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1.
Plant Biotechnol J ; 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33236499

RESUMO

Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat (Triticum aestivum L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high-amylose wheat through targeted mutagenesis of TaSBEIIa in a modern winter wheat cv Zhengmai 7698 (ZM) and a spring wheat cv Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene-free mutant lines either with partial or triple null TasbeIIa alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple null alleles were dosage dependent with triple null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin, and physiochemical and nutritional properties. The flours of triple null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high-amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of TaSBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties, and generated transgene-free high-amylose wheat. Our finding provides deep insights on the role of TaSBEIIa in determining starch composition, structure, properties and end-use quality in different genetic backgrounds, and improving RS content with multiple breeding and end-use applications in cereal crop species through genome editing for health benefits.

2.
Mol Plant ; 13(6): 811-813, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32353627
5.
Mol Plant Pathol ; 20(10): 1463-1474, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31273916

RESUMO

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.


Assuntos
Vírus de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/virologia , Mutagênese , Vírus de Plantas/fisiologia , RNA Guia/metabolismo , Triticum/metabolismo , Triticum/virologia , Zea mays/metabolismo , Zea mays/virologia
7.
Nat Biotechnol ; 37(4): 445-450, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886437

RESUMO

One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.


Assuntos
Marcação de Genes/métodos , Genes de Plantas , Recombinação Homóloga , Oryza/genética , Acetolactato Sintase/genética , Sequência de Bases , Biotecnologia , Sistemas CRISPR-Cas , DNA de Plantas/genética , Mutação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Proteínas Recombinantes/genética , Reparo de DNA por Recombinação , Moldes Genéticos
9.
J Exp Bot ; 69(20): 4715-4721, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-29955893

RESUMO

The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.


Assuntos
Quebras de DNA de Cadeia Dupla , Edição de Genes , Oryza/genética , Reparo de DNA por Recombinação/genética , Sistemas CRISPR-Cas
11.
J Integr Plant Biol ; 60(7): 536-540, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29575650

RESUMO

Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.


Assuntos
Alelos , Edição de Genes , Genes de Plantas , Oryza/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , RNA Guia/metabolismo
12.
Front Plant Sci ; 8: 298, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28326091

RESUMO

Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use CRISPR/Cas9 technology to generate targeted mutagenesis in SBEI and SBEIIb in rice. The frequencies of obtained homozygous or bi-allelic mutant lines with indels in SBEI and SBEIIb in T0 generation were from 26.7 to 40%. Mutations in the homozygous T0 lines stably transmitted to the T1 generation and those in the bi-allelic lines segregated in a Mendelian fashion. Transgene-free plants carrying only the frame-shifted mutagenesis were recovered in T1 generation following segregation. Whereas no obvious differences were observed between the sbeI mutants and wild type, sbeII mutants showed higher proportion of long chains presented in debranched amylopectin, significantly increased AC and RS content to as higher as 25.0 and 9.8%, respectively, and thus altered fine structure and nutritional properties of starch. Taken together, our results demonstrated for the first time the feasibility to create high-amylose rice through CRISPR/Cas9-mediated editing of SBEIIb.

14.
Int J Mol Sci ; 17(12)2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27983619

RESUMO

RNA interference (RNAi) has been widely used in functional genomics of insects and received intensive attention in the development of RNAi-based plants for insect control. Ecdysone receptor (EcR) and ultraspiracle protein (USP) play important roles in molting, metamorphosis, and reproduction of insects. EcR and USP orthologs and their function in grain aphid (Sitobion avenae F.) have not been documented yet. Here, RT-PCR, qRT-PCR, dsRNA feeding assay and aphid bioassay were employed to isolate EcR and USP orthologs in grain aphid, investigate their expression patterns, and evaluate the effect of RNAi on aphid survival and fecundity, and its persistence. The results indicated that SaEcR and SaUSP exhibited similar expression profiles at different developmental stages. Oral administration of dsRNAs of SaEcR and dsSaUSP significantly decreased the survival of aphids due to the down-regulation of these two genes, respectively. The silencing effect was persistent and transgenerational, as demonstrated by the reduced survival and fecundity due to knock-down of SaEcR and SaUSP in both the surviving aphids and their offspring, even after switching to aphid-susceptible wheat plants. Taken together, our results demonstrate that SaEcR and SaUSP are essential genes in aphid growth and development, and could be used as RNAi targets for wheat aphid control.


