High Throughput Sequencing of circRNAs in Tomato Leaves Responding to Multiple Stresses of Drought and Heat

Our aim is to study the roles of a new emerging group of non-coding RNAs, circRNAs, in tomato(Solanum lycopersicum L.) plants grown at the combination of drought and heat, two of the most usual stress conditions known to frequently happen in field. Tomato seedlings from cultivar‘Jinling Meiyu’ were treated without stresses(control), at water shortage, high temperature and subjected the multiple stresses. In total, 467 circRNAs were identified with 87.82% from exon using high throughput sequencing technology. Among the circRNAs, 70 were from chr1 with the range from 23 to 49 from the other chromosomes. In detail, 156 circRNAs were shared in the four libraries, while 21, 17 and 36 circRNAs were only shown in drought, heat and multiple stresses libraries, respectively. Through a differential expression analysis, four, seven and nine circRNAs were differentially regulated in tomato at drought, heat and multiple stresses as compared with control. These circRNAs played roles on photosynthesis, starch and sucrose metabolism, RNA transport, RNA degradation, spliceosome, ribosome, etc. Our study underlined the potential role of circRNAs involved in the abiotic stress response in tomato, which might pave the way for studying biological roles of circRNAs responding to multiple stresses in plants.

Expression of long non-coding RNAs in complete transection spinal cord injury: a transcriptomic analysis

Long non-coding RNAs(lncRNAs)are abundantly expressed in the central nervous system and exert a critical role in gene regulation via multiple biological processes.To uncover the functional significance and molecular mechanisms of lncRNAs in spinal cord injury(SCI),the expression signatures of lncRNAs were profiled using RNA sequencing(RNA-seq)technology in a Sprague-Dawley rat model of the 10th thoracic vertebra complete transection SCI.Results showed that 116 of 14,802 detected lncRNAs were differentially expressed,among which 16—including eight up-regulated(H19,Vof16,Hmox2-ps1,LOC100910973,Ybx1-ps3,Nnat,Gcgr,LOC680254)and eight down-regulated(Rmrp,Terc,Ngrn,Ppp2r2b,Cox6a2,Rpl37a-ps1,LOC360231,Rpph1)—demonstrated fold changes>2 in response to transection SCI.A subset of these RNA-seq results was validated by quantitative real-time PCR.The levels of 821 mRNAs were also significantly altered post-SCI;592 mRNAs were up-regulated and 229 mRNAs were down-regulated by more than 2-fold.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that differentially expressed mRNAs were related to GO biological processes and molecular functions such as injury and inflammation response,wound repair,and apoptosis,and were significantly enriched in 15 KEGG pathways,including cell phagocytosis,tumor necrosis factor alpha pathway,and leukocyte migration.Our results reveal the expression profiles of lncRNAs and mRNAs in the rat spinal cord of a complete transection model,and these differentially expressed lncRNAs and mRNAs represent potential novel targets for SCI treatment.We suggest that lncRNAs may play an important role in the early immuno-inflammatory response after spinal cord injury.This study was approved by the Administration Committee of Experimental Animals,Guangdong Province,China.

Structural shift of gut microbiota during chemopreventive effects of epigallocatechin gallate on colorectal carcinogenesis in mice

AIM To investigate the effect of epigallocatechin gallate(EGCG) on structural changes of gut microbiota in colorectal carcinogenesis.METHODS An azoxymethane(AOM)/dextran sodium sulfate(DSS)-induced colitis mouse model was established. Fortytwo female FVB/N mice were randomly divided into the following three groups: group 1(10 mice, negative control) was treated with vehicle, group 2(16 mice, positive control) was treated with AOM plus vehicle, and group 3(16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci(ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16 S r RNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16 S r RNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed.RESULTS Treatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS(5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85%(33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria(Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics(Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG's effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group(EG) remained relatively stable.CONCLUSION Gut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.

