Stable Gene Expression Research Articles

Interference inhibition of Plum pox virus, induced by a hairpin-RNA of viral origin, provides long-term resistance to PPV infection in adult plants of the Startovaya (Prunus domestica L.) variety

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum `Startovaya` (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, Immuno Strip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene

Development of Reference Genes for Horticultural Plants

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is extensively applied technique to investigate the transcript abundance of target genes in various organisms. Selection of appropriate reference genes (RGs) for qRT-PCR normalization is a crucial prerequisite for accurately quantifying gene expression level. RGs should exhibit minimal variation in gene expression. However, the actual expression stability of RGs fluctuates greatly in different species or under different experimental conditions. Due to rapid advancements in next-generation sequencing (NGS) technology, it is no longer difficult to get massive transcriptome data, which has greatly promoted the development of RGs. In this review, we elaborate on the strategies for developing RGs using Northern blotting, expressed sequence tags (ESTs), qRT-PCR, and high-throughput technologies such as microarray and RNA-sequencing (RNA-Seq). The process for developing RGs based on RNA-Seq is further addressed, including processing and normalization of RNA-Seq data, evaluation of gene expression stability, and screening and validation of RGs. The most frequently used RGs in horticultural plants are summarized, and the strategies for developing these RGs are introduced in detail. The information provided here will help to design effective strategies for the development of RGs in horticultural plants, with a focus on using big data generated by RNA-Seq.

Selection and validation of reference genes for quantitative real-time PCR of Quercus mongolica Fisch. ex Ledeb under abiotic stresses

Quercus mongolica Fisch. ex Ledeb is the main species of coniferous and broadleaved mixed forests in northeast and north China, which has high ornamental, economic, and ecological value. The appropriate reference genes must be selected for quantitative real-time PCR to reveal the molecular mechanisms of stress responses and their contribution to breeding of Q. mongolica. In the present study, we chose 11 candidate reference genes (TUA, CYP18, HIS4, RPS13, ACT97, TUB1, UBQ10, UBC5, SAND, PP2A, and SAMDC) and used four programs (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of the above genes in roots, stems, and leaves under five abiotic stress factors (cold, salt, drought, weak light, and heavy metal). The findings revealed that under various experimental environments, the most stable genes were different; CYP18, ACT97, and RPS13 ranked the highest under most experimental environments. Moreover, two genes induced by stress, CMO and P5CS2, were chosen to demonstrate the reliability of the selected reference genes in various tissues under various stress conditions. Our research provides a significant basis for subsequent gene function studies of Q. mongolica.

Selection of Appropriate Housekeeping Genes for Gene Expression Normalization in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Since lots of researches using qPCR technique have addressed gene expression in groupers, the suitable housekeeping genes are needed to obtain reliable and precise results. In this study, by using different mathematical algorithms (Genorm, normFinder, BestKeeper and comparative ΔCt method) and the comprehensive ranking software (RefFinder), the stability of seventeen housekeeping genes were assessed in eleven tissues of the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Results showed that the stability of housekeeping genes were various in different tissues and the newly identified housekeeping genes were likely more stable than that of commonly used. In conclusion, the expression stability of housekeeping genes showed a tissue-specific manner, and the conserved oligomeric Golgi complex subunit 5 (cog5) and peroxisomal biogenesis factor 14 (pex14) could be universally used as appropriate housekeeping genes across different tissues in hybrid grouper. The present study provides the foundation for more accurate results of qPCR assay in hybrid grouper and other groupers.

Expression stability of ACTB, 18S, and GAPDH in human placental tissues from subjects with PCOS and controls: GAPDH expression is increased in PCOS.

The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls. mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools. ACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05). ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS.

The effect of the PLIN1 gene on the metabolism and mitochondria of porcine skeletal muscle satellite cells.

