Increased complexity of gene structure and base composition in vertebrates

How the structure and base composition of genes changed with the evolution of vertebrates remains a puzzling question. Here we analyzed 895 orthologous protein-coding genes in six multicellular animals： human, chicken, zebrafish, sea squirt, fruit fly, and worm. Our analyses reveal that many gene regions, particularly intron and 3~ UTR, gradually expanded throughout the evolution of vertebrates from their invertebrate ancestors, and that the number of exons per gene increased. Studies based on all protein-coding genes in each genome provide consistent results. We also find that GC-content increased in many gene regions （especially 5＇ UTR） in the evolution of endotherms, except in coding-exons. Analysis of individual genomes shows that 3t UTR demonstrated stronger length and GC-content correlation with intron than 5~ UTR, and gene with large intron in all six species demonstrated relatively similar GC-content. Our data indicates a great increase in complexity in vertebrate genes and we propose that the requirement for morphological and functional changes is probably the driving force behind the evolution of structure and base composition complexity in multicellular animal genes.

Genome-wide identification and characterization of ACBP gene family in Populus reveal salinity alkali-responsive profiles

Acyl-CoA-binding proteins(ACBPs)are important for the transport of acyl groups for macro molecular biosynthesis involved in plant growth,development,and diverse stress(e.g.,cold,drought,salinity,and heavy metals)responses.Here,we report the phylogeny and characteristics of the ACBP family in the woody plant Populus trichocarpa.Eight genes encoding ACBP proteins were identified,and they are distributed on eight chromosomes in P.trichocarpa.These PtACBP genes were divided into four subgroups according to gene structure,conserved motifs and phylogenetic relationship.Promoter analysis revealed that cis-elements were related to stress response,phytohormone response,and physical and reproductive growth regulation.Expression levels of PtACBP genes varied among different organs,with the highest expression in leaves and the lowest in stems.Quantitative real-time PCR(qRT-PCR)analysis showed that under salinity-alkali stresses(i.e.,200 mM NaCl,75 mM Na2CO3,and 100 mM NaHCO3),four(PtACBP1,PtACBP3,PtACBP4 and PtACBP8)of eight PtACBP genes were significantly induced in roots and leaves.These data provide a comprehensive analysis of the ACBPs family in P.trichocarpa,which could be useful for gene function analyses.

Sequence and Bioinformatics Analysis on MSTN Gene of the Hybrid Grouper Derived from(Epinephelus fuscoguttatus×Epinephelus polyphekadion)

[Objectives]This study aimed to investigate the sequence structure and function of Myostatin(MSTN)gene in the hybrid grouper(Epinephelus fuscoguttatus,♀×Epinephelus polyphekadion,♂).[Methods]Genetic DNA samples were extracted from the caudal fins of the hybrid grouper and its parents to amplify their MSTN genes.Then,MSTN gene sequences were analyzed using bioinformatics tools to predict their protein structures and functions.[Results]The hybrid grouper and its parents shared the same MSTN gene structure,consisting of three exons and two introns.Nucleotide sequence of the gene could be translated into 376 amino acids,including an N-terminal signal peptide,a proteolytic processing site(RXXR motif),and nine conserved cysteine residues at C-terminal,which were the typical features of transforming growth factor beta(TGF-β)superfamily proteins.Alignment of protein sequence showed that MSTN was highly conserved between the hybrid grouper and its parents.Especially,exon 3,an important functional domain,exhibited a sequence similarity of 100%among them.In addition,four variable amino acid residues were detected in exon 2 at positions 141,153,185 and 186 in the hybrid grouper,but they did not affect the secondary structure of the protein.[Conclusion]These results will provide molecular information for future investigation on the growth and heterosis of hybrid grouper species,and on the roles of MSTN gene in regulating the growth traits of the hybrid grouper.

