Conserved Sequence Domains Research Articles

The genome sequencing and comparative genomics analysis of Rhizoctonia solani reveals a novel effector family owning a uinque domain in Basidiomycetes

Rhizoctonia solani is a soil-borne pathogen with 14 anastomosis groups (AGs), and different subgroups are genetically diverse. However, the genetic factors contributing to the pathogenicity of the fungus have not been well characterized. In this study, the genome of R. solani AG1-ZJ was sequenced. As the result, a 41.57 Mb draft genome containing 12,197 putative coding genes was obtained. Comparative genomic analysis of 11 different AGs revealed conservation and unique characteristics between the AGs. Furthermore, a novel effector family containing a 68 amino acid conserved domain unique in basidiomycetous fungi was characterized. Two effectors containing the conserved domain in AG4-JY were identified, and named as RsUEB1 and RsUEB2. Furthermore, the spray-induced gene silencing strategy was used to generate a dsRNA capable of silencing the conserved domain sequence of RsUEB1 and RsUEB2. This dsRNA can significantly reduce the expression of RsUEB1 and RsUEB2 and the pathogenicity of AG4-JY on foxtail millet, maize, rice and wheat. In conclusion, this study provides significant insights into the pathogenicity mechanisms of R. solani. The identification of the conserved domain and the successful use of dsRNA silencing of the gene containing the conserved domain will offer a new strategy for controlling sheath blight in cereal crops.

LncRNA CCRR maintains Ca2+ homeostasis against myocardial infarction through the FTO-SERCA2a pathway.

Cardiac conduction regulatory RNA (CCRR) has been documented as an antiarrhythmic lncRNA in our earlier investigation. This study aimed to evaluate the effects of CCRR on SERCA2a and the associated Ca2+ homeostasis in myocardial infarction (MI). Overexpression of CCRR via AAV9-mediated delivery not only partially reversed ischemia-induced contractile dysfunction but also alleviated abnormal Ca2+ homeostasis and reduced the heightened methylation level of SERCA2a following MI. These effects were also observed in CCRR over-expressing transgenic mice. A conserved sequence domain of CCRR mimicked the protective function observed with the full length. Furthermore, silencing CCRR in healthy mice led to intracellular Ca2+ overloading of cardiomyocytes. CCRR increased SERCA2a protein stability by upregulating FTO expression. The direct interaction between CCRR and FTO protein was characterized by RNA-binding protein immunoprecipitation (RIP) analysis and RNA pulldown experiments. Activation of NFATc3 was identified as an upstream mechanism responsible for CCRR downregulation in MI. This study demonstrates that CCRR is a protective lncRNA that acts by maintaining the function of FTO, thereby reducing the m6A RNA methylation level of SERCA2a, ultimately preserving calcium homeostasis for myocardial contractile function in MI. Therefore, CCRR may be considered a promising therapeutic strategy with a beneficial role in cardiac pathology.

Cloning and Analysis of T-DNA Targeted Gene of Colletotrichum gloeosporioides Pathogenicity-Defective Strain Cs16

In preliminary stage, the nucleotide sequence of Cs16 Colletotrichum gloeosporioides associated gene obtained from database of (NCBI), analyzed with Clustalx 1.83 in order to assess the deduced amino acid sequences. The full-length sequences of Cs16 gene is including with specific Open Reading Frame (ORF) having 3’ and 5’ UTR was obtained. In order to assess the relationship of C. gloeosporioides associated CS16 gene with other members, a combined rooted neighbor-joining (NJ) tree was generated through MEGA 4.5 by following 1000 bootstrap replicates. The resulted alignment predicts that Cs16 gene showed entirely diverse sequences from other species and has no any conserved domain sequences between them. In order to scrutinize the full length nucleotide sequences, the gene functional cloning of Cs16 gene has been carried out by PCR amplification. To examine the functional analysis of Cs16 gene in C. gloeosporioides and its regulatory mechanisms, the over expressing vector (pGapneoR26) was transformed into Agrobacterium strain LBA4404 that resulted successful detection of GFP-Cs16 expression into C. gloeosporioides transformants and it was also substantially detected by fluorescence microscopy. The T-DNA intentional mutants of C. gloeosporioides mycelia illustrated phonotypic different intensification, colour, colony growth rate than wild type isolated C. gloeosporioides STIJ6 strain.