Effect of glucose on the soil bacterial diversity and function in the rhizosphere of Cerasus sachalinensis

Most cherry orchards in China have low organic carbon content,though carbon is very important for plant growth.The changes in soil carbon and bacterial diversity were determined after different amounts of 12C-glucose were added to the rhizosphere of Cerasus sachalinensis.Soil bacteria diversity was measured using high throughput sequencing,and bacteria containing 13C-glucose were identified using DNA-SIP methods.The results demonstrated that soil microbial biomass carbon(MBC)content and the soil respiratory rate were increased at 3 and 7 days after adding glucose.The soil organic carbon(SOC)content was decreased on the 7th day in the treatment where the added glucose-C was equivalent to the MBC content.SOC content was decreased on the 15th day after adding glucose-C equivalent to five times that of the soil MBC.Compared to the controls,the relative abundance of taxa at the phylum level displayed no significant change in the treatments with glucose-C added as 10%and equal amount of soil MBC 3–30 days after treatment.However,the relative abundance of Proteobacteria increased significantly in the treatment with the addition of glucose-C equivalent to five times of soil MBC.The main changes were observed in the bacteria in several genera including A4,Flavisolibacter,Aquicella,and Candidatus Solibacter.DNA-SIP results indicated that the relative abundance of the Proteobacteria and Pseudomonas was the highest;these were the primary bacteria phylum and genus,respectively,from day 3 to day 15.In conclusion,the changing pattern demonstrated that with the addition of more glucose,the range of the bacterial communities changed more.Proteobacteria and Pseudomonas may be the bacteria promoting priming effect.

Characterization of maize leaf pyruvate orthophosphate dikinase using high throughput sequencing

In C4 photosynthesis, pyruvate orthophosphate dikinase （PPDK） catalyzes the regeneration of phospho- enolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Nlutator and Ds to generate multiple mutant alleles of PPDK. Loss-of- function mutants are seedling lethal, even when plants were grown under 2% CO2, and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we exam- ined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression ofenzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

Diversity and community structure of eukaryotic microalgae in surface sediments in the central Bohai Sea,China,based on a metabarcoding approach

Sediment samples were collected at 17 stations in the central Bohai Sea,China,and the diversity and community structure of eukaryotic microalgae were assessed by metabarcoding the V4 region of 18S r DNA.A total of 930 operational taxonomic units(OTUs)were detected for microeukaryotes,including 98algal OTUs.The algal communities comprised 42 genera belonging to 19 classes of six phyla,and they were dominated by chrysophytes and dinoflagellates.Dinoflagellates were also the most diverse microalgal group.The nano-sized dinoflagellates Biecheleria halophila and Azadinium trinitatum occurred abundantly in the study area;however,they have not been reported previously,as they may be overlooked or misidentified in light microscopy.Many pico-sized chlorophytes were detected in the sediment samples.Sixteen of the detected OTUs were assigned to potentially harmful and/or bloom-forming microalgae,suggesting some potential risks of harmful algal blooms in the central Bohai Sea.The capacity of metabarcoding to detect morphologically cryptic and small species makes this method a sufficiently sensitive means of detection for assessing eukaryotic microalgae in sediments.

Particle size shapes the prokaryotic microbial communities in mangrove sediments:A case study of Sanya,China

The prokaryotic microbial communities in the sediments play crucial roles in the ecological functions of mangrove ecosystems.Therefore,the environmental factors that affect the structures of these prokaryotic microbial communities could indirectly participate in the regulation of mangrove functions,which is of great value for mangrove studies.The particle size(PS)of soils is recently demonstrated as a key environmental factor for shaping the microbial communities;however,this hypothesis has rarely been tested for mangrove environments.A case study of three tropical mangroves from Sanya,China was performed in this work to assess the influence of PS on the prokaryotic microbial community structures of bacteria,archaea,diazotrophs,and denitrifiers in the sediments.Results showed the variability in the spatial scale and the stability in the temporal scale for the prokaryotic communities,indicating that the tropical mangrove sediments could be a versatile but stable environment.Among the collected environmental factors,PS,salinity,and humidity had the greatest impacts,and PS mostly affected the structures of these prokaryotic communities based on its highest R^2 values of canonical correspondence analysis,Mental test,and linear fitting(p≤0.05).Furthermore,PS was positively correlated with the diversity and abundance of diazotrophic communities and negatively correlated with the abundances of methanogenic communities including Methanobacteriaceae,Methanospirillaceae,Methanoregulaceae,and Methanosaetaceae.Former studies show the increasing trend of PS caused by the rise of sea level and the intensification of human activities.Therefore,our findings indicate that PS could be a potential intermediate that links climate change and human activities with the possible ecological function migration of mangroves;meanwhile,the increase of PS could in turn release the stress of these environmental changes by increasing the abundance and diversity of the diazotrophic community and decreasing the abundances of methanogens.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