Bioinformatics analysis of structure and function in the MRP gene family and its expression in response to various drugs in Bursaphelenchus xylophilus

Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance.To further explore the molecular mechanism of drug resistance in pine wood nematode(Bursaphelenchus xylophilus),we used bioinformatics approaches to analyze genomic data for B.xylophilus and identified Bx-MRP genes.We predicted the structure and function of the genes and encoded proteins.Using bioinformatics programs to predict and analyze various properties of the predicted proteins,including hydrophobicity,transmembrane regions,phosphorylation sites,and topologically isomeric structures,of these Bx-MRP genes,we determined that they function in transmembrane transport.From the results of RT-qPCR,the Bx-MRP family members confer significant differential resistance to different drug treatments.After treatment with different concentrations of emamectin benzoate,avermectin and matrine,the expression of each gene increased with increasing drug concentrations,indicating that the family members play a positive role in the regulation of multidrug resistance.

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes (FBEs) in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.

Amplification and function analysis of N6-adenine-specific DNA methyltransferase gene in Nilaparvata lugens

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase（N6AMT） is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens（Nlu-N6AMT） and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenine Mlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding ds RNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

The evolution of resistance gene in plants

Resistance genes enable plants to fight against plant pathogens. Plant resistance genes （R gene） are organized complexly in genome. Some resistance gene sequence data enable an insight into R gene structure and gene evolution. Some sites like Leucine-Rich Repeat （LRR） are of specific interest since homologous recombination can happen. Crossing over, transposon insertion and excision and mutation can produce new specificity. Three models explaining R gene evolution were discussed. More information needed for dissection of R gene evolution though some step can be inferred from genetic and sequence analysis.

Long-term rice-rice-green manure rotation changing the microbial communities in typical red paddy soil in South China

On the basis of a long-term（30 years） field experiment that involved four rotation systems, rice-rice-winter fallow（RRF）, rice-rice-ryegrass（RRG）, rice-rice-rape（RRP）, and rice-rice-milk vetch（RRV）, this study described the effects of green manure on the microbial communities in the red paddy soils using 454 pyrosequencing for the 16 S r RNA gene. The Chao1 richness and non-parametric Shannon＇s index increased in all soil samples that received green manure treatments. The communities＇ structures with the green manure applications were significantly dissimilar from that under the winter fallow. Using Metastats tests, many genera in the RRG, RRP and RRV soils were significantly different from those in the RRF soil, including a number of genera that functioned in the nitrogen and sulfur cycles. Analyses of the genera with these functions revealed the shifts in microbial ecosystem functions after long-term green manuring. Changes in the microbial communities increased the ammonium supply and decreased the soil acidification in green-manure-amended soils. Together, these data suggested powerful effects of green manure on both the microbial communities and the biogeochemical cycle driven by the shifts in bacterial functional groups.

STRUCTURE IN THE PRECORE REGION OF HEPATITIS B CORE GENE AFFECTING ITS EXPRESSION IN E.coil

Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E.coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert’s chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter,SD sequence and identical N-terminus.The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants.The findings

A Combined Computational and Experimental Study on the Structure-Regulation Relationships of Putative Mammalian DNA Replication Initiator GINS

GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized. In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS mRNA levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17β-Estradiolstimulated estrogen receptor a, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

Genome-wide Identification and Expression of ARF Gene Family during Adventitious Root Development in Hot Pepper(Capsicum annuum)

Auxin response factors(ARFs) are transcription factors that activate or repress the expression of primary/early auxin response genes by binding to auxin-responsive elements(Aux REs) in their promoter regions. The ARFs play important roles in diverse developmental processes.To explore the ARF gene family in hot pepper(Capsicum annuum L.), we performed a genome-wide identification and expression analysis. In this study, 19 pepper ARF genes(Ca ARFs) clustered into three phylogenetic groups(I, II, and III) were comprehensively analyzed. Conserved domain analysis showed that all Ca ARFs contained a B3 DNA-binding domain and a middle domain, but two members lacked the carboxyterminal dimerization(CTD) domain. The number of introns in Ca ARF genes ranged from 1 to 13 and the gene structure was similar among genes in the same phylogenetic group. Additionally, prediction of Ca ARFs promoter elements and putative targets for micro RNAs suggested that the regulation of Ca ARFs may occur at both transcriptional and posttranscriptional levels. Most Ca ARFs were expressed in more than one tested tissue, and most Ca ARFs were identified as being responsive to exogenous auxin. Moreover, time-course transcription profiles of Ca ARFs revealed their roles in adventitious rooting of hypocotyl cuttings from pepper seedlings. Therefore, our results will provide a foundation for better understanding the regulatory mechanisms and molecular functions of Ca ARFs in hot pepper.

