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BACKGROUND: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. RESULTS: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, "6251AB" and "6284AB". At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, "4001AB" and "4006AB". Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between "4001A" and "4001B", two differentially expressed miRNAs between "4006A" and "4006B", four differentially expressed miRNAs between "6251A" and "6251B", and ten differentially expressed miRNAs between "6284A" and "6284B". The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5' modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. CONCLUSIONS: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.
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Brassica napus/genética , MicroARNs/fisiología , Infertilidad Vegetal/genética , Polen/genética , ARN de Planta/fisiología , Brassica napus/crecimiento & desarrollo , Biblioteca de Genes , Desarrollo de la Planta/genética , TranscriptomaRESUMEN
Given the breadth of currently arising opportunities and concerns associated with nanoparticles for biomedical imaging, various types of nanoparticles have been widely exploited, especially for cellular/subcellular level probing. However, most currently reported nanoparticles either have inefficient delivery into cells or lack specificity for intracellular destinations. The absence of well-defined nanoplatforms remains a critical challenge hindering practical nano-based bio-imaging. Herein, the authors elaborate on a tailorable membrane-penetrating nanoplatform as a carrier with encapsulated actives and decorated surfaces to tackle the above-mentioned issues. The tunable contents in such a versatile nanoplatform offer huge flexibility to reach the expected properties and functions. Aggregation-induced emission luminogen (AIEgen) is applied to achieve sought-after photophysical properties, specific targeting moieties are installed to give high affinity towards different desired organelles, and critical grafting of cell-penetrating cyclic disulfides (CPCDs) to promote cellular uptake efficiency without sacrificing the specificity. Hereafter, to validate its practicability, the tailored nano products are successfully applied to track the dynamic correlation between mitochondria and lysosomes during autophagy. The authors believe that the strategy and described materials can facilitate the development of functional nanomaterials for various life science applications.
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Nanopartículas , Nanoestructuras , Lisosomas , Mitocondrias , Orgánulos/metabolismoRESUMEN
Intracellular lipid metabolism occurs in lipid droplets (LDs), which is critical to the survival of cells. Imaging LDs is an intuitive way to understand their physiology in live cells. However, this is limited by the availability of specific probes that can properly visualize LDs in vivo. Here, an LDs-specific red-emitting probe is proposed to address this need, which is not merely with an ultrahigh signal-to-noise (S/N) ratio and a large Stokes shift (up to 214 nm) but also with superior resistance to photobleaching. The probe has been successfully applied to real-time tracking of intracellular LDs behaviors, including fusion, migration, and lipophagy processes. We deem that the proposed probe here offers a new possibility for deeper understanding of LDs-associated behaviors, elucidation of their roles and mechanisms in cellular metabolism, and determination of the transition between adaptive lipid storage and lipotoxicity as well.
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Colorantes Fluorescentes/química , Luz , Gotas Lipídicas/química , Animales , Transporte Biológico , Color , Transporte de Electrón , Colorantes Fluorescentes/metabolismo , Células HeLa , Células Hep G2 , Humanos , Gotas Lipídicas/metabolismo , Imagen Molecular , Pez CebraRESUMEN
Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.
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Brassica/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Agrobacterium/genética , Cruzamiento/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Genes de Plantas/genética , Homocigoto , Mutación/genética , Tasa de Mutación , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
BACKGROUND/AIMS: To correlate the endoscopic characteristics with the histopathology of specimens of esophageal high-grade intraepithelial neoplasia obtained by endoscopic submucosal dissection (ESD). METHODS: This was a retrospective study developed from January 2010 to December 2015. The study included 169 patients who underwent ESD and were diagnosed with esophageal high-grade intraepithelial neoplasia according to endoscopic forceps biopsy, Lugol staining, endoscopic ultrasonography, computed tomography, and Narrow-Band Imaging. The demographic, endoscopic, and histopathologic characteristics were analyzed. RESULTS: A total of 19 cases (11.2%) had a change in diagnosis after histopathology exam and 16 (9.5%) needed a change in established treatment. An increase in the severity of disease was correlated with a lesion size > 2 cm, less than 4 samples in biopsy, and depressed or excavated patterns (p < 0.05). One hundred forty patients (82.8%) underwent curative resection. Lesions with leukoplakia (p < 0.001) and negative Lugol staining (p = 0.028) were independent risk factor for non-curative resection. CONCLUSION: This study confirms that lesion size > 2 cm, depressed and excavated patterns, and ≤4 biopsy samples are independent risk factors for histological grade changes compared to pre-endoscopic treatment diagnosis. Similarly, leukoplakia and no Lugol staining of lesions are independent risk factors for non-curative resection.
