Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.
Agaricales ; Gastrodia/genetics* ; Protoplasts ; Symbiosis/genetics* ; Transformation, Genetic

YWHAE gene is located on chromosome 17p13.3, and its product 14-3-3epsilon protein belongs to 14-3-3 protein family. As a molecular scaffold, YWHAE participates in biological processes such as cell adhesion, cell cycle regulation, signal transduction and malignant transformation, and is closely related to many diseases. Overexpression of YWHAE in breast cancer can increase the ability of proliferation, migration and invasion of breast cancer cells. In gastric cancer, YWHAE acts as a negative regulator of MYC and CDC25B, which reduces their expression and inhibits the proliferation, migration, and invasion of gastric cancer cells, and enhances YWHAE-mediated transactivation of NF-κB through CagA. In colorectal cancer, YWHAE lncRNA, as a sponge molecule of miR-323a-3p and miR-532-5p, can compete for endogenous RNA through direct interaction with miR-323a-3p and miR-532-5p, thus up-regulating K-RAS/ERK/1/2 and PI3K-AKT signaling pathways and promoting the cell cycle progression of the colorectal cancer. YWHAE not only mediates tumorigenesis as a competitive endogenous RNA, but also affects gene expression through chromosome variation. For example, the FAM22B-YWHAE fusion gene caused by t(10; 17) (q22; p13) may be associated with the development of endometrial stromal sarcoma. At the same time, the fusion transcript of YWHAE and NUTM2B/E may also lead to the occurrence of endometrial stromal sarcoma. To understand the relationship between YWHAE, NUTM2A, and NUTM2B gene rearrangement/fusion and malignant tumor, YWHAE-FAM22 fusion gene/translocation and tumor, YWHAE gene polymorphism and mental illness, as well as the relationship between 17p13.3 region change and disease occurrence. It provides new idea and basis for understanding the effect of YWHAE gene molecular mechanism and genetic variation on the disease progression, and for the targeted for the diseases.
14-3-3 Proteins/metabolism* ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Transformation, Neoplastic/genetics* ; Colorectal Neoplasms/genetics* ; Endometrial Neoplasms ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/genetics* ; Phosphatidylinositol 3-Kinases/metabolism* ; Sarcoma, Endometrial Stromal/pathology* ; Stomach Neoplasms/genetics* ; Transcription Factors/genetics* ; Translocation, Genetic

Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.
CRISPR-Cas Systems ; Citrullus/genetics* ; Cucurbitaceae/genetics* ; Gene Editing ; Plant Breeding ; Transformation, Genetic

The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.
Agrobacterium ; genetics ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Roots ; genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic ; genetics ; Stress, Physiological ; Tobacco ; genetics ; growth & development ; Transformation, Genetic

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.
Agrobacterium tumefaciens ; metabolism ; Breeding ; Cucumis sativus ; genetics ; microbiology ; Plants, Genetically Modified ; microbiology ; Research ; Transformation, Genetic

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.
Arabidopsis ; genetics ; Arabidopsis Proteins ; genetics ; Asarum ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Leaves ; Plant Proteins ; genetics ; Transcription Factors ; Transformation, Genetic

ObjectiveThe objective of the study was to summarize the role of DNA methylation in the development and metastasis of uveal melanoma (UM).

Data SourcesThe relevant studies in MEDLINE were searched.

Study SelectionIn this review, we performed a comprehensive literature search in MEDLINE using "uveal melanoma" AND ("DNA methylation" OR "epigenetics") for original research/review articles published before February 2018 on the relationship between DNA methylation and UM. References of the retrieved studies were also examined to search for potentially relevant papers.

