RESUMO
The precise genome editing has not been well established in plants, largely because of the limited frequency of homology recombination and the delivery barrier of donor templates. Recently, Dr. Caixia Gao's group from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed a series of plant prime editors (PPEs), which mediats the prime editing in the genomes of rice and wheat. The PPE systems are able to generate all 12 kinds of programmable base substitutions, as well desired multiplex nucleotide substitutions and small deletions or insertions without DNA double-strand breaks, thus providing versatile tools for precise plant genome editing. Herein, we introduce the structure and the editing capacity of the PPEs. The attemp on efficiency enhancements of PPEs and other PPEs are also discussed, which may provide a reference for appropriate application of PPEs in plants and also for continuous optimization of the editing tools.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Oryza , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Genoma de Planta/genética , Oryza/genéticaRESUMO
Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.
Assuntos
Oryza/genética , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Cloreto de Sódio/farmacologia , Adaptação Fisiológica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Tolerância ao Sal , Estresse FisiológicoRESUMO
KEY MESSAGE: A novel rice constitutive promoter (P OsCon1 ) was isolated. The molecular mechanism of the promoter activity was investigated. P OsCon1 could be used as an alternative constitutive promoter for crop transgenic engineering. Monocot constitutive promoter is an important resource for crop transgenic engineering. In this report, we isolated a novel promoter, Oscon1 promoter (P OsCon1 ), from the 5' upstream region of a constitutively expressed rice gene OsDHAR1. In P OsCon1 ::GUS transgenic rice, we showed that P OsCon1 had a broad expression spectrum in all tested tissues. The expression of the promoter was further analyzed in comparison with the previously characterized strong constitutive promoters. P OsCon1 exhibited comparable activity to OsCc1, OsAct1 or ZmUbi promoters in most tissues, and more active than 35S promoter in roots, seeds, and calli. Further quantitative assays indicated that P OsCon1 activity was not affected by developmental stages or by environmental factors. Further, 5'-deletions analysis indicated that the distinct regions might contribute to the strong expression of P OsCon1 in different tissues. Overall, our results suggest that P OsCon1 is a novel constitutive promoter, which could potentially use in transgenic crop development.
Assuntos
Oryza/genética , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2·(-) )-generating chemical methyl viologen was stronger than that of hydrogen peroxide (H2 O2 ). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O2·(-) . Furthermore, the stress induction did not rely on chloroplast- or cytosol-derived O2·(-) . Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O2·(-) is involved in the process.
Assuntos
Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Oryza/fisiologia , Oxirredutases/metabolismo , Paraquat/farmacologia , Proteínas de Plantas/metabolismo , Transdução de Sinais , Estresse Fisiológico , Cálcio/metabolismo , Núcleo Celular/metabolismo , Temperatura Baixa , Secas , Sequestradores de Radicais Livres/farmacologia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Oryza/efeitos dos fármacos , Oryza/enzimologia , Oryza/genética , Estresse Oxidativo , Oxirredutases/efeitos dos fármacos , Oxirredutases/genética , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/farmacologia , Salinidade , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos/análise , Superóxidos/farmacologiaRESUMO
Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non-nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH), which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT) but not abscisic acid- (ABA) regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (P OsUAH ). Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of P OsUAH . Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE) element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s) subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.
RESUMO
The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and "transgene clean" targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.
Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Genoma de Planta , Oryza/genética , Transgenes , Genes de Plantas , Instabilidade Genômica , Padrões de Herança , Mutagênese , Mutação , Plantas Geneticamente Modificadas , Edição de RNARESUMO
Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5' distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat.
Assuntos
Aconitato Hidratase/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Proteínas de Plantas/genética , Aconitato Hidratase/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Genes Reporter/genética , Temperatura Alta , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Nicotiana/genéticaRESUMO
Cadmium (Cd) is an important soil pollutant. Developing genetically engineered crops might be a feasible strategy for Cd decontamination and damage prevention. Both genes and promoters are critical for the effective construction of genetically modified plants. Although many functional genes for Cd tolerance and accumulation have been identified, few reports have focused on plant Cd-inducible promoters. Here, we identified three Cd-inducible genes in the rice genome: two tau class glutathione S-transferase (GSTU) genes, OsGSTU5 and OsGSTU37, and an HSP20/alpha crystallin family protein gene, OsHSP18.6. The promoter sequences were isolated and tested in transgenic rice lines using a GUSplus reporter gene. All of the promoters exhibited low background expression under normal conditions and could be strongly induced by Cd stress. Although their strength was comparable to that of the constitutive OsACTIN promoter under Cd stress, their time-dependent expression patterns under both short- and long-term Cd exposure were markedly different. The responses of the three promoters to other heavy metals were also examined. Furthermore, heavy metal-responsive cis elements in the promoters were computationally analyzed, and regions determining the Cd stress response were analyzed using a series of truncations. Our results indicate that the three Cd-inducible rice promoters described herein could potentially be used in applications aimed at improving heavy metal tolerance in crops or for the bio-monitoring of environmental contamination.