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Carcinoma in Situ/diagnóstico por imagen , Carcinoma in Situ/patología , Acalasia del Esófago/diagnóstico por imagen , Acalasia del Esófago/patología , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Carcinoma in Situ/cirugía , Colorantes , Resección Endoscópica de la Mucosa , Endoscopía Gastrointestinal , Endosonografía , Acalasia del Esófago/cirugía , Mucosa Esofágica/patología , Femenino , Humanos , Yoduros , Leucoplasia/diagnóstico por imagen , Leucoplasia/patología , Masculino , Persona de Mediana Edad , Imagen de Banda Estrecha , Clasificación del Tumor , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Carga TumoralRESUMEN
BACKGROUND: Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexpress Arabidopsis HTT2 to improve thermotolerance in the absence of ta-siR255-mediated gene silencing and the weak potential of coexpression. RESULTS: To test the potential application of HTT2 in improving crop thermotolerance, we transferred p35S::HTT2 to heading Chinese cabbage. We tested the leaf electrical conductivity, hypocotyl elongation, and survival percentage of p35S::HTT2 plants subjected to high-temperature (38 °C) and heat-shock (46 °C) treatment. The leaf electrical conductivity of p35S::HTT2 seedlings under high temperature decreased but did negligibly change under heat shock. The hypocotyl length of p35S::HTT2 seedlings increased under high temperature and heat shock. The survival rate of p35S::HTT2 seedlings increased under heat shock. BrpHsfs, a subset of heat-shock factor genes, were upregulated in p35S::HTT2 plants under high-temperature and heat shock conditions. In the field, transgenic plants with HTT2 appeared greener and formed leafy heads earlier than wild-type plants. CONCLUSIONS: Exogenous HTT2 increased the survival rates of heat-shocked heading Chinese cabbage by promoting thermotolerance through decreasing electrical conductivity and extending hypocotyl length. Our work provides a new approach to the genetic manipulation of thermotolerance in crops through the introduction of exogenous thermotolerance genes.
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Proteínas de Arabidopsis/genética , Brassica rapa/fisiología , Genes de Plantas , Proteínas Nucleares/fisiología , Proteínas de Plantas/genética , Termotolerancia/genética , Arabidopsis , Proteínas de Arabidopsis/fisiología , Brassica rapa/genética , Respuesta al Choque Térmico , Calor , Hipocótilo/crecimiento & desarrollo , Proteínas Nucleares/genética , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Plantones/genética , Plantones/fisiología , Termotolerancia/fisiologíaRESUMEN
KEY MESSAGE: We identified and cloned the two precursors of miR158 and its target gene in Brassica campestris ssp. chinensis, which both had high relative expression in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility, which was caused by the degradation of pollen contents from the binucleate microspore stage. These results first suggest the role of miR158 in pollen development of Brassica campestris ssp. chinensis. MicroRNAs (miRNAs) play crucial roles in many important growth and development processes both in plants and animals by regulating the expression of their target genes via mRNA cleavage or translational repression. In this study, miR158, a Brassicaceae specific miRNA, was functionally characterized with regard to its role in pollen development of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Two family members of miR158 in B. campestris, namely bra-miR158a1 and bra-miR158a2, and their target gene bra027656, which encodes a pentatricopeptide repeat (PPR) containing protein, were identified. Then, qRT-PCR analysis and GUS-reporter system revealed that both bra-miR158 and its target gene had relatively high expression levels in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility and pollen germination ratio, and the degradation of pollen contents from the binucleate microspore stage was also found in those deformed pollen grains, which led to pollen shrinking and collapse in later pollen development stage. These results first shed light on the importance of miR158 in pollen development of Brassica campestris ssp. chinensis.