Perilipin 1 (PLIN1) is a lipid droplet scaffolding protein that plays a regulatory role in fat decomposition and mitochondrial function. In this study, the effects of PLIN1 gene knockout (PLIN1-KO) and PLIN1 gene overexpression (PLIN1-EX) on cell metabolism and mitochondrial function in porcine skeletal muscle satellite cells were assessed. Porcine skeletal muscle satellite cells were used as the control group (NC). The expression of mitochondrial function-related proteins was detected by western blot. Apoptosis, cell cycle, mitochondrial function-related indices, mitochondrial structure, and morphology were measured by flow cytometry. Our results demonstrated that stable expression of the PLIN1 gene in skeletal muscle satellite cells is critical to maintaining cell metabolism and mitochondrial function. After knockout and overexpression of the PLIN1 gene, the anti-apoptotic ability of cells was enhanced, and the metabolic activity of the cells was accelerated, but at the cost of mitochondrial structural damage, reduction in the number of mitochondria, and decreased mitochondrial function. This study explored the effect of the PLIN1 gene on the mitochondria and metabolism of porcine skeletal muscle satellite cells and provided a theoretical basis for the subsequent study of the effects of PLIN1 on muscle tissue development and meat quality.

Marker-Free Rice (Oryza sativa L. cv. IR 64) Overexpressing PDH45 Gene Confers Salinity Tolerance by Maintaining Photosynthesis and Antioxidant Machinery.

Helicases function as key enzymes in salinity stress tolerance, and the role and function of PDH45 (pea DNA helicase 45) in stress tolerance have been reported in different crops with selectable markers, raising public and regulatory concerns. In the present study, we developed five lines of marker-free PDH45-overexpressing transgenic lines of rice (Oryza sativa L. cv. IR64). The overexpression of PDH45 driven by CaMV35S promoter in transgenic rice conferred high salinity (200 mM NaCl) tolerance in the T1 generation. Molecular attributes such as PCR, RT-PCR, and Southern and Western blot analyses confirmed stable integration and expression of the PDH45 gene in the PDH45-overexpressing lines. We observed higher endogenous levels of sugars (glucose and fructose) and hormones (GA, zeatin, and IAA) in the transgenic lines in comparison to control plants (empty vector (VC) and wild type (WT)) under salt treatments. Furthermore, photosynthetic characteristics such as net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 (Ci), and chlorophyll (Chl) content were significantly higher in transgenic lines under salinity stress as compared to control plants. However, the maximum primary photochemical efficiency of PSII, as an estimated from variable to maximum chlorophyll a fluorescence (Fv/Fm), was identical in the transgenics to that in the control plants. The activities of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (GPX), were significantly higher in transgenic lines in comparison to control plants, which helped in keeping the oxidative stress burden (MDA and H2O2) lesser on transgenic lines, thus protecting the growth and photosynthetic efficiency of the plants. Overall, the present research reports the development of marker-free PDH45-overexpressing transgenic lines for salt tolerance that can potentially avoid public and biosafety concerns and facilitate the commercialization of genetically engineered crop plants.

METTL3-stabilized lncRNA SNHG7 accelerates glycolysis in prostate cancer via SRSF1/c-Myc axis

BackgroundLong non-coding RNAs (lncRNAs) have emerged as novel players in cancer metabolism. lncRNA small nucleolar RNA host gene 7 (SNHG7) plays an oncogenic role in prostate cancer (PCa). However, the role and mechanism of SNHG7 in PCa metabolism remain largely undefined. MethodsA cohort of 30 PCa tumors and their counterparts were collected. qRT-PCR was employed to detect target gene expression and RNA stability. CCK-8 assay was used to assess cell viability. N6‐methyladenosine (m6A) level was measured by a commercial kit. Cell glycolysis was evaluated by measuring glucose uptake, lactate, ATP production and Extracellular acidification rate (ECAR). Bioinformatics analysis and RNA immunoprecipitation (RIP) assay were used to verify the interactions among SNHG7, serine/arginine-rich splicing factor 1 (SRSF1) and c-Myc. ResultsSNHG7 and c-Myc were highly expressed in PCa tissues and cells. Methyltransferase-like 3 (METTL3)-mediated m6A modification of SNHG7 and enhanced its stability. Silencing of SNHG7 suppressed proliferation and glycolysis in PCa cells. Mechanistically, SNHG7 regulated c-Myc via interacting with SRSF1. Gain- and loss-of function experiments revealed that SNHG7 promoted glycolysis via SRSF1/c-Myc axis in PC-3 and DU-145 cells. ConclusionMETTL3-stabilized lncRNA SNHG7 accelerates glycolysis in PCa via SRSF1/c-Myc axis and inspires the understanding of m6A roles in lncRNA metabolism and tumor progression.