ResultsPrevious studies on the relationship between DNA methylation and UM covered many genes including tumor suppressor genes (TSGs), cyclin-dependent kinase genes, and other genes. Among them, the TSG genes such as RASSF1A and p16INK4a, which encodes a cyclin-dependent kinase inhibitor, are relatively well-studied genes. Specifically, a high percentage of promoter methylation of RASSF1A was observed in UM cell lines and/or patients with UM. Promoter methylation of RASSF1A was also associated with the development of metastasis. Similarly, a high percentage of promoter hypermethylation of p16INK4a was found in UM cell lines. DNA promoter methylation can control the expression of p16INK4a, which affect cell growth, migration, and invasion in UM. Many other genes might also be involved in the pathogenesis of UM such as the Ras and EF-hand domain containing (RASEF) gene, RAB31, hTERT, embryonal fyn-associated substrate, and deleted in split-hand/split-foot 1.

ConclusionsOur review reveals the complex mechanisms underlying the tumorigenesis of UM and highlights the great needs of future studies to discover more genes/5'-C-phosphate-G-3' sites contributing to the development/metastasis of UM and explore the mechanisms through which epigenetic changes exert their function in UM.

Cell Transformation, Neoplastic ; genetics ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Humans ; Melanoma ; genetics ; Promoter Regions, Genetic ; genetics ; Uveal Neoplasms ; genetics

BACKGROUNDHuman myxovirus resistant protein A (MxA), encoded by the myxovirus resistance 1 (Mx1) gene, is an interferon (IFN)-triggered dynamin-like multi-domain GTPase involved in innate immune responses against viral infections. Recent studies suggest that MxA is associated with several human cancers and may be a tumor suppressor and a promising biomarker for IFN therapy. Mx1 gene mutations in the coding region for MxA have been discovered in many types of cancer, suggesting potential biological associations between mutations in MxA protein and corresponding cancers. In this study, we performed a systematic analysis based on the crystal structures of MxA and elucidated how these mutations specifically affect the structure and therefore the function of MxA protein.

METHODSCancer-associated Mx1 mutations were collected and screened from the COSMIC database. Twenty-two unique mutations that cause single amino acid alterations in the MxA protein were chosen for the analysis. Amino acid sequence alignment was performed using Clustal W to check the conservation level of mutation sites in Mx proteins and dynamins. Structural analysis of the mutants was carried out with Coot. Structural models of selected mutants were generated by the SWISS-MODEL server for comparison with the corresponding non-mutated structures. All structural figures were generated using PyMOL.

RESULTSWe analyzed the conservation level of the single-point mutation sites and mapped them on different domains of MxA. Through individual structural analysis, we found that some mutations severely affect the stability and function of MxA either by disrupting the intra-/inter-molecular interactions supported by the original residues or by incurring unfavorable configuration alterations, whereas other mutations lead to gentle or no interference to the protein stability and function because of positions or polarity features. The potential clinical value of the mutations that lead to drastic influence on MxA protein is also assessed.

CONCLUSIONSAmong all of the reported tumor-associated single-point mutations, seven of them notably affect the structure and function of MxA and therefore deserve more attention with respect to potential clinical applications. Our research provides an example for systematic analysis and consequence evaluation of single-point mutations on a given cancer-related protein.

Amino Acid Sequence ; Cell Transformation, Neoplastic ; genetics ; Crystallography, X-Ray ; Databases, Genetic ; Humans ; Myxovirus Resistance Proteins ; genetics ; Neoplasm Proteins ; genetics ; Neoplasms ; genetics ; Point Mutation ; Protein Domains ; genetics ; Protein Folding ; Sequence Alignment ; Stereoisomerism ; Structure-Activity Relationship

Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.
Arabidopsis ; genetics ; Arabidopsis Proteins ; biosynthesis ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Pichia ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Thiolester Hydrolases ; biosynthesis ; Transformation, Genetic

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
DNA, Recombinant ; Electroporation ; Genetic Engineering ; Genetic Vectors ; Hygromycin B ; Protoplasts ; Real-Time Polymerase Chain Reaction ; Rhizopus ; genetics ; Transformation, Genetic