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Brassica/genética , Regulación de la Expresión Génica de las Plantas , MicroARNs/metabolismo , Polen/genética , Secuencia de Bases , Brassica/crecimiento & desarrollo , Brassica/ultraestructura , Genes de Plantas , Germinación/genética , MicroARNs/genética , Especificidad de Órganos/genética , Plantas Modificadas Genéticamente , Polen/crecimiento & desarrollo , Polen/ultraestructura , Supervivencia TisularRESUMEN
BACKGROUND: microRNAs (miRNAs) are endogenous, noncoding, small RNAs that have essential regulatory functions in plant growth, development, and stress response processes. However, limited information is available about their functions in sexual reproduction of flowering plants. Pollen development is an important process in the life cycle of a flowering plant and is a major factor that affects the yield and quality of crop seeds. RESULTS: This study aims to identify miRNAs involved in pollen development. Two independent small RNA libraries were constructed from the flower buds of the male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) of Brassica campestris ssp. chinensis. The libraries were subjected to high-throughput sequencing by using the Illumina Solexa system. Eight novel miRNAs on the other arm of known pre-miRNAs, 54 new conserved miRNAs, and 8 novel miRNA members were identified. Twenty-five pairs of novel miRNA/miRNA* were found. Among all the identified miRNAs, 18 differentially expressed miRNAs with over two-fold change between flower buds of male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) were identified. qRT-PCR analysis revealed that most of the differentially expressed miRNAs were preferentially expressed in flower buds of the male fertile line (Bcajh97-01B). Degradome analysis showed that a total of 15 genes were predicted to be the targets of seven miRNAs. CONCLUSIONS: Our findings provide an overview of potential miRNAs involved in pollen development and interactions between miRNAs and their corresponding targets, which may provide important clues on the function of miRNAs in pollen development.
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Brassica/genética , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Polen/genética , Flores/genética , Perfilación de la Expresión Génica , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/química , Conformación de Ácido Nucleico , Estabilidad del ARNRESUMEN
Aqueous zinc-ion batteries (AZIBs) based on vanadium oxides or sulfides are promising candidates for large-scale rechargeable energy storage due to their ease of fabrication, low cost, and high safety. However, the commercial application of vanadium-based electrode materials has been hindered by challenging problems such as poor cyclability and low-rate performance. To this regard, sophisticated nanostructure engineering technology is used to adeptly incorporate VS2 nanosheets into the MXene interlayers to create a stable 2D heterogeneous layered structure. The MXene nanosheets exhibit stable interactions with VS2 nanosheets, while intercalation between nanosheets effectively increases the interlayer spacing, further enhancing their stability in AZIBs. Benefiting from the heterogeneous layered structure with high conductivity, excellent electron/ion transport, and abundant reactive sites, the free-standing VS2/Ti3C2Tz composite film can be used as both the cathode and the anode of AZIBs. Specifically, the VS2/Ti3C2Tz cathode presents a high specific capacity of 285 mAh g-1 at 0.2 A g-1. Furthermore, the flexible Zn-metal free in-plane VS2/Ti3C2Tz//MnO2/CNT AZIBs deliver high operation voltage (2.0 V) and impressive long-term cycling stability (with a capacity retention of 97% after 5000 cycles) which outperforms almost all reported Vanadium-based electrodes for AZIBs. The effective modulation of the material structure through nanocomposite engineering effectively enhances the stability of VS2, which shows great potential in Zn2+ storage. This work will hasten and stimulate further development of such composite material in the direction of energy storage.
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Fertilization is controlled by a complex gene regulatory network. To study the fertilization mechanism, we determined time courses of the four developmental stages of fertilization in Chinese cabbage pak-choi (Brassica campestris subsp. chinensis) by cytological observation. We then used the Arabidopsis ATH1 microarray to characterize the gene expression profiles of pollinated and unpollinated pistils in B. campestris subsp. chinensis. The result showed 44 up-regulated genes and 33 down-regulated genes in pollinated pistils compared with unpollinated pistils. Gene ontology analysis identified 20% of the up-regulated genes as belonging to the category of cell wall metabolism. We compared the up-regulated genes in pollinated pistils with previously identified pollen development related genes. Ten genes were found to be in common, which were termed as continuously expressed genes, in the two processes in the present article. Their expression patterns during pollen development and fertilization processes were then verified by RT-PCR. One of the continuously expressed genes, the homologous gene of At3g01270 in B. campestris subsp. chinensis, was confirmed as specifically expressed in microspores and pollinated pistils by using in situ hybridization. The potential biological functions of the other continuously expressed genes were also discussed.