Chromosome-level genome assembly of Mentha longifolia L. reveals gene organization underlying disease resistance and essential oil traits

Mentha longifolia (L.) Huds., a wild, diploid mint species, has been developed as a model for mint genetic and genomic research to aid breeding efforts that target Verticillium wilt disease resistance and essential oil monoterpene composition. Here, we present a near-complete, chromosome-scale mint genome assembly for M. longifolia USDA accession CMEN 585. This new assembly is an update of a previously published genome draft, with dramatic improvements. A total of 42,107 protein-coding genes were annotated and placed on 12 chromosomal scaffolds. One hundred fifty-three genes contained conserved sequence domains consistent with nucleotide binding site-leucine-rich-repeat plant disease resistance genes. Homologs of genes implicated in Verticillium wilt resistance in other plant species were also identified. Multiple paralogs of genes putatively involved in p-menthane monoterpenoid biosynthesis were identified and several cases of gene clustering documented. Heterologous expression of candidate genes, purification of recombinant target proteins, and subsequent enzyme assays allowed us to identify the genes underlying the pathway that leads to the most abundant monoterpenoid volatiles. The bioinformatic and functional analyses presented here are laying the groundwork for using marker-assisted selection in improving disease resistance and essential oil traits in mints.

Evolutionary History of RNA Modifications at N6-Adenosine Originating from the R-M System in Eukaryotes and Prokaryotes.

Simple SummaryThe m6A is the most abundant and well-studied modification of mRNA, and plays an important role in transcription and translation. It is known to be evolutionarily conserved machinery present in the last eukaryotic common ancestor (LECA). The writers and erasers responsible for adding or removing m6A belong to specific protein families, respectively, suggesting that these members are evolutionarily related. However, only some of these mRNA m6A modification-associated proteins have been studied from an evolutionary perspective, while there has been no comprehensive and systematic analysis of the distributions and evolutionary history of N6mA-associated proteins in the three kingdoms of life. In this study, we identified orthologues of all the reported N6mA-associated proteins in 88 organisms from three kingdoms of life and comprehensively reconstructed the evolutionary history of the RNA N6mA modification machinery. The results demonstrate that RNA N6mA-MTases are derived from at least two different types of prokaryotic DNA MTases (class α and β MTases). As the m6A reader, YTH proteins may be acquired by LECA from an individual prokaryotic YTH-domain protein that evolved from the N-terminals of an R-M system endonuclease. In addition, the origin of eukaryotic ALKBH family proteins is inferred to be driven by at least two occasions of independent HTG from the bacterial ALKB family.Methylation at the N6-position of adenosine (N6mA) on mRNA (m6A) is one of the most widespread, highly selective and dynamically regulated RNA modifications and plays an important role in transcription and translation. In the present study, a comprehensive analysis of phylogenetic relationships, conserved domain sequence characteristics and protein structure comparisons were employed to explore the distribution of RNA N6mA modification (m6A, m6,6A, m6Am, m6, 6Am and m6t6A)-associated proteins (writers, readers and erasers) in three kingdoms of life and reveal the evolutionary history of these modifications. These findings further confirmed that the restriction-modification (R-M) system is the origin of DNA and RNA N6mA modifications. Among them, the existing mRNA m6A modification system derived from the last eukaryotic common ancestor (LECA) is the evolutionary product of elements from the last universal common ancestor (LUCA) or driven by horizontal gene transfer (HGT) from bacterial elements. The subsequent massive gene gains and losses contribute to the development of unique and diverse functions in distinct species. Particularly, RNA methyltransferases (MTases) as the writer responsible for adding N6mA marks on mRNA and ncRNAs may have evolved from class α and β prokaryotic “orphan” MTases originating from the R-M system. The reader, YTH proteins that specifically recognize the m6A deposit, may be acquired by LECA from an individual prokaryotic YTH-domain protein that evolved from N-terminals of an R-M system endonuclease. The eraser, which emerged from the ALKB family (ALKBH5 and FTO) in eukaryotes, may be driven by independent HTG from bacterial ALKB proteins. The evolutionary history of RNA N6mA modifications was inferred in the present study, which will deepen our understanding of these modifications in different species.

Genome-Wide Analysis of the Fatty Acid Desaturase Gene Family Reveals the Key Role of PfFAD3 in α-Linolenic Acid Biosynthesis in Perilla Seeds.