Bladder Malignant Granular Cell Tumor with EP300 Gene Mutation: A Case Report and Literature Review

Background: Malignant granular cell tumor (GCT) is extremely rare. Malignant GCT with EP300 gene mutation in the bladder has not been reported in the literature. Case Presentation: We report a special case of 45-year-old female with malignant GCT of the bladder. Pathological examination showed that the mass was 11 × 11 × 4.5 cm in size, involved in the bladder’s posterior wall. Under the microscope, the tumor cells were arranged in the shape of a nest or cord to infiltrate the bladder’s wall. The tumor cells were pleomorphic, red-stained granular within the cytoplasm, with increased nuclear/cytoplasmic ratio, vacuolar nuclei, and obvious nucleoli. The tumor cells were showed obvious nuclear atypia, and the mitosis was more than 5/50HPF. Coagulative necrosis was widely showed within the tumor. Immunohistochemistry (IHC) showed that S-100, NSE, CD68, CR, α-AT, and TFE-3 were strongly positive, and the Ki-67 proliferation index was around 15%. The next-generation high throughput sequencing indicated that EP300 gene was missense mutated (c.457A > G) with 33% mutation abundance, and genes of DPYD (c.1627A > G), ERCC1 (c.354T > C), NQO1 (c.559C > T), TPMT (c.719A > G) and XRCC1 (c.1196A > G) were polymorphic mutated. The patient died after three months of the second surgical treatment. Conclusions: We report for the first time a primary bladder malignant GCM accompanied by mutations in special driving genes such as EP300. We also conducted a comprehensive literature review and an in-depth discussion.

Whole-genome sequencing in medicinal plants:current progress and prospect

Advancements in genomics have dramatically accelerated the research on medicinal plants,and the development of herbgenomics has promoted the“Project of 1K Medicinal Plant Genome”to decipher their genetic code.However,it is difficult to obtain their high-quality whole genomes because of the prevalence of polyploidy and/or high genomic heterozygosity.Whole genomes of 123 medicinal plants were published until September 2022.These published genome sequences were investigated in this review,covering their classification,research teams,ploidy,medicinal functions,and sequencing strategies.More than 1,000 institutes or universities around the world and 50 countries are conducting research on medicinal plant genomes.Diploid species account for a majority of sequenced medicinal plants.The whole genomes of plants in the Poaceae family are the most studied.Almost 40%of the published papers studied species with tonifying,replenishing,and heat-cleaning medicinal effects.Medicinal plants are still in the process of domestication as compared with crops,thereby resulting in unclear genetic backgrounds and the lack of pure lines,thus making their genomes more difficult to complete.In addition,there is still no clear routine framework for a medicinal plant to obtain a high-quality whole genome.Herein,a clear and complete strategy has been originally proposed for creating a high-quality whole genome of medicinal plants.Moreover,whole genome-based biological studies of medicinal plants,including breeding and biosynthesis,were reviewed.We also advocate that a research platform of model medicinal plants should be established to promote the genomics research of medicinal plants.

Digitalizing river aquatic ecosystems

Traditional river health assessment relies on limited water quality indices and representative organism activity,but does not comprehensively obtain biotic and abiotic information of the ecosystem.Here,we propose a new approach to evaluate the ecological and health risks of river aquatic ecosystems.First,detailed physicochemical and biological characterization of a river ecosystem can be obtained through pollutant determination(especially emerging pollutants)and DNA/RNA sequencing.Second,supervised machine learning can be applied to perform classification analysis of characterization data and ascertain river ecosystem ecology and health.Our proposed methodology transforms river ecosystem health assessment and can be applied in river management.