Effect of Metformin on HIF-1α Signaling and Postoperative Adhesion Formation.

Peritoneal adhesion formation is common after abdominal surgery and results in severe complications. Tissue hypoxia is one of the main drivers of peritoneal adhesions. Thus, we determined the clinical role of hypoxia-inducible factor (HIF)-1 signaling in peritoneal adhesions and investigated whether the biguanide antidiabetic drug metformin shows HIF-inhibitory effects and could be repurposed to prevent adhesion formation. As part of the ReLap study (DRKS00013001), adhesive tissue from patients undergoing relaparotomy was harvested and graded using the adhesion grade score. HIF-1 signaling activity within tissue biopsies was determined and correlated with adhesion severity. The effect of metformin on HIF-1 activity was analyzed by quantification of HIF target gene expression and HIF-1 protein stabilization in human mesothelial cells and murine fibroblast under normoxia and hypoxia. Mice were treated with vehicle or metformin 3 days before and until 7 days after induction of peritoneal adhesions; alternatively, metformin treatment was discontinued 48 hours before induction of peritoneal adhesions. HIF-1 signaling activity correlated with adhesion severity in patient biopsies. Metformin significantly mitigated HIF-1 activity in vitro and in vivo. Oral treatment with metformin markedly prevented adhesion formation in mice even when the treatment was discontinued 48 hours before surgery. Although metformin treatment did not alter macrophage polarization, metformin reduced proinflammatory leucocyte infiltration and attenuated hypoxia-induced profibrogenic expression patterns and myofibroblast activation. Metformin mitigates adhesion formation by inhibiting HIF-1-dependent (myo)fibroblast activation, conferring an antiadhesive microenvironment after abdominal surgery. Repurposing the clinically approved drug metformin might be useful to prevent or treat postoperative adhesions.

Modulation of RNA stability regulates gene expression in two opposite ways: through buffering of RNA levels upon global perturbations and by supporting adapted differential expression.

The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. Here, we explore the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. We identify two distinct RNA-stability mediated responses involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, we show that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

Biallelic PAN2 variants in individuals with a syndromic neurodevelopmental disorder and multiple congenital anomalies.

PAN2 encodes a subunit of a deadenylation complex with important functions in mRNA stability and post-transcriptional regulation of gene expression. A homozygous frameshift deletion in PAN2 was reported in a single affected individual with developmental delay and multiple congenital anomalies. Here, we describe five additional individuals from three unrelated families with homozygous predicted loss-of-function variants in PAN2. The affected individuals presented with significant overlap in their clinical features, including mild-moderate intellectual disability, hypotonia, sensorineural hearing loss, EEG abnormalities, congenital heart defects (tetralogy of Fallot, septal defects, dilated aortic root), urinary tract malformations, ophthalmological anomalies, short stature with other skeletal anomalies, and craniofacial features including flat occiput, ptosis, long philtrum, and short neck. Our data confirm that biallelic predicted loss-of-function variants in PAN2 cause a syndrome with multiple congenital anomalies, and suggest an important role of mRNA polyA tail length for proper organ formation.

Comprehensive analysis of expression and prognosis for LMNB family genes in human sarcoma.