Perilla (Perilla frutescens), a traditional medicinal and oilseed crop in Asia, contains extremely high levels of polyunsaturated α-linolenic acid (ALA) (up to 60.9%) in its seeds. ALA biosynthesis is a multistep process catalyzed by fatty acid desaturases (FADs), but the FAD gene family in perilla has not been systematically characterized. Here, we identified 42 PfFADs in the perilla genome and classified them into five subfamilies. Subfamily members of PfFADs had similar exon/intron structures, conserved domain sequences, subcellular localizations, and cis-regulatory elements in their promoter regions. PfFADs also possessed various expression patterns. PfFAD3.1 was highly expressed in the middle stage of seed development, whereas PfFAD7/8.3 and PfFAD7/8.5 were highly expressed in leaf and later stages of seed development, respectively. Phylogenetic analysis revealed that the evolutionary features coincided with the functionalization of different subfamilies of PUFA desaturase. Heterologous overexpression of PfFAD3.1 in Arabidopsis thaliana seeds increased ALA content by 17.68%–37.03%. These findings provided insights into the characteristics and functions of PfFAD genes in perilla.

Nitric Oxide Negatively Regulates Larval Metamorphosis in Hard-Shelled Mussel (Mytilus coruscus)

Marine invertebrates undergo distinct life cycles and development processes, and some undergo a metamorphic transition from pelagic larvae to benthic juvenile. Previous studies have shown that the second messenger nitric oxide (NO) plays important roles in regulating larval metamorphosis in marine invertebrates. However, this regulatory function is not conserved. Herein, we investigated the regulatory role of NO in larval metamorphosis in Hard-shelled mussel (Mytilus coruscus). Pharmacological experiments revealed that after exposure to nitric oxide synthase (NOS) inhibitors, the larval metamorphosis rate was increased significantly. By contrast, exposure to NO donors significantly reduced the larval metamorphosis rate. To further investigate the function of NO in metamorphosis, we cloned the NOS cDNA sequence from M. coruscus (McNOS), analyzed amino acid sequences of McNOS from different species, and measured McNOS mRNA temporal expression alongside the molecular chaperone heat shock protein 90 (HSP90) in different larval stages. The results showed that NOS proteins share a highly conserved domain sequence in different species, even in invertebrates and vertebrates, but their classification among invertebrates and vertebrates is not the same. Except for the umbo veliger stage, McNOS mRNA was expressed at low levels in pediveliger larval and post-larvae stage, indicating a potential inhibitory function for NO in larval metamorphosis. Meanwhile, HSP90 mRNA expression was correspondingly high in the umbo veliger stage and lower in the pediveliger larval and post-larvae stage, indicating potential functional synergism. Interestingly, McNOS enzyme activity in different larval stages was inconsistent with its mRNA expression. Metamorphosis assays of pharmacological treatments combined with McNOS and HSP90 gene expression and NOS enzyme activity analyses indicated that NO acts as a suppressor to regulate metamorphosis in M. coruscus, regardless of whether nitric oxide is endogenous or exogenous.

De novo transcriptome analysis of stressed blood clam (Anadara broughtonii) and identification of genes associated with hemoglobin.

Blood clam (Anadara broughtonii) is a commercially important marine bivalve characterised by the red blood. Recently, the clams have been subjected to severe resource recession. Multiple environmental stressors are indispensable for the recession. We aimed to investigate the transcriptome information of blood clam under environmental stressors. Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Several public databases were introduced for gene annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathways analyses. The Open Reading Frame of annotated hemoglobin (Hb) was predicted and validated by DNAMAN 6.0 and NCBI BLASTx, respectively. A total of 242,919 transcripts were generated, 116,264 unigenes were subsequently assembled with an average length of 747 base pairs, and 33,776 unigenes were successfully annotated. Gene Ontology (GO) categories indicated that the terms of cellular processes, metabolic processes, cell, cell part, binding, and catalytic activity were dominant. KEGG pathway analyses suggested ribosome, oxytocin, focal adhesion, Ras, and PI3K-Akt were the largest signaling pathway groups, and many presented pathways (Ras, Rap1, and MAPK, etc.) were related to apoptosis, immune and stress response. In addition, a total of 19,306 potential simple sequence repeats (SSRs) were detected in 15,852 sequences. Six hemoglobin-related genes with complete conserved domain sequences were identified and 3 of them were predicted as HbI, HbIIα, and HbIIβ. This study provides transcriptome responses to multiple environmental stressors in blood clams and would provide interesting hints for further studies.