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Brassica/genética , Fertilización/genética , Regulación de la Expresión Génica de las Plantas , Perfilación de la Expresión Génica , Genes de Plantas , Polinización/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensis cv. 'Aijiaohuang' genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Of these, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of 'Bcajh97-01A' sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5' modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development.
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Brassica/genética , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Bases , Brassica/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Infertilidad Vegetal/genética , Proteínas de Plantas/genética , Polen/genética , Polen/crecimiento & desarrolloRESUMEN
Seed density per silique (SDPS) and valid silique length (VSL) are two important yield-influencing traits in rapeseed. SDPS has a direct or indirect effect on rapeseed yield through its effect on seed per silique. In this study, a quantitative trait locus (QTL) for SDPS was detected on chromosome A09 using the QTL-seq approach and confirmed via linkage analysis in the mapping population obtained from 4263 × 3001 cross. Furthermore, one major QTL for SDPS (qSD.A9-1) was mapped to a 401.8 kb genomic interval between SSR markers Nys9A190 and Nys9A531. In the same genomic region, a QTL (qSL.A9) linked to VSL was also detected. The phenotypic variation of qSD.A9-1 and qSL.A9 was 53.1% and 47.6%, respectively. Results of the additive and dominant effects demonstrated that the expression of genes controlling SDPS and VSL were derived from a different parent in this population. Subsequently, we identified 56 genes that included 45 specific genes with exonic (splicing) variants. Further analysis identified specific genes containing mutations that may be related to seed density as well as silique length. These genes could be used for further studies to understand the details of these traits of rapeseed.
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Brassica napus , Brassica rapa , Brassica napus/genética , Mapeo Cromosómico/métodos , Sitios de Carácter Cuantitativo/genética , Fenotipo , Brassica rapa/genética , Semillas/genéticaRESUMEN
Rapeseed is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been widely used successfully for rapeseed hybrid production. The physiological and molecular mechanism of pollen development in GMS lines of rapeseed (Brassica napus L.) need to be determined for the creation of hybrids and cultivation of new varieties. However, limited studies have focused on systematically mining genes that regulate the pollen development of GMS lines in B. napus. In the present study, to determine the stage at which pollen development begins to show abnormality in the GMS lines, we performed semi-thin section analysis of the anthers with five pollen development stages. The results indicated that the abnormal pollen development in DGMS lines might start at the meiotic stage, and abnormal pollen development in RGMS lines probably occurred before the tetrad stage. To investigate the critical genes and pathways involved in pollen development in GMS lines, we constructed and sequenced 24 transcriptome libraries for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines (6251AB and 6284AB) and two dominant GMS (DGMS) lines (4001AB and 4006AB). A total of 23,554 redundant DEGs with over two-fold change between sterile and fertile lines were obtained. A total of 346 DEGs were specifically related to DGMS, while 1,553 DEGs were specifically related to RGMS. A total of 1,545 DEGs were shared between DGMS and RGMS. And 253 transcription factors were found to be differentially expressed between the sterile and fertile lines of GMS. In addition, 6,099 DEGs possibly related to anther, pollen, and microspore development processes were identified. Many of these genes have been reported to be involved in anther and microspore developmental processes. Several DEGs were speculated to be key genes involved in the regulation of fertility. Three differentially expressed genes were randomly selected and their expression levels were verified by quantitative PCR (qRT-PCR). The results of qRT-PCR largely agreed with the transcriptome sequencing results. Our findings provide a global view of genes that are potentially involved in GMS occurrence. The expression profiles and function analysis of these DEGs were provided to expand our understanding of the complex molecular mechanism in pollen and sterility development in B. napus.