Previous studies indicated that lamin proteins were thought to be related to gene expression, chromatin structure, and unclear stability. There are 2 types of vertebrate lamins, including A and B. The 2 B type proteins are encoded by lamin B1 (LMNB1) and lamin B2 (LMNB2). The LMNBs factor has been found to be associated with the development of multiple tumors, but its association with sarcoma has been barely mentioned.The transcription levels of LMNBs were analyzed via Oncomine database. Gene Expression Profiling Interactive Analysis (GEPIA) dataset was adopted to analyze the differential expression of LMNBs in sarcoma. Cancer Cell Line Encyclopedia dataset was used to explore the expression of LMNBs in sarcoma cell line. We analyzed the prognostic value of LMNBs in GEPIA and Kaplan-Meier Plotter. Oncomine and GEPIA datasets were also used to detect the relationship between LMNBs and their co-expressed genes. We used the Database for Annotation, Visualization and Integrated Discovery to conduct the Gene Ontology analysis of LMNBs and their co-expressed genes. Kyoto Encyclopedia of Genes and Genomes was also used to analyze the pathway of LMNBs.LMNB1 and LMNB2 were reported to be hyperexpressed in sarcoma. The expression of LMNBs was elevated in various sarcoma cell lines. According to the results, we observed that LMNBs were connected to the poor overall survival, recurrence-free survival, and disease-free survival of sarcoma patients.This study indicated that hyperexpression of LMNBs was significantly related to worse outcome of sarcoma, LMNB1 and LMNB2 were expected to become potential biomarkers for human.

Gene Body Methylation in Plants: Mechanisms, Functions, and Important Implications for Understanding Evolutionary Processes.

Gene body methylation (gbM) is an epigenetic mark where gene exons are methylated in the CG context only, as opposed to CHG and CHH contexts (where H stands for A, C, or T). CG methylation is transmitted transgenerationally in plants, opening the possibility that gbM may be shaped by adaptation. This presupposes, however, that gbM has a function that affects phenotype, which has been a topic of debate in the literature. Here, we review our current knowledge of gbM in plants. We start by presenting the well-elucidated mechanisms of plant gbM establishment and maintenance. We then review more controversial topics: the evolution of gbM and the potential selective pressures that act on it. Finally, we discuss the potential functions of gbM that may affect organismal phenotypes: gene expression stabilization and upregulation, inhibition of aberrant transcription (reverse and internal), prevention of aberrant intron retention, and protection against TE insertions. To bolster the review of these topics, we include novel analyses to assess the effect of gbM on transcripts. Overall, a growing body of literature finds that gbM correlates with levels and patterns of gene expression. It is not clear, however, if this is a causal relationship. Altogether, functional work suggests that the effects of gbM, if any, must be relatively small, but there is nonetheless evidence that it is shaped by natural selection. We conclude by discussing the potential adaptive character of gbM and its implications for an updated view of the mechanisms of adaptation in plants.

Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris.

Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways has proven to be exceedingly difficult, as the cofactors, cellular conditions, and energy sources necessary for this pathway to function may not be present in the host organism. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris, including origins of replication, fluorescent reporters, terminators, and 5′ untranslated regions, and employed the microbe’s endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris’ many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.

Optogenetic Control of Engrafted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Live Mice: A Proof-of-Concept Study.

Background: Cellular transplantation has emerged as promising approach for treating cardiac diseases. However, a poor engraftment rate limits our understanding on how transplanted cardiomyocytes contribute to cardiac function in the recipient’s heart. Methods: The CRISPR/Cas9 technique was employed for stable and constitutive gene expression in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Myocardial infarction was induced in adult immunodeficient mice, followed by intramyocardial injection of hiPSC-CMs expressing either CCND2/channelrhodopsin 2 (hiPSC-CCND2OE/ChR2OECMs) or CCND2/luciferase (hiPSC-CCND2OE/LuciOECMs). Six months later, hemodynamics and intramural electrocardiogram were recorded upon blue light illuminations in anesthetized, open-chest mice. Results: Blue light resets automaticity of spontaneously beating hiPSC-CCND2OE/ChR2OECMs in culture, but not that of hiPSC-CCND2OE/LuciOECMs. Response to blue light was also observed in mice carrying large (>106 cells) intracardiac grafts of hiPSC-CCND2OE/ChR2OECM but not in mice carrying hiPSC-CCND2OE/LuciOECMs. The former exhibited single premature ventricular contractions upon light illumination or ventricular quadrigeminy upon second-long illuminations. At the onset of premature ventricular contractions, maximal systolic ventricular pressure decreased while ventricular volume rose concomitantly. Light-induced changes reversed upon resumption of sinus rhythm. Conclusions: We established an in vivo model for optogenetic-based modulation of the excitability of donor cardiomyocytes in a functional, reversible, and localized manner. This approach holds unique value for studying electromechanical coupling and molecular interactions between donor cardiomyocytes and recipient hearts in live animals.

