A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants

Pu, Yan; Tuo, Decai; Shen, Wei; Deng, Haida; Zhou, Peng; Gao, Xin

doi:10.1016/j.xplc.2022.100471

Cited by 12 publications

(8 citation statements)

References 59 publications

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“…To mitigate this concern, mutations were exclusively performed on selected sites of a stem, with degenerate sites strategically interspersed by unmutated sites to minimize potential disruptions to the local structure ( Fig 1A ). A pNC-Green vector expressing full-length PSTVd intermediate strain RNA (from nucleotide 88 to 87) surrounded by two hammerhead self-cleaving ribozymes was used to prepare mutant pools using degenerate primers [ 39 ]. Briefly, the experimental procedure involved conducting two separate PCR reactions ( S1 Fig ).…”

Section: Resultsmentioning

confidence: 99%

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies

Wu,

Zhang,

Nie

et al. 2024

PLoS Pathog

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RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

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Section: Resultsmentioning

confidence: 99%

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies

Wu,

Zhang,

Nie

et al. 2024

PLoS Pathog

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“…However, a systematic study of the NAC family in Malvales has not been conducted, especially in A. sinensis. The following numbers of NAC transcription factors were identified from nine Malvales plants in this study (Figure 1), including C. Capsularis (86), C. Olitorius (81), D. Turbinatus (126), D. Zibethinus (226), G. raimondii (153), H. Cannabinus (243), H. haianensis (127) and T. cacao (103). In particular, 111 NAC transcription factors were identified from A. sinensis, which were named AsNAC001-AsNAC111 (Figure 4).…”

Section: Identification and Evolution Of Nac Transcription Factorsmentioning

confidence: 87%

“…The sequences of three AsNAC plasmids, verified by the method of Sanger sequencing, were used to construct a recombinant vector of pNC-Green-SubN-AsNAC [103]. The recombinant vector was transferred into Agrobacterium tumefaciens GV3101 receptor cells, which was then infiltrated into the cells of the onion epidermis [39,41].…”

Section: Gene Cloning and Subcellular Localization Of Asnac Genesmentioning

confidence: 99%

Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis

Yang,

Mei,

Wang

et al. 2023

IJMS

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NAC is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in Aquilaria sinensis, a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the NAC gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of NAC gene families in Malvales plants. Then, 111 NAC genes were systemically characterized in A. sinensis. The phylogenetic analysis suggests that NAC genes in A. sinensis can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that AsNACs presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 AsNACs in A. sinensis after injury treatment indicated that AsNAC019 and AsNAC098 were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, AsNAC019 and AsNAC098 were also found to bind with the AsPKS07 promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the NAC gene family in Malvales plants and highlights the potential mechanisms of AsNACs for regulating secondary metabolite biosynthesis in A. sinensis, especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.

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“…Transactivation activity assay was performed as previously described with minor modifications [ 42 ]. The BD-PbWRKY31 vector was constructed by inserting the coding sequence of PbWRKY31 into pNC-GBKT7 using the Nimble Cloning method [ 43 ]. Approximately 500 ng of the BD-PbWRKY31 vector was transformed into yeast strain Y187 using the Super Yeast Transformation Kit II (Coolaber, Beijing, China) and screened sequentially on SD/-Trp and SD/-Trp-His plates at 30 °C incubator for three days before photographing.…”

Section: Methodsmentioning

confidence: 99%

Time-Series Transcriptome Analysis Reveals the Molecular Mechanism of Ethylene Reducing Cold Sensitivity of Postharvest ‘Huangguan’ Pear

Wei

et al. 2023

IJMS

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‘Huangguan’ pear (Pyrus bretschneideri Rehd) fruit is susceptible to cold, characterized by developing peel browning spots (PBS) during cold storage. Additionally, ethylene pretreatment reduces chilling injury (CI) and inhibits PBS occurrence, but the mechanism of CI remains unclear. Here, we deciphered the dynamic transcriptional changes during the PBS occurrence with and without ethylene pretreatment via time-series transcriptome. We found that ethylene suppressed the cold-signaling gene expression, thereby decreasing the cold sensitivity of the ‘Huangguan’ fruit. Moreover, the “Yellow” module closely correlated with PBS occurrence was identified via weighted gene co-expression network analysis (WGCNA), and this module was related to plant defense via Gene Ontology (GO) enrichment analysis. Local motif enrichment analysis suggested that the “Yellow” module genes were regulated by ERF and WRKY transcription factors. Functional studies demonstrated that PbWRKY31 has a conserved WRKY domain, lacks transactivation activity, and localizes in the nucleus. PbWRKY31-overexpressed Arabidopsis were hypersensitive to cold, with higher expression levels of cold signaling and defense genes, suggesting that PbWRKY31 participates in regulating plant cold sensitivity. Collectively, our findings provide a comprehensive transcriptional overview of PBS occurrence and elucidate the molecular mechanism by which ethylene reduces the cold sensitivity of ‘Huangguan’ fruit as well as the potential role of PbWRKY31 in this process.

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A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants

Cited by 12 publications

References 59 publications

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies

Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis

Time-Series Transcriptome Analysis Reveals the Molecular Mechanism of Ethylene Reducing Cold Sensitivity of Postharvest ‘Huangguan’ Pear

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