Transient Expression Research Articles

Development of a Csy4-Processing TRV-Based CRISPR/Cas9 Genome Editing System in Nicotiana benthamiana

AbstractThe CRISPR/Cas9 system is a site-specific genome editing tool that has been widely used in various plant species. The plant virus-based gRNA (guide RNA) delivery system, which differs from the typical Agrobacterium-mediated transformation, is an attractive method to facilitate the application of CRISPR/Cas9. The virally delivered gRNA is usually driven by heterologous plant U6 or viral promoters (e.g., pea early-browning virus, PEBV; barley stripe mosaic virus, BSMV). However, heterologous promoters may have poor performance in some cases. In this paper, a feasible option to detach gRNA(s) from the virus genome is employed. Specifically, the Csy4-RNA processing system is used to release gRNA(s) from the tobacco rattle virus (TRV). The coding sequences of Cas9 and Csy4 nucleases are cloned into a single polycistronic expression cassette under an estrogen-inducible promoter in a binary vector, and the gRNA is cloned into the TRV genome flanked by two 20 bp Csy4 recognition sites. The results show that the Csy4-processing TRV-based delivery system works effectively in targeting single and multiple sites, nucleotide replacement, and large fragment deletion in Cas9-mediated genome editing via transient expression in Nicotiana benthamiana. The Csy4-TRV is a promising gRNA(s) processing and delivery system for CRISPR/Cas9 genome editing. This method can be easily adapted to other plant RNA viruses, facilitating the application of the CRISPR/Cas9 system in plants.

Two putative calcium-dependent protein kinases are involved in the regulation of sugarcane defense genes

Sugarcane is a primary sugar crop and an important source of bioenergy. Pathogens are the major factors affecting sugarcane yield and sugar content. However, the mechanisms of sugarcane defense regulation remain largely unknown, and research on prospective genetic targets for modification is scarce. As the main class of calcium sensors, calcium-dependent protein kinases (CDPKs/CPKs) play a crucial role in the immune regulatory network. Using sugarcane genomic data, we identified 229 putative ScCDPKs in primordial specie Saccharum officinarum. Searching the putative CDPKs in sugarcane cultivars from National Center for Biotechnology Information (NCBI), 12 putative ScCDPKs in cultivars were identified. Phylogenetic analysis revealed evolutionary relationships among these CDPKs in sugarcane cultivars and those in S. officinarum, Arabidopsis thaliana, and rice. Truncation mutants of ScCDPKs were introduced into the reporter system and examined for activity in inducing Sugarcane Pathogenesis Related Protein 1 (ScPR1). The results showed that truncated ScCDPK1 and ScCDPK8 induce higher expression of ScPR1 than full-length ScCDPK1 and ScCDPK8. Additionally, transient expression of truncated ScCDPK1 and ScCDPK8 exhibit stronger activity in sugarcane protoplasts for activation of ScPR1 and sugarcane Serine Protease Inhibitor (ScSPI). These results demonstrate that ScCDPK1 and ScCDPK8 possess auto-inhibitory activity. The findings of this study provide a basis for an in-depth study of the sugarcane CDPK gene family and lay the foundation for further genetic improvement.

Comparative Physio-Biochemical and Transcriptome Analyses Reveal Contrasting Responses to Magnesium Imbalances in Leaves of Mulberry (Morus alba L.) Plants

Magnesium (Mg) deficiency is a major factor limiting the growth and development of plants. Mulberry (Morus alba L.) is an important fruit tree crop that requires Mg for optimal growth and yield, especially in acid soils. However, the molecular mechanism of Mg stress tolerance in mulberry plants remains unknown. In this study, we used next-generation sequencing technology and biochemical analysis to profile the transcriptome and physiological changes of mulberry leaves under different Mg treatments (deficiency: 0 mM, low: 1 mM, moderate low: 2 mM, sufficiency: 3 mM, toxicity: 6 mM, higher toxicity: 9 mM) as T1, T2, T3, CK, T4, T5 treatments, respectively, for 20 days. The results showed that Mg imbalance altered the antioxidant enzymatic activities, such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and non-enzymatic, including soluble protein, soluble sugar, malondialdehyde (MDA), and proline (PRO), contents of the plant. The Mg imbalances disrupted the ultrastructures of the vital components of chloroplast and mitochondria relative to the control. The transcriptome data reveal that 11,030 genes were differentially expressed (DEGs). Genes related to the photosynthetic processes (CAB40, CAB7, CAB6A, CAB-151, CAP10A) and chlorophyll degradation (PAO, CHLASE1, SGR) were altered. Antioxidant genes such as PER42, PER21, and PER47 were downregulated, but DFR was upregulated. The carbohydrate metabolism pathway was significantly altered, while those involved in energy metabolism processes were perturbed under high Mg treatment compared with control. We also identified several candidate genes associated with magnesium homeostasis via RT-qPCR validation analysis, which provided valuable information for further functional characterization studies such as promoter activity assay or gene overexpression experiments using transient expression systems.

Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke.

Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.

Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta

BackgroundEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs.ResultIn this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool).ConclusionsThis set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Functional characterization of fatty acyl-coenzyme A reductase, RcFAR1, specially expressed by Rosa chinensis consequent to infection by rose powdery mildew, Podosphaera pannosa

Rose (Rosa chinensis), as the most popular cut flowers in the world, is perennially attacked by rose powdery mildew (Podosphaera pannosa) and beet armyworm (Spodoptera exigua). The indirect plant-mediated interactions between these two pests always exist in the ternary system consisting of these three organisms, but little is known about the molecular mechanisms for such interactions. In this paper, we investigated the function of an insect-resistance-related gene in rose, RcFAR1, encoding a fatty acyl coenzyme A reductase. The gene was expressed in the roses when the plants were infected by P. pannosa, and as a consequence, the resistance of the roses against S. exigua was significantly induced. The results from heterologous expression of RcFAR1 in Saccharomyces cerevisiae showed that RcFAR1 primarily accumulated primary alcohols, including C12:0, C16:0, and C18:0 by using GC-MS analyses. Overexpression in Arabidopsis thaliana resulted in 4.0-, 3.6-, 8.2-, and 3.1-fold increases in the contents of fatty alcohols in T1 generation plants of A. thaliana for C12:0, C14:0, C16:0, and C18:0, respectively, suggesting that RcFAR1 catalyzes the biosynthesis of primary alcohols of C12:0, C14:0, C16:0, and C18:0 in the plants. Transient expression in the tobacco showed that RcFAR1 localized to the cytoplasm or the cell membrane. Our study has elucidated the molecular mechanism for the synthesis of dodecanol, tetradecanol, hexadecanol and other primary alcohols in P. pannosa-infected roses, and thus revealed one of the important molecular regulatory mechanisms by which the resistances of the roses against S. exigua are induced by the infection of the roses by P. pannosa.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Generation of a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line via CRISPR base editing

The KCNQ1 gene encodes a voltage-gated potassium channel, which plays an important role in the repolarization of myocardial action potentials. Mutations in this gene often result in type 1 long QT syndrome (LQT1). Here, we generated a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line (WAe009-A-1D) based on the transient expression adenine base editing system that converts base A to G. The WAe009-A-1D cell maintains the morphology, pluripotency, and normal karyotype of the stem cells and is capable of differentiating into all three germ layers in vivo.

Transcriptomic analysis – Transient expression of CYP714C2 improves resistance in pears

Postharvest pear fruit is highly susceptible to infection by pathogens, particularly by Penicillium expansum. The cytochrome P450 family plays a crucial role in the plant's defense response against pathogen invasion. However, the mechanism underlying CYP714C2 defense in pear fruit remains unclear. In this study, we investigated the molecular and physiological changes in pears after overexpressing CYP714C2 using transient expression technology and transcriptomics analysis. Our findings demonstrate that the up-regulation of CYP714C2 significantly enhances the expression of genes related to pear resistance, including CDPK, CNGC, FLS2, WRKY22, WRKY29, PYL1, ETR, ERS, EIL3, ERF1, NPR, GST, BGLU and others. Moreover, CYP714C2 overexpression altered starch and sucrose metabolism, pentose and glucuronate interconversions, glutathione metabolism, plant hormone signal transduction, MAPK signaling pathway - Plant, plant-pathogen interaction and other resistance-related pathways. Meanwhile, SOD, POD and GST activities of pear were increased. The transient expression of CYP714C2 also resulted in reduced incidence and severity of pathogen infection in pears. It was also found that CYP714C2 protein can interact with GA2ox1 which is a key protein in gibberellin metabolic pathway. As a result, the research showed that CYP714C2 played a role in enhancing resistance in pear fruit and detailed its molecular mechanism for enhancing fruit resistance. This provides a theoretical foundation for future exploration of fruit disease resistance.