Assuntos
Cádmio/toxicidade , DNA de Plantas/isolamento & purificação , Oryza/genética , Regiões Promotoras Genéticas , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Oryza/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Deleção de Sequência , Especificidade da Espécie , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
Histone deacetylases (HDACs) are important in chromatin remodeling and epigenetic regulation of gene expression. Histone deacetylase inhibitors (HDACi) have highly effective anti-metastatic and anti-angiogenic activity in various types of cancer, while the molecular mechanisms involved in this process are not fully understood. In the present study, trichostatin A (TSA), a HDACi, was found to suppress MCF-7 breast carcinoma cell invasion and upregulate TET1 expression in a dose-dependent manner. TET1, a dioxygenase involved in cytosine demethylation, is downregulated during breast cancer progression. TET1 knockdown in MCF-7 cells facilitates cell invasion, inhibits the expression of tissue inhibitors of metalloproteinase 2/3 (TIMP2/3) and promotes matrix metalloproteinases (MMP) 2/9 transcriptional activity. Importantly, TET1 depletion impaired the inhibitory effect of TSA on breast cancer cell invasion. Together, these results illustrated a mechanism by which TET1 partially mediates HDACi elicited suppression of breast cancer invasion.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Adulto , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Oxigenases de Função Mista , Invasividade Neoplásica , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
The standard surgery for early-stage endometrial cancer is total abdominal hysterectomy (TAH), while total laparoscopic hysterectomy (TLH) is less invasive and assumed to be associated with lower morbidity. This meta- analysis was performed to investigate the effects of TLH versus TAH in women with early-stage endometrial cancer. We searched the PubMed, EMBASE, CBM and Cochrane Review databases for randomized trials assessing the effects of TLH versus TAH in women with early-stage endometrial cancer. The relative risks (RR) with 95% confidence intervals (CIs) from each study were pooled using meta-analysis. In our study, 9 randomized trials with a total of 1,263 patients were included. Meta-analyses showed that TLH was associated with lower risks of major complications (RR = 0.53, 95%CI 0.29-0.98, P = 0.042), total complications (RR = 0.59, 95%CI 0.42-0.82, P = 0.002) and postoperative complications (RR = 0.57, 95%CI 0.40-0.83, P = 0.003). However, there were no obvious differences in risks of intra-operative complications (RR = 0.98, 95%CI 0.62-1.55, P = 0.919) and mortality (RR = 0.96, 95%CI 0.66-1.40, P = 0.835). In conclusion, our results provide new evidence of a benefit for TLH over TAH in terms of major complications, total complications and postoperative complications in endometrial cancer patients.
Assuntos
Neoplasias da Mama/mortalidade , Obesidade/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Feminino , Seguimentos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Obesidade/fisiopatologia , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
The objective of this paper was to study the in vitro and in vivo inhibitory effect of Bidens bipinnata L. extract on growth of cervical carcinoma U14 cells. MTT method was used to determine the inhibitory effect of Bidens bipinnata L. extract on U14 tumour cells, and the effects of Bidens bipinnata L. extract on inhibition rate of solid tumour and life prolongation rate of ascites tumour were observed through the establishment of two animal models of mouse cervical carcinoma U14 solid tumour and ascites tumour. In the in vitro MTT assay, the inhibition rate gradually increased with the increase of dose of Bidens bipinnata L. and the extension of time. Its inhibition rate was 70.44% at a concentration of 80µg/L. Solid tumour inhibition rates in the high- and low-dose groups and cisplatin group were 49.13%, 2.26% and 75.72% respectively; life prolongation rates in each ascites tumour group were 63.63%, 34.86% and 87.34% respectively. The Bidens bipinnata L. extract has a certain inhibitory effect on growth of mouse cervical carcinoma U14.