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[Cloning and expression of the AeMYB4 gene associated with the development of Amana edulis stolons]

The stolon is an important organ for the reproduction and regeneration of Amana edulis. Previous transcription analysis showed that MYB was one of the most active transcription factor families during the evolution of A. edulis stolon. To study the potential role of MYB transcription factors in rhesus development, the authors selected a MYB regulator gene called AeMYB4 by analyzing the expression profile of MYB transcription factors. In this study, sequence analysis showed that AeMYB4 contains a 756 bp open reading frame encoding 251 amino acids, and domain analysis revealed that the predicted amino acid sequences contain two highly conserved SANT domains and binding sites for the cold stress factor CBF. Multiple sequence alignment and genetic analysis indicate that AeMYB4 co-localized with AtMYB15 from Arabidopsis thaliana, belongs to the S2 subgroup of R2 R3-MYB. Most of the transcription factors in this subfamily are associated with low temperature stress. The GFP-AeMYB4 fusion protein expression vector was generated for subcellular localization and transferred to Agrobacterium tumefaciens to infect Nicotiana benthamiana leaves, and the results showed that the protein was present in the nucleus. To investigate transcriptional activation, the pGBKT7-AeMYB4 fusion expression vector was transfected into startling Y2 H cells, showing that AeMYB4 was a transcriptional activator with strong transcriptional activity. Quantitative real-time polymerase chain reaction was used to detect AeMYB4 gene expression at three different stages of stamen development and in leaves, flowers, and bulbs of A. edulis, indicating that the transcription factor AeMYB4 was tissue-specific in expression. Primate evolution, and this expression was most active in the middle stage of primate development, suggesting that the AeMYB4 gene may play an important role in primate development. This study contributes to further investigation of the role of the transcription factor AeMYB4 in the development of A. edulis stolons.

Overexpression of the Ammopipptanthus nanus phospholipase Dα gene enhances the salt tolerance of a phospholipase Dα1-deficient Arabidopsis mutant.

The phospholipase Dα (AnPLDα) gene was cloned from the dry desert plant Ammopiptanthus nanus and its over-salt tolerance resulted in the PLDα1-deficient Arabidopsis mutant. Phospholipase Dα (PLDα) degrades phosphatidylcholine to produce phosphatidic acid and plays an important role in plant tolerance to abiotic stress. In this study, the phospholipase Dα (AnPLDα) gene was cloned from the plant Ammopiptanthus nanus xerophyte by homologous cloning and rapid cDNA-end amplification methods, and its role in stress tolerance was evaluated. The full-length cDNA was 2832 bp in length and contained an open reading frame of 2427 bp coding for 808 amino acids. The putative protein was predicted to be localized in the cytoplasm and this was confirmed by transient expression of a fluorescent fusion protein. The endogenous expression of the AnPLDα gene was induced by the high content of salt, dehydration, cold and abscisic acid. Heterogeneous expression of the AnPLDα gene enhanced the salt tolerance of the Arabidopsis pldα1 inactivating mutant and upregulated the expression of the AtABI, AtNCED, AtRD29A, AtRD29B and AtADH genes. Therefore, it was concluded that the AnPLDα gene was involved in the abiotic stress response. The AnPLDα gene is a promising candidate for transgenic applications to improve stress tolerance of commercial crops.

Chimeric virus regulatory factor-2 (IRF-2) binds IRF-E (IRF-binding element), VREβ (viral response element), but not VREα1.