Intraocular mRNA delivery with endogenous MmPEG10-based virus-like particles

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo.

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.

A systemic approach to identifying sequence frameworks that decrease mAb production in a transient Chinese hamster ovary cell expression system.

Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their "developability" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these "difficult-to-express" (DTE) mAbs has shown that these phenotypes are driven by post-translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG1κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines.

Functional characterization of Fagopyrum tataricum ZIP gene family as a metal ion transporter.

The zinc/iron-regulated transporter-like proteins (ZIP) family acts as an important transporter for divalent metal cations such as Zn, Fe, Mn, Cu, and even Cd. However, their condition is unclear in Tartary buckwheat (Fagopyrum tataricum). Here, 13 ZIP proteins were identified and were predicted to be mostly plasma membrane-localized. The transient expressions of FtZIP2 and FtZIP6 in tobacco confirmed the prediction. Multiple sequence alignment analysis of FtZIP proteins revealed that most of them had 8 putative transmembrane (TM) domains and a variable region rich in histidine residues between TM3 and TM4, indicating the reliable affinity to metal ions. Gene expression analysis by qRT-PCR showed that FtZIP genes were markedly different in different organs, such as roots, stems, leaves, flowers, fruits and seeds. However, in seedlings, the relative expression of FtZIP10 was notably induced under the CdCl2 treatment, while excessive Zn2+, Fe2+, Mn2+ and Cd2+ increased the transcript of FtZIP5 or FtZIP13, in comparison to normal conditions. Complementation of yeast mutants with the FtZIP family genes demonstrate that FtZIP7/10/12 transport Zn, FtZIP5/6/7/9/10/11 transport Fe, FtZIP12 transports Mn and FtZIP2/3/4/7 transport Cd. Our data suggest that FtZIP proteins have conserved functions of transportation of metal ions but with distinct spatial expression levels.

Stationary analysis of a constrained Markov fluid model with two buffers

. We consider Markov fluid models with two infinite buffers, whose fluid rates ensure that the fluid level of buffer 1 is never larger than the one of buffer 2. For the model, we derive the system of PDEs describing the transient behavior and expressions for the stationary analysis in the Laplace domain. Examples for the application of the model in the context of network access control are presented. Numerical results are compared with detailed simulation results to show that the numerical approach computes the required measures with sufficient accuracy.

P53 promotes revival stem cells in the regenerating intestine after severe radiation injury

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

ACL5 acquired strict thermospermine synthesis activity during the emergence of vascular plants.

Norspermine (Nspm), one of the uncommon polyamines (PAs), was detected in bryophytes and lycophytes; therefore, the aminopropyltransferases involved in the synthesis of Nspm were investigated. The enzymatic activity was evaluated by the transient high expression of various aminopropyltransferase genes in Nicotiana benthamiana, followed by quantification of PA distribution in the leaves using gas chromatography-mass spectrometry. The bryophyte orthologues of ACL5, which is known to synthesise thermospermine (Tspm) in flowering plants, were found to have strong Nspm synthesis activity. In addition, two ACL5 orthologous with different substrate specificities were conserved in Selaginella moellendorffii, one of which was involved in Tspm synthesis and the other in Nspm synthesis. Therefore, further detailed analysis using these two factors revealed that the β-hairpin structural region consisting of β-strands 1 and 2 at the N-terminus of ACL5 is involved in substrate specificity. Through functional analysis of a total of 40 ACL5 genes in 33 organisms, including algae, it was shown that ACL5 has changed its substrate specificity several times during plant evolution and diversification. Furthermore, it was strongly suggested that ACL5 acquired strict Tspm synthesis activity during the emergence of vascular plants, especially through major changes around the β-hairpin structural region.