Genome-wide analysis of the Catalpa bungei caffeic acid O-methyltransferase (COMT) gene family: identification and expression profiles in normal, tension, and opposite wood.

Caffeic acid O-methyltransferase (COMT) is an important protein that participates in lignin synthesis and is associated with the ratio of G-/S-type lignin in plants. COMTs are associated with the wood properties of forest trees; however, little known about the COMT family in Catalpa bungei, a valuable timber tree species in China . We performed a comprehensive analysis of COMT genes in the C. bungei genome by describing the gene structure and phylogenetic relationships of each family member using bioinformatics-based methods. A total of 23 putative COMT genes were identified using the conserved domain sequences and amino acid sequences of COMTs from Arabidopsis thaliana and Populus trichocarpa as probes. Phylogenetic analysis showed that 23 CbuCOMTs can be divided into three groups based on their structural characteristics; five conserved domains were found in the COMT family. Promoter analysis indicated that the CbuCOMT promoters included various cis-acting elements related to growth and development. Real-time quantitative polymerase chain reaction (PCR) analysis showed differential expression among CbuCOMTs. CbuCOMT2, 7, 8, 9, 10, 12, 13, 14, 21, and 23 were mainly expressed in xylem. Only CbuCOMT23 was significantly downregulated in tension wood and upregulated in opposite wood compared to normal wood. Our study provides new information about the CbuCOMT gene family and will facilitate functional characterisation in further research.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci

BackgroundThe mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality.ResultsWe identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other’s expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers.ConclusionsThis work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Diversification of the Histone Acetyltransferase GCN5 through Alternative Splicing in Brachypodium distachyon.

The epigenetic modulatory SAGA complex is involved in various developmental and stress responsive pathways in plants. Alternative transcripts of the SAGA complex's enzymatic subunit GCN5 have been identified in Brachypodium distachyon. These splice variants differ based on the presence and integrity of their conserved domain sequences: the histone acetyltransferase domain, responsible for catalytic activity, and the bromodomain, involved in acetyl-lysine binding and genomic loci targeting. GCN5 is the wild-type transcript, while alternative splice sites result in the following transcriptional variants: L-GCN5, which is missing the bromodomain and S-GCN5, which lacks the bromodomain as well as certain motifs of the histone acetyltransferase domain. Absolute mRNA quantification revealed that, across eight B. distachyon accessions, GCN5 was the dominant transcript isoform, accounting for up to 90% of the entire transcript pool, followed by L-GCN5 and S-GCN5. A cycloheximide treatment further revealed that the S-GCN5 splice variant was degraded through the nonsense-mediated decay pathway. All alternative BdGCN5 transcripts displayed similar transcript profiles, being induced during early exposure to heat and displaying higher levels of accumulation in the crown, compared to aerial tissues. All predicted protein isoforms localize to the nucleus, which lends weight to their purported epigenetic functions. S-GCN5 was incapable of forming an in vivo protein interaction with ADA2, the transcriptional adaptor that links the histone acetyltransferase subunit to the SAGA complex, while both GCN5 and L-GCN5 interacted with ADA2, which suggests that a complete histone acetyltransferase domain is required for BdGCN5-BdADA2 interaction in vivo. Thus, there has been a diversification in BdGCN5 through alternative splicing that has resulted in differences in conserved domain composition, transcript fate and in vivo protein interaction partners. Furthermore, our results suggest that B. distachyon may harbor compositionally distinct SAGA-like complexes that differ based on their histone acetyltransferase subunit.

Genome-Wide Analysis of the RAV Family in Soybean and Functional Identification of GmRAV-03 Involvement in Salt and Drought Stresses and Exogenous ABA Treatment.