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Background: The endoscopic resection of suspected gastric high-grade intraepithelial neoplasia (HGIN) may incidentally cause the patient to suffer from early gastric cancer (EGC), complicating the subsequent clinical management. Identifying the risk factors for such misstaging may help guide the clinical management. Methods: The information obtained from 123,460 patients, who underwent conventional upper gastrointestinal endoscopy at the First Affiliated Hospital of Nanjing Medical University from January 2010 to December 2015, were retrospectively reviewed. Patients with an initial diagnosis of HGIN underwent endoscopic submucosal dissection (ESD), and received a final diagnosis of EGC. The risk factors for the upgraded pathology and noncurative resection were analyzed. Results: Among the 134 patients initially diagnosed with HGIN, 35 (26.12%) patients were finally diagnosed with EGC after ESD. A lesion size of ≥2 cm (odds ratio [OR] = 5.16, 95% confidence interval [CI] = 2.04-13.05; P < .01), ≤4 biopsies taken (OR = 2.73, 95% CI = 1.15-6.48; P < .05), and the presence of upper gastrointestinal bleeding (UGIB; OR = 15.64, 95% CI = 1.29-189.75; P < .05) were the independent risk factors for upgraded pathology. In addition, patients >65 years old (OR = 0.022, 95% CI = 0.901-6.549; P < .05) or with a lesion size of ≥2 cm (OR = 4.237, 95% CI = 1.650-10.878; P < .01) were more likely to endure the noncurative resection. Conclusion: For suspected gastric HGIN patients, age, lesion size, the number of biopsies, and UGIB should be taken into account before deciding on the ESD.
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Carcinoma in Situ , Resección Endoscópica de la Mucosa , Neoplasias Gástricas , Anciano , Carcinoma in Situ/etiología , Carcinoma in Situ/patología , Carcinoma in Situ/cirugía , Resección Endoscópica de la Mucosa/efectos adversos , Mucosa Gástrica/patología , Mucosa Gástrica/cirugía , Humanos , Estudios Retrospectivos , Neoplasias Gástricas/patología , Resultado del TratamientoRESUMEN
MicroRNAs (miRNAs) and their target genes play vital roles in crops. However, the genetic variations in miRNA-targeted sites that affect miRNA cleavage efficiency and their correlations with agronomic traits in crops remain unexplored. On the basis of a genome-wide DNA re-sequencing of 210 elite rapeseed (Brassica napus) accessions, we identified the single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in miRNA-targeted sites complementary to miRNAs. Variant calling revealed 7.14 million SNPs and 2.89 million INDELs throughout the genomes of 210 rapeseed accessions. Furthermore, we detected 330 SNPs and 79 INDELs in 357 miRNA target sites, of which 33.50% were rare variants. We also analyzed the correlation between the genetic variations in miRNA target sites and 12 rapeseed agronomic traits. Eleven SNPs in miRNA target sites were significantly correlated with phenotypes in three consecutive years. More specifically, three correlated SNPs within the miRNA-binding regions of BnSPL9-3, BnSPL13-2, and BnCUC1-2 were in the loci associated with the branch angle, seed weight, and silique number, respectively; expression profiling suggested that the variation at these 3 miRNA target sites significantly affected the expression level of the corresponding target genes. Taken together, the results of this study provide researchers and breeders with a global view of the genetic variations in miRNA-targeted sites in rapeseed and reveal the potential effects of these genetic variations on elite agronomic traits.
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Rapeseed (Brassica napus L.) is an important oil crop with a huge genome. This study used next generation sequencing technology to develop SSR markers in rapeseed. A total of 213,876 sequence reads were obtained in 58.8 Mb. For these reads, 21,523 SSRs were recovered from 18,575 microsatellites sequences and 8,964 SSR primer pairs were identified. Di- and mono-nucleotides were the most abundant, accounting for 47.5% and 30.7% of all SSRs, respectively. A total of 8,776 SSRs were designed from contigs and 100 SSR primers were tested for validation of SSR locus amplification. Nearly all (94%) of the markers were found to produce clear amplicons and to be reproducible. For these markers, forty-three SSRs showed polymorphic bands in eight rapeseed accessions. Thirty-four SSRs were then applied to 78 rapeseed accessions from China to evaluate the genetic diversity. Result showed that the allele number varied from two to seven, with a mean value of 3.59. The effective allele number of ranged from 1.14 to 3.25, with an average of 2.09. The average values of observed heterozygosity and expected heterozygosity were 0.54 and 0.49, respectively. The Nei's gene diversity varied from 0.12 to 0.69, with a mean value of 0.48. Resulting of the markers testing showed that the identified genome-wide SSRs were useful in rapeseed genetic studies, including genetic diversity, QTL mapping and marker-assisted selection for breeding.