Identification and Characterization of ABA-Responsive MicroRNAs in Rice

MicroRNAs （miRNAs） are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid （ABA） is essential for plant development and adaptation to abiotic and biotic stresses. In Arabidopsis, miRNAs are implicated in ABA functions. However, ABA-responsive miRNAs have not been systematically studied in rice. Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant, Osabal. Of these, 13 were confirmed by stem-loop RT-PCR. Among them, miR1425-5P, miR169a, miR169n, miR390-5P, miR397a and miR397b were up-regulated, but miR162b reduced in expression in Osabal. The targets of these 13 miRNAs were predicted and validated by gene expression profiling. Interestingly, the expression levels of these miRNAs and their targets were regulated by ABA. Cleavage sites were detected on 7 of the miRNA targets by 5＇-Rapid Amplification of cDNA Ends （5＇-RACE）. Finally, miR162b and its target OsTRE1 were shown to affect rice resistance to drought stress, suggesting that miR162b increases resistance to drought by targeting OsTRE1. Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.

Dietary protein levels and amino acid supplementation patterns alter the composition and functions of colonic microbiota in pigs

Different dietary nitrogen(N) patterns may have different effects on gut microbiota.To investigate the effects of different crude protein(CP) levels or essential amino acids(EAA) supplementation patterns on the structure and functions of colonic microbiota,42 barrows(25±0.39 kg) were randomly assigned to 7 dietary treatments including:diet 1,a high CP diet with balanced 10 EAA;diet 2,a medium CP diet with approximately 2% decreased CP level from diet 1 and balanced 10 EAA;diets 3,4,5,6 and 7,low CP diets with 4% decreased CP level from diet 1.Specifically,diet 3 was only balanced for Lys,Met,Thr and Trp;diets 4,5 and 6 were further supplemented with IIe,Val and IIe+Val on the basis of diet 3,respectively;and diet 7 was balanced for 10 EAA.Results over a 110-d trial showed that reducing the CP level by 2% or4% dramatically decreased N intake and excretion(P <0.05) in the presence of balanced 10 EAA,which was not observed when altering the EAA supplementation patterns in low CP diet(-4%).With balanced10 EAA,2% reduction in dietary CP significantly reduced Firmicutes-to-Bacteroidetes(F:B) ratio and significantly elevated the abundance of Prevotellaceae NK3 B31(P <0.05);whereas 4% reduction evidently increased the abundances of Proteobacteria,Succinivibrio and Lachnospiraceae XPB1014(P <0.05).Among the 5 low CP diets(-4%),supplementation with Ile,or Val+Ile,or balanced 10 EAA increased F:B ratio and the abundance of Proteobacteria.In addition,the predicted functions revealed that different CP levels and EAA balanced patterns dramatically altered the mRNA expression profiles of N-metabolizing genes,the "N and energy metabolism" pathways or the metabolism of some small substances,such as amino acids(AA) and vitamins.Our findings suggested that reducing the dietary CP levels by 2% to 4% with balancing 10 EAA,or only further supplementation with Ile or Val+Ile to a low protein diet(-4%) reduced the N contents entering the hindgut to various degrees,altered the abundances of N-metabolizing bacteria,and improved the abilities of N utilization.

Profiling kidney microRNAs from juvenile grass carp(Ctenopharyngodon idella) after 56 days of oral exposure to decabromodiphenyl ethane

Grass carp（Ctenopharyngodon idella） is one of the most important species in China.Decabromodiphenyl ethane（DBDPE） is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs（miRNAs） are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags（ESTs） of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated（FDR 〈 0.001,1.5-fold change） after DBDPE exposure. Real-time quantitative PCR（RT-qPCR） was performed on 4 miRNAs from the two samples, and the sequencing and RT-qPCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.