Transcription factors play vital roles in plant growth and in plant responses to abiotic stresses. The RAV transcription factors contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. Although genome-wide analyses of RAV family genes have been performed in several species, little is known about the family in soybean (Glycine max L.). In this study, a total of 13 RAV genes, named as GmRAVs, were identified in the soybean genome. We predicted and analyzed the amino acid compositions, phylogenetic relationships, and folding states of conserved domain sequences of soybean RAV transcription factors. These soybean RAV transcription factors were phylogenetically clustered into three classes based on their amino acid sequences. Subcellular localization analysis revealed that the soybean RAV proteins were located in the nucleus. The expression patterns of 13 RAV genes were analyzed by quantitative real-time PCR. Under drought stresses, the RAV genes expressed diversely, up- or down-regulated. Following NaCl treatments, all RAV genes were down-regulated excepting GmRAV-03 which was up-regulated. Under abscisic acid (ABA) treatment, the expression of all of the soybean RAV genes increased dramatically. These results suggested that the soybean RAV genes may be involved in diverse signaling pathways and may be responsive to abiotic stresses and exogenous ABA. Further analysis indicated that GmRAV-03 could increase the transgenic lines resistance to high salt and drought and result in the transgenic plants insensitive to exogenous ABA. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in soybean.

Micropropagation and Androgenesis in coconut: an assessment of Sri Lankan implication

The coconut palm is an important cash crop mostly in the sub-tropical countries which provides almost all the necessities of life to the mankind. Genetic improvement for increased productivity is a prioritized research area of coconut palm. In vitro cloning of elite palms via somatic embryogenesis seems to be a promising method due to its potential for massive propagation. Based on the Sri Lankan experience, this paper summarizes the notable achievements towards developing a reliable protocol for clonal propagation of coconut and also dihaploid plant production. The newel explants described by Coconut Research Institute Sri Lanka (CRISL), unfertilized ovaries dissected from immature inflorescences collected from -4 stage (inflorescence to be opened in 4 months time), is a somatic tissue which causes minimal damage to the mother palm while collecting. Limited number of female flowers in an inflorescence which limits large scale culturing could overcome by callus multiplication phase to produce higher number of plants. Somatic embryogenesis and germination of somatic embryos was achieved by reduction of auxin concentration in the culture medium followed by an addition of cytokinin. AINTEGUMENTA-LIKE (ANT-like) gene isolated from coconut genome which shows higher conserved domain sequence similarities with BABYBOOM gene will assist development of suitable molecular markers to assess the morphogenic potential of in vitro cultured coconut tissues and callus. Successful plant regeneration from cultured anthers was reported by culturing anthers at three weeks before splitting (3-WBS) inflorescence stage which contains late uninucleate microspores. Heat pretreatment of anthers for six days at 38°C induced androgenesis in coconut upon culturing in 2, 4-D and 1-naphthyl acetic acid (NAA) added medium. Current clonal propagation protocol is efficient when responsive genotype is in use.Further improvement to current protocol will enable commercially viable protocol for coconut micropropagation.

Co-infection of a hypovirulent isolate of Sclerotinia sclerotiorum with a new botybirnavirus and a strain of a mitovirus.

BackgroundSclerotinia sclerotiorum, a notorious plant fungal pathogen, causes yield loss of many crops and vegetables, and is a natural host of a diverse viruses with positive-sense RNA (+ssRNA), negative-sense RNA (−ssRNA), double-stranded RNA (dsRNA), or DNA genomes. Mixed-infection with multiple related or unrelated mycoviruses is a common phenomenon in S. sclerotiorum. However, a single strain co-infected with dsRNA and + ssRNA viruses has not been reported in S. sclerotiorum.ResultsWe report two unrelated viruses, Sclerotinia sclerotiorum botybirnavirus 2 (SsBRV2) with a bipartite dsRNA genome and Sclerotinia sclerotiorum mitovirus 4 (SsMV4/AH16) with a + ssRNA genome, which were originally detected in a single hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 has a typical genome of mitovirus and is a strain of mitovirus SsMV4. The genome of SsBRV2 consists of two separated dsRNA segments. The large dsRNA segment is 6159 bp in length and only has a single open reading frame (ORF) encoding a putative 1868-aa polyprotein with a conserved RNA dependent RNA polymerase (RdRp) domain. The small dsRNA segment is 5872 bp in length and encodes a putative 1778-aa protein. Phylogenetic analysis using RdRp conserved domain sequences revealed that SsBRV2 is phylogenetically related to the previously reported three bipartite viruses SsBRV1, Botrytis porri RNA virus 1 (BpRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1). Electron microscopy demonstrated that SsBRV2 forms rigid spherical virions with a diameter of approximately 40 nm in infected mycelia. The virion of SsBRV2 was successfully introduced into a virus-free strain, which provides conclusive evidence that SsBRV2 confers hypovirulence on phytopathogenic fungus S. sclerotiorum.ConclusionsA bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented + ssRNA virus (SsMV4/AH16) were characterized in a hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 is a strain of a reported mitovirus, whereas SsBRV2 is a new botybirnavirus. SsBRV2 is the causal agent of hypovirulence on S. sclerotiorum. Our findings supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and + ssRNA mycoviruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0550-2) contains supplementary material, which is available to authorized users.