Assuntos
Afídeos/genética , Fertilidade/genética , Genes de Insetos , Herbivoria/genética , Proteínas de Insetos/genética , Interferência de RNA , Receptores de Esteroides/genética , Triticum/parasitologia , Animais , Afídeos/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Filogenia , RNA de Cadeia Dupla/metabolismo , Receptores de Esteroides/metabolismo
15.
Front Plant Sci ; 7: 1324, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27708648

RESUMO

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

16.
Pest Manag Sci ; 72(6): 1090-8, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26888776

RESUMO

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant-mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant-mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant-mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid-resistant plants through plant-mediated RNAi strategy. © 2016 Society of Chemical Industry.


Assuntos
Afídeos , Produtos Agrícolas , Controle de Insetos/métodos , Plantas Geneticamente Modificadas , Interferência de RNA , Animais , Produtos Agrícolas/genética , Produtos Agrícolas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/parasitologia
18.
Front Plant Sci ; 7: 1928, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28066481

RESUMO

Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes' encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired "safe" harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant cells. We then address the challenges and propose future perspectives in order to facilitate the implementation of precise genome modification through SSNs-mediated GT for crop improvement in a global context.

19.
New Phytol ; 206(3): 1101-15, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25644034

RESUMO

Aphids are important pests of wheat (Triticum aestivum) that affect crop production globally. Herbivore-induced emission of sesquiterpenes can repel pests, and farnesyl pyrophosphate synthase (FPS) is a key enzyme involved in sesquiterpene biosynthesis. However, fps orthologues in wheat and their functional roles in sesquiterpene synthesis and defence against aphid infestation are unknown. Here, two fps isoforms, Tafps1 and Tafps2, were identified in wheat. Quantitative real-time polymerase chain reaction (qRT-PCR) and in vitro catalytic activity analyses were conducted to investigate expression patterns and activity. Heterologous expression of these isoforms in Arabidopsis thaliana, virus-induced gene silencing (VIGS) in wheat and aphid behavioural assays were performed to understand the functional roles of these two isoforms. We demonstrated that Tafps1 and Tafps2 played different roles in induced responses to aphid infestation and in sesquiterpene synthesis. Heterologous expression in A. thaliana resulted in repulsion of the peach aphid (Myzus persicae). Wheat plants with these two isoforms transiently silenced were significantly attractive to grain aphid (Sitobion avenae). Our results provide new insights into induced defence against aphid herbivory in wheat, in particular, the different roles of the two Tafps isoforms in both sesquiterpene biosynthesis and defence against aphid infestation.


Assuntos
Afídeos/fisiologia , Geraniltranstransferase/química , Sesquiterpenos/metabolismo , Triticum/enzimologia , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Inativação Gênica , Geraniltranstransferase/genética , Herbivoria , Interações Hospedeiro-Parasita/genética , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise de Sequência de Proteína , Triticum/genética
20.
J Integr Plant Biol ; 57(9): 770-82, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25644472

RESUMO

(E)-ß-Farnesene (EßF) synthase catalyses the production of EßF, which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies. Many plants possess EßF synthase genes and can release EßF to repel aphids. In order to effectively recruit the plant-derived EßF synthase genes for aphid control, by using chloroplast transit peptide (CTP) of the small subunit of Rubisco (rbcS) from wheat (Triticum aestivum L.), we targeted AaßFS1, an EßF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP + AaßFS1 transgenic lines. The CTP + AaßFS1 transgenic tobacco plants could emit EßF at a level up to 19.25 ng/day per g fresh tissues, 4-12 fold higher than the AaßFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bioassays demonstrated that the CTP + AaßFS1 transgenic tobacco showed enhanced repellence to green peach aphid (Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae, thus affecting aphid infestation at two trophic levels. These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EßF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.


Assuntos
Afídeos/fisiologia , Cloroplastos/enzimologia , Regulação da Expressão Gênica de Plantas , Pirofosfatases/genética , Pirofosfatases/metabolismo , Tabaco/enzimologia , Tabaco/genética , Animais , Interações Hospedeiro-Parasita , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
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