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Brassica napus/genética , Marcadores Genéticos , Repeticiones de Microsatélite , Genoma de Planta , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
BACKGROUND: Pathogenesis of Helicobacter Pylori (HP) vacuolating toxin A (vacA) depends on polymorphic diversity within the signal (s), middle (m), intermediate (i), deletion (d) and c-regions. These regions show distinct allelic diversity. The s-region, m-region and the c-region (a 15 bp deletion at the 3'-end region of the p55 domain of the vacA gene) exist as 2 types (s1, s2, m1, m2, c1 and c2), while the i-region has 3 allelic types (i1, i2 and i3). The locus of d-region of the vacA gene has also been classified into 2 genotypes, namely d1 and d2. We investigated the "d-region"/"loop region" through bioinformatics, to predict its properties and relation to disease. One thousand two hundred fifty-nine strains from the NCBI nucleotide database and the dryad database with complete vacA sequences were included in the study. The sequences were aligned using BioEdit and analyzed using Lasergene and BLAST. The secondary structure and physicochemical properties of the region were predicted using PredictProtein. RESULTS: We identified 31 highly polymorphic genotypes in the "d-region", with a mean length of 34 amino acids (9 ~ 55 amino acids). We further classified the 31 genotypes into 3 main types, namely K-type (strains starting with the KDKP motif in the "d-region"), Q-type (strains starting with the KNQT motif), and E-type (strains starting with the ESKT motif) respectively. The most common type, K-type, is more prevalent in cancer patients (80.87%) and is associated with the s1i1m1c1 genotypes (P < .01). Incidentally, a new region expressing sequence diversity (2 aa deletion) at the C-terminus of the p55 domain of vacA was identified during bioinformatics analysis. CONCLUSIONS: Prediction of secondary structures shows that the "d-region" adopts a loop conformation and is a disordered region.
Asunto(s)
Proteínas Bacterianas/genética , Helicobacter pylori/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Genotipo , Humanos , Prevalencia , Estructura Secundaria de Proteína , Solventes , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/microbiologíaRESUMEN
Piwi like RNA-mediated gene silencing 1 (Hiwi) is a human homolog of the Piwi gene family that has been reported to be upregulated in hepatocellular carcinoma (HCC). The present study aimed to investigate the role of Hiwi in the initiation and development of HCC in vitro and in vivo. Adenovirus-mediated Hiwi overexpression was established in primary murine hepatocytes and SMMC7721 HCC cells. Cell viability and proliferation were assessed using MTT and EdU assays, respectively. Cell migration was measured using a scratch migration assay. The cell cycle was assessed using flow cytometry, and the expression of genes associated with the epithelial mesenchymal transition (EMT) was assessed using reverse transcription-quantitative polymerase chain reaction. SMMC7721 cells that stably express Hiwi were also generated and injected subcutaneously into the nude mice, and tumor growth was examined. Recombinant adenovirus encoding green fluorescent protein or Hiwi was delivered by injection into the tail vein, and its effect on murine hepatocyte gene expression was studied. The present study revealed that the overexpression of Hiwi did not affect the proliferation or migration of liver cancer cells and failed to suppress perifosine- or doxorubicin-induced apoptosis in vitro. The tumors of mice that were injected with Hiwi-expressing SMMC7721 cells were not significantly larger compared with mice that were injected with control SMMC7721 cells. Hiwi overexpression did not noticeably alter the expression of genes involved in EMT, either in vitro or in vivo. The results of the present study indicate that although expression of Hiwi is associated with HCC development and progression in the clinic, it does not act as an oncogene in liver cancer cells.