Mining of Blast Resistance Gene, Pi2 and Its Novel Allelic Variant from Landraces of Rice from Karnataka

Rice blast is one of the important fungal diseases of rice, caused by Magnaporthe oryzae. Rice blast can be effectively managed by the deployment of R-genes. Pi2 (Piz-5) is one of the major blast resistant genes effective against pathogen populations in various parts of India. In the present study, Pi2 gene was mined from 83 native landraces of rice from Karnataka state, India which revealed the presence of Pi2 in 60 landraces with the frequency of 72.28 %. Further, by using sequencing based allele mining approach, allelic variants of Pi2 from 16 landraces were analysed. High nucleotide variations (InDels) in the middle region of the conserved domain sequence of Pi2 in all variants were obtained. 469 polymorphic sites were observed with a total 570 mutations and 191 InDels among 16 Pi2 variants. Among Pi2 resistant and susceptible variants, 206 mutations with 29 InDel events were observed. Comparative modeling and Ramachandran plot of Pi2 protein variants from resistant and susceptible landraces were studied which revealed the significant differences among them. One potential novel Pi2 allelic variant from landrace Vanasurya was identified for the first time. This novel effective allelic variant can be used in rice breeding programmes to enhance blast resistance.

Alternatively spliced, spliceosomal twin introns in Helminthosporium solani

Spliceosomal twin introns, “stwintrons”, have been defined as complex intervening sequences that carry a second intron (“internal intron”) interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an “internal intron” interrupted the donor sequence of an “external intron”. Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg2+ ions lead to the production of FPP, while the presence of Co2+ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg2+ the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways.

Complete mitochondrial genome of the Onychostruthus taczanowskii (Aves, Passeridae) and its phylogenetic analysis

The complete mitochondrial genome sequence of Onychostruthus taczanowskii consisted 16 917 bp, the genome harbored the same gene order with that of other birds, contained 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 non-coding control region. The all tRNAs formed typical cloverleaf secondary structures, except tRNASer (AGN) and tRNAPhe. The control region was located between tRNAGlu and tRNAPhe with 1310 bp in length, which consist of three domains: highly variable domain I, central conserved domain II and conserved sequence domain III. According to the phylogenetic analysis, Onychostruthus taczanowskii had a closer relationship with Pyrgilauda ruficollis, and Passer montanus were sister to them.

Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

Genome-wide analysis of the AP2/ERF gene family in maize waterlogging stress response

Members of the APETALA2/ethylene response factor (AP2/ERF) transcription factor superfamily are widely present in plants and play important roles in the plant cell cycle, growth and development, as well as the response to biotic and abiotic stresses. The maize genome project has been completed; therefore, it is possible to identify all of the AP2/ERF genes in the maize genome. In this study, 184 AP2/ERF genes were identified by an in silico cloning method, and were compared with AP2/ERF genes from Arabidopsis, rice, grape and poplar. The 184 AP2/ERF maize genes were classified into four subfamilies: DREB (51), ERF (107), AP2 (22) and RAV (3), as well as one soloist. The amino acid sequence composition, physical and chemical characteristics, phylogenetic trees, conserved domain sequences, functional domains, and chromosomal location of the genes were predicted and analyzed. The 184 AP2/ERF genes are distributed on maize chromosomes 1–10 (31, 21, 13, 19, 22, 18, 21, 16, 11 and 12 genes, respectively). Under 0, 1, 2, 4 h waterlogging stress, the expression of 184 AP2/ERF genes in root of Hz32 inbred line (tolerance to waterlogging) were performed using RNA-sequence, and the result showed that 38 genes were responsive to waterlogging stress. This study lays the foundation for subsequent cloning and investigation of the function of the AP2/ERF genes responding to waterlogging stress in maize.