A Dual-Purpose Real-Time Indicator and Transcriptional Integrator for Calcium Detection in Living Cells.

Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator that combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides a real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts calcium-generated bioluminescence into stable gene expression. We also show that LuCID's modular design enables it to read out other cellular events such as protein-protein interactions. LuCID adds to the arsenal of tools for studying cells and cell populations that utilize calcium for signaling.

Decoding the sorghum methylome: understanding epigenetic contributions to agronomic traits.

DNA methylation is a chromatin modification that plays an essential role in regulating gene expression and genome stability and it is typically associated with gene silencing and heterochromatin. Owing to its heritability, alterations in the patterns of DNA methylation have the potential to provide for epigenetic inheritance of traits. Contemporary epigenomic technologies provide information beyond sequence variation and could supply alternative sources of trait variation for improvement in crops such as sorghum. Yet, compared with other species such as maize and rice, the sorghum DNA methylome is far less well understood. The distribution of CG, CHG, and CHH methylation in the genome is different compared with other species. CG and CHG methylation levels peak around centromeric segments in the sorghum genome and are far more depleted in the gene dense chromosome arms. The genes regulating DNA methylation in sorghum are also yet to be functionally characterised; better understanding of their identity and functional analysis of DNA methylation machinery mutants in diverse genotypes will be important to better characterise the sorghum methylome. Here, we catalogue homologous genes encoding methylation regulatory enzymes in sorghum based on genes in Arabidopsis, maize, and rice. Discovering variation in the methylome may uncover epialleles that provide extra information to explain trait variation and has the potential to be applied in epigenome-wide association studies or genomic prediction. DNA methylation can also improve genome annotations and discover regulatory elements underlying traits. Thus, improving our knowledge of the sorghum methylome can enhance our understanding of the molecular basis of traits and may be useful to improve sorghum performance.

Identification of Suitable Reference Genes for qRT-PCR Normalization in Kiwifruit

Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes ACT1, ACT2, GAPDH, 18S rRNA, UBQ, TUB and CYP were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. GAPDH, ACT1 and ACT2 were selected as reference genes for different genotypes of kiwifruit. GAPDH and UBQ were the best combinations of reference genes for root, stem, leaf, flower and fruit. GAPDH and ACT1 could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of ACT1 and UBQ constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit.

Avian Orthoavulavirus Type-1 as Vaccine Vector against Respiratory Viral Pathogens in Animal and Human.

Avian orthoavulaviruses type-1 (AOaV-1) have recently transitioned from animal vaccine vector to a bona fide vaccine delivery vehicle in human. Owing to induction of robust innate and adaptive immune responses in mucus membranes in both birds and mammals, AOaVs offer an attractive vaccine against respiratory pathogens. The unique features of AOaVs include over 50 years of safety profile, stable expression of foreign genes, high infectivity rates in avian and mammalian hosts, broad host spectrum, limited possibility of recombination and lack of pre-existing immunity in humans. Additionally, AOaVs vectors allow the production of economical and high quantities of vaccine antigen in chicken embryonated eggs and several GMP-grade mammalian cell lines. In this review, we describe the biology of AOaVs and define protocols to manipulate AOaVs genomes in effectively designing vaccine vectors. We highlighted the potential and established portfolio of AOaV-based vaccines for multiple respiratory and non-respiratory viruses of veterinary and medical importance. We comment on the limitations of AOaV-based vaccines and propose mitigations strategies. The exploitation of AOaVs vectors is expanding at an exciting pace; thus, we have limited the scope to their use as vaccines against viral pathogens in both animals and humans.