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Transcriptome analysis reveals various genes involved in the regulation of potato to late blight

BackgroundPotato (Solanum tuberosum L.) production is seriously threatened by the oomycete Phytophthora infestans (P. infestans). However, it remains unclear how the two potato cultivars, Q9 (moderately resistant) and Atl (susceptible), initiate distinct defense responses after inoculation with P. infestans. To uncover the underlying process, we investigated the dynamic gene expression profiles and the functions of the differentially expressed genes (DEGs).ResultsThe results showed that the numbers of up-regulated DEGs were 1345, 1063 and 1694 in Q9 and 272, 603 and 2554 in Atl at 1 day post inoculation (dpi), 3 dpi and 5 dpi, respectively. Meanwhile, the down-regulated DEGs were 1557, 1417 and 389 in Q9 and 114, 364 and 2528 in Atl simultaneously. KEGG enrichment analysis revealed that plant-pathogen interaction, phenylpropanoid biosynthesis and MAPK signaling pathway were significantly enriched in Q9 at the later stage. Furthermore, DEGs associated with plant-pathogen interaction and phenylpropanoid biosynthesis showed higher expression levels in Q9 compared to Atl, especially at the later stage. The expressions of eight DEGs were validated by quantitative real-time PCR (qRT-PCR), which further verified the accuracy of the transcriptomics analysis. In addition, DEGs that were exclusively up-regulated and down-regulated in Q9 were analyzed, which may contribute to the resistance of Q9. Transient expression analysis revealed that four DEGs, including StHP1, StMYB2, StHSP3 and StNAC5, exhibited increased tolerance to P. infestans, indicating a positive role in enhancing the resistance of Q9.ConclusionTherefore, the DEGs associated with plant-pathogen interaction, phenylpropanoid biosynthesis and MAPK signaling pathway were involved in regulating late blight, and the expression level of the related genes significantly increased in Q9. In addition, StHP1, StHSP3 and the transcription factors, including StMYB2 and StNAC5, played positive roles in improving the resistance of potato Q9 to P. infestans. These findings provide useful information for further understanding the molecular mechanism of potato resistance to late blight. Furthermore, our study identified novel resistance genes that can be used in resistance breeding and functional research.Graphical abstract

Comparative transcriptome analysis identifies candidate genes related to sucrose accumulation in longan (Dimocarpus longan Lour.) pulp.

Sucrose content is one of the important factors to determine longan fruit flavor quality. To gain deep insight of molecular mechanism on sucrose accumulation in longan, we conducted comparative transcriptomic analysis between low sucrose content longan cultivar 'Qingkebaoyuan' and high sucrose content cultivar 'Songfengben'. A total of 12,350 unique differentially expressed genes (DEGs) were detected across various development stages and different varieties, including hexokinase (HK) and sucrose-phosphate synthase (SPS), which are intricately linked to soluble sugar accumulation and metabolism. Weighted gene co-expression network analysis (WGCNA) identified magenta module, including DlSPS gene, was significantly positively correlated with sucrose content. Furthermore, transient expression unveiled DlSPS gene play crucial role in sucrose accumulation. Moreover, 5 transcription factors (MYB, ERF, bHLH, C2H2, and NAC) were potentially involved in DlSPS regulation. Our findings provide clues for sucrose metabolism, and lay the foundation for longan breeding in the future.

MRNA therapies: Pioneering a new era in rare genetic disease treatment

Rare genetic diseases, often referred to as orphan diseases due to their low prevalence and limited treatment options, have long posed significant challenges to our medical system. In recent years, Messenger RNA (mRNA) therapy has emerged as a highly promising treatment approach for various diseases caused by genetic mutations. Chemically modified mRNA is introduced into cells using carriers like lipid-based nanoparticles (LNPs), producing functional proteins that compensate for genetic deficiencies. Given the advantages of precise dosing, biocompatibility, transient expression, and minimal risk of genomic integration, mRNA therapies can safely and effectively correct genetic defects in rare diseases and improve symptoms. Currently, dozens of mRNA drugs targeting rare diseases are undergoing clinical trials. This comprehensive review summarizes the progress of mRNA therapy in treating rare genetic diseases. It introduces the development, molecular design, and delivery systems of mRNA therapy, highlighting their research progress in rare genetic diseases based on protein replacement and gene editing. The review also summarizes research progress in various rare disease models and clinical trials. Additionally, it discusses the challenges and future prospects of mRNA therapy. Researchers are encouraged to join this field and collaborate to advance the clinical translation of mRNA therapy, bringing hope to patients with rare genetic diseases.