Effect of reclaimed water effluent on bacterial community structure in the Typha angustifolia L. rhizosphere soil of urbanized riverside wetland, China

In order to evaluate the impact of reclaimed water on the ecology of bacterial communities in the Typha angustifolia L. rhizosphere soil, bacterial community structure was investigated using a combination of terminal restriction fragment length polymorphism and 16S rRNA gene clone library. The results revealed significant spatial variation of bacterial communities along the river from upstream and downstream. For example, a higher relative abundance of γ-Proteobacteria, Firmicutes, Chloroflexi and a lower proportion of β-Proteobacteria and ε-Proteobacteria was detected at the downstream site compared to the upstream site. Additionally, with an increase of the reclaimed water interference intensity, the rhizosphere bacterial community showed a decrease in taxon richness, evenness and diversity. The relative abundance of bacteria closely related to the resistant of heavy-metal was markedly increased, while the bacteria related for carbon/nitrogen/phosphorus/sulfur cycling wasn＇t strikingly changed. Besides that, the pathogenic bacteria markedly increased in the downstream rhizosphere soil since reclaimed water supplement, while the possible plant growth-promoting rhizobacteria obviously reduced in the downstream sediment. Together these data suggest cause and effect between reclaimed water input into the wetland, shift in bacterial communities through habitat change, and alteration of capacity for biogeochemical cycling of contaminants.

Molecular features and expression of DAZAP2 in human multiple myeloma

Background In our previous study, we found that DAZAP2 was the most significantly down regulated gene when differential screening of complementary DNA （cDNA） chips were used to analyze mRNA isolated from bone marrow mononuclear cells from newly diagnosed multiple myeloma （MM） patients without anticancer treatment. In this study, we observed DAZAP2 mRNA and protein expression in the mononuclear cells from MM bone marrow and investigated its role in the pathogenesis of MM. Methods The full-length cDNA of DAZAP2 was cloned and sequenced from mononuclear cells from human bone marrow. The nucleotide and amino acid sequences of DAZAP2 were analyzed using the ClustalW program. A dendrogram was constructed by multiple sequence alignment using ClustalW and amino acid sequence identity/similarity was derived based on comparisons attained using the MegAlign software. The recombinant pEGFP expression vector was constructed and the confocal microscopy was used for the localization of the DAZAP2 protein in transfected COS7 cells. The expression of DAZAP2 mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） and the expression level of DAZAP2 protein was detected by Western blotting analysis in MM samples. Results DAZAP2 proteins of vertebrates is highly conserved in evolution. It contains a proline-rich region, several potential SH2 and SH3 domain-binding motifs and a possible protein kinase C （PKC） phosphorylation site. We showed by confocal microscopy that the DAZAP2 protein predominantly resides in the cytoplasm with a discrete pattern of punctuated distribution. The expression of DAZAP2 was not detected in 24 of 36 MM samples by semi-quantitative RT-PCR. In contrast, DAZAP2 expression was detected in all 30 normal controls. The expression level of DAZAP2 protein was assayed by Western blotting analysis, showing a robust down-regulation in MM patients （P〈0.001） that matched with the results of the RT-PCR. Conclusions DAZAP2 is downregulated in MM samples and it may be a signal molecule in MM cells. DAZAP2 is involved in the pathogenesis of MM and could be used as a genetic marker for MM.

2020 update on human coronaviruses:One health,one world

Since human coronavirus(HCoVs)was first described in the 1960s,seven strains of respiratory human coronaviruses have emerged and caused human infections.After the emergence of severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV),a pneumonia outbreak of coronavirus disease 2019(COVID-19)caused by a novel coronavirus(SARS-CoV-2)has represented a pandemic threat to global public health in the 21st century.Without effectively prophylactic and therapeutic strategies including vaccines and antiviral drugs,these three coronaviruses have caused severe respiratory syndrome and high case-fatality rates around the world.In this review,we detail the emergence event,origin and reservoirs of all HCoVs,compare the differences with regard to structure and receptor usage,and summarize therapeutic strategies for COVID-19 that cause severe pneumonia and global pandemic.