Interferon regulatory factor-2 (IRF-2) is a multifunctional transcription factor with epigenetic activation, repression, and a synergistic effect together with IRF-1. IRF-2 is also involved in type I IFN signaling by suppressing the INFβ gene. To date, the molecular mechanism of DNA-binding activity remains elusive. We have performed a microsubcloning, expression, and electrophoresis study to examine IRF-2 chimeric mice. Here, we report the expression of murine chimeric IRF-2 as a GST-IRF-2 fusion protein in Escherichia coli/BL21 cells and demonstrate DNA-binding activity by radiogel retardation technique (32) IRF-E with marked P motif (GAAAGT) 4, viral response element (VRE) of the human INFβ gene and of the IFNα1 gene. We observed five different blocks of DNA/GST-IRF-2(1-5) complexes with the IRF-E motif, three different blocks of DNA/GST-IRF-2(1-3) complexes with VREß, but could not observe any complicated. DNA/GST-IRF-2 with VREα1. Specific binding of the IRF-E motif was confirmed by performing a 100-fold cold competition with (32) P-labelled IRF-E motif. In contrast to the specific binding on VREß, we used the negative control as we did not observe any complex. . but we did observe complexes with clonal extracts induced by IPTG. Regarding the binding to VREα1, we could not observe any complex in the negative control or in the extract of clones inducible by IPTG. Chimeric IRF-2 binds to the IRF-E element and VREβ but not to VREα1. This study is the first of its kind and paves the way for understanding the differential DNA-binding mechanism and the molecular mechanism of the DNA-binding activity of the IRF-2 molecule, which is critical to its function(s). ). ).

Cold Fusion Cloning Kit with Competent Cells (20 rxns) International Sales Only

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Cold Fusion Cloning Kit [96X] with 96-well format for Competent Cells -- International Sales Only

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1041-24 Intact Genomics 24/PK 242 EUR

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1011-12 Intact Genomics 12/PK 175 EUR

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1012-48 Intact Genomics 24/PK 383 EUR

DH10B Chemically Competent Cells (ig10B) - 6x200µl

1014-24 Intact Genomics 6/PK 216 EUR

Faster Experimental Validation of MicroRNA Targets Using Cold Fusion Cloning and the Firefly-Renilla Luciferase Dual Reporter Assay.

Various target prediction algorithms have been developed to provide a list of candidate microRNA (miRNA) target genes from a given animal. However, these computational methods provide false positive and false negative predictions. Therefore, target genes for a specific miRNA identified in silico need to be experimentally validated. In this chapter, we describe a step-by-step protocol for experimental validation of a direct miRNA target using the faster Dual Firefly-Renilla Luciferase reporter assay. We describe how to construct reporter plasmids using a simple, rapid, and highly efficient cold fusion cloning technique, which does not require ligase, phosphatase, or restriction enzymes. In addition, we present a protocol for cotransduction of reporter plasmids with miRNAs or miRNAs into human embryonic kidney 293 (HEK293) cells, as well as a description of how to measure Firefly and Renilla luciferase activity using the Dual-Glo luciferase assay. Equipment. As an example of the use of this technique, we will validate glucose-6-phosphate dehydrogenase (G6PD) as a direct target of miR-1207-5p.

Mutations in the atypical resistance protein TIR-NB-LRR-LIM lead to autoimmunity.

To defend against microbial infection, plants use a complex immune system that relies in part on resistance (R) proteins that initiate complex signaling cascades upon detection of pathogens. The resistance signaling network used by plants is only partially characterized. Genetic screening to identify new defense regulators involved in this network resulted in the isolation of the snc6-1D mutant. In situ cloning revealed that this mutant harbored a molecular lesion in the quench-sensitive gene 3 (CHS3), and thus the allele was renamed chs3-2D. CHS3 encodes a TIR-NB-LRR R protein containing a LIM domain (Lin-11, Isl-1, Mec-3). Although this protein has previously been implicated in cold stress and defense response, the role of the LIM domain in modulating protein activity is unclear. The chs3-2D allele contains a mutation from G to A causing substitution of C1340 for Y1340 near the LIM domain. It codes for a prevailing boom in job creation. The chs3-2D mutant is severely stunted and shows a rolled leaf morphology. In addition, it constitutively expresses germline disease (PR)-related genes, accumulates salicylic acid, and shows increased resistance to the virulent oomycete isolate Hyaloperonospora arabidopsidis (H.a) Noco2. Cellular localization assays using GFP fusion constructs indicate that both CHS3 and chs3-2D localize to the nucleus. A third allele of the mutant chypothesis that the LIM domain suppresses the activity of the CHS3-like protein R. This suppression may occur either through auto-blocking or binding to a negative defense regulator.

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