza/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromossomos de Plantas , Biologia Computacional , Canais de Cátion Regulados por Nucleotídeos Cíclicos/classificação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Duplicação Gênica , Dados de Sequência Molecular , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Elementos Reguladores de Transcrição , Plântula/metabolismo , Alinhamento de Sequência , Transcriptoma
16.
Theor Appl Genet ; 127(11): 2491-501, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25208645

RESUMO

KEY MESSAGE: A γ-ray-induced xantha trait is epigenetically controlled by the genomes uncoupled 4 gene with enhanced promoter segment methylation and down-regulated expression in rice. For easy testing and to increase varietal purity, a xantha mutation (xnt), which turns plants yellow and makes them visually distinguishable from normal green rice, has been generated and bred into male sterile lines for hybrid rice production. The xnt locus was previously fine mapped to a ~100-kb interval on chromosome 11, but its identity was unknown. In this study, xnt was further narrowed down to a 57-kb fragment carrying eight opening reading frames (ORFs). All eight ORFs had identical genomic sequences and all but ORF2 (g enomes uncoupled 4, OsGUN4) had similar transcript abundance in the xantha mutant Huangyu B (HYB) and its parental variety Longtefu B (LTB). The expression of OsGUN4, however, was significantly reduced in HYB compared with LTB in terms of both transcript abundance (0.2% that of LTB) and expressed protein level (barely detectable in HYB but greater than the heat shock protein reference in LTB). Therefore, OsGUN4 was identified as the candidate gene underlying the xantha trait. The function of OsGUN4 in the xantha phenotype was confirmed by identification and characterization of new allelic OsGUN4 mutations. Comparative bisulfite genomic sequencing of OsGUN4 revealed increased methylation in a promoter region in the mutant, and the correlation between increased methylation and the xantha phenotype was further verified by demethylation treatment. In summary, we have identified an epi-allele of OsGUN4 as the causal gene of the xantha marker trait and revealed that enhanced methylation in its promoter down-regulated its expression in rice.


Assuntos
Epigênese Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oryza/genética , Proteínas de Plantas/genética , Alelos , Carotenoides/metabolismo , Clorofila/metabolismo , Mapeamento Cromossômico , Metilação de DNA , Análise Mutacional de DNA , DNA de Plantas/genética , Regulação para Baixo , Raios gama , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos , Fases de Leitura Aberta , Fenótipo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas
17.
Transgenic Res ; 23(4): 585-99, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24648215

RESUMO

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.


Assuntos
Germinação/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Oryza/metabolismo , Ácido Fítico/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Peso Corporal , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Fósforo/análise , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sementes/química
18.
Rice (N Y) ; 7(1): 13, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26055995

RESUMO

The exploitation of male sterility systems has enabled the commercialization of heterosis in rice, with greatly increased yield and total production of this major staple food crop. Hybrid rice, which was adopted in the 1970s, now covers nearly 13.6 million hectares each year in China alone. Various types of cytoplasmic male sterility (CMS) and environment-conditioned genic male sterility (EGMS) systems have been applied in hybrid rice production. In this paper, recent advances in genetics, biochemistry, and molecular biology are reviewed with an emphasis on major male sterility systems in rice: five CMS systems, i.e., BT-, HL-, WA-, LD- and CW- CMS, and two EGMS systems, i.e., photoperiod- and temperature-sensitive genic male sterility (P/TGMS). The interaction of chimeric mitochondrial genes with nuclear genes causes CMS, which may be restored by restorer of fertility (Rf) genes. The PGMS, on the other hand, is conditioned by a non-coding RNA gene. A survey of the various CMS and EGMS lines used in hybrid rice production over the past three decades shows that the two-line system utilizing EGMS lines is playing a steadily larger role and TGMS lines predominate the current two-line system for hybrid rice production. The findings and experience gained during development and application of, and research on male sterility in rice not only advanced our understanding but also shed light on applications to other crops.

19.
Theor Appl Genet ; 126(12): 3009-20, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24042572

RESUMO

The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding.


Assuntos
Rearranjo Gênico , Mutação/genética , Oryza/genética , Ácido Fítico/metabolismo , Proteínas de Plantas/genética , Retroelementos/genética , Southern Blotting , Western Blotting , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Metilação de DNA , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Mutagênese Insercional , Oryza/metabolismo , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Theor Appl Genet ; 125(7): 1413-23, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22733447

RESUMO

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.


Assuntos
Éxons/genética , Mutação/genética , Ácido Fítico/metabolismo , Sítios de Splice de RNA/genética , Homologia de Sequência de Aminoácidos , Soja/enzimologia , Soja/genética , Sequência de Bases , Cruzamentos Genéticos , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Homozigoto , Dados de Sequência Molecular , Fenótipo , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Mapeamento Físico do Cromossomo , Sementes/genética , Sementes/metabolismo , Soja/embriologia , Transcrição Genética
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