Large Gene Family Research Articles

Comparative analysis of basic helix\u2013loop\u2013helix gene family among Brassica oleracea, Brassica rapa, and Brassica napus

BackgroundThe basic helix–loop–helix (bHLH) is the second largest gene family in the plant, some members play important roles in pistil development and response to drought, waterlogging, cold stress and salt stress. The bHLH gene family has been identified in many species, except for Brassica oleracea and B. napus thus far. This study aims to identify the bHLH family members in B. oleracea, B. rapa and B. napus, and elucidate the expression, duplication, phylogeny and evolution characters of them.ResultA total of 268 bHLH genes in B. oleracea, 440 genes in B. napus, and 251 genes in B. rapa, including 21 new bHLH members, have been identified. Subsequently, the analyses of the phylogenetic trees, conserved motifs and gene structures showed that the members in the same subfamily were highly conserved. Most Ka/Ks values of homologous gene were < 1, which indicated that these genes suffered from strong purifying selection for retention. The retention rates of BrabHLH and BolbHLH genes were 51.6 and 55.1%, respectively. The comparative expression patterns between B. rapa and B. napus showed that they had similar expression patterns in the root and contrasting patterns in the stems, leaves, and reproductive tissues. In addition, there were 41 and 30 differential expression bHLH genes under the treatments of ABA and JA, respectively, and the number of down regulation genes was significantly more than up regulation genes.ConclusionIn the present study, we identified and performed the comparative genomics analysis of bHLH gene family among B. oleracea, B. rapa and B. napus, and also investigated their diversity. The expression patterns between B. rapa and B. napus shows that they have the similar expression pattern in the root and opposite patterns in the stems, leaves, and reproduction tissues. Further analysis demonstrated that some bHLH gene members may play crucial roles under the abiotic and biotic stress conditions. This is the first to report on the bHLH gene family analysis in B. oleracea and B. napus, which can offer useful information on the functional analysis of the bHLH gene in plants.

Transcriptional analysis of arogenate dehydratase genes identifies a link between phenylalanine biosynthesis and lignin biosynthesis.

Biogenesis of the secondary cell wall in trees involves the massive biosynthesis of the phenylalanine-derived polymer lignin. Arogenate dehydratase (ADT) catalyzes the last, and rate-limiting, step of the main pathway for phenylalanine biosynthesis. In this study, we found that transcript levels for several members of the large ADT gene family, including ADT-A and ADT-D, were enhanced in compression wood of maritime pine, a xylem tissue enriched in lignin. Transcriptomic analysis of maritime pine silenced for PpMYB8 revealed that this gene plays a critical role in coordinating the deposition of lignin with the biosynthesis of phenylalanine. Specifically, it was found that ADT-A and ADT-D were strongly down-regulated in PpMYB8-silenced plants and that they were transcriptionally regulated through direct interaction of this transcription factor with regulatory elements present in their promoters. Another transcription factor, PpHY5, exhibited an expression profile opposite to that of PpMYB8 and also interacted with specific regulatory elements of ADT-A and ADT-D genes, suggesting that it is involved in transcriptional regulation of phenylalanine biosynthesis. Taken together, our results reveal that PpMYB8 and PpHY5 are involved in the control of phenylalanine formation and its metabolic channeling for lignin biosynthesis and deposition during wood formation in maritime pine.

CsCYT75B1, a Citrus CYTOCHROME P450 Gene, Is Involved in Accumulation of Antioxidant Flavonoids and Induces Drought Tolerance in Transgenic Arabidopsis.

CYTOCHROME P450s genes are a large gene family in the plant kingdom. Our earlier transcriptome data revealed that a CYTOCHROME P450 gene of Citrus sinensis (CsCYT75B1) was associated with flavonoid metabolism and was highly induced after drought stress. Here, we characterized the function of CsCYT75B1 in drought tolerance by overexpressing it in Arabidopsis thaliana. Our results demonstrated that the overexpression of the CsCYT75B1 gene significantly enhanced the total flavonoid contents with increased antioxidant activity in transgenic Arabidopsis. The gene expression results showed that several genes that are responsible for the biosynthesis of antioxidant flavonoids were induced by 2–12 fold in transgenic Arabidopsis lines. After 14 days of drought stress, all transgenic lines displayed an enhanced tolerance to drought stress along with accumulating antioxidant flavonoids with lower superoxide radicals and reactive oxygen species (ROS) than wild type plants. In addition, drought-stressed transgenic lines possessed higher antioxidant enzymatic activities than wild type transgenic lines. Moreover, the stressed transgenic lines had significantly lower levels of electrolytic leakage than wild type transgenic lines. These results demonstrate that the CsCYT75B1 gene of sweet orange functions in the metabolism of antioxidant flavonoid and contributes to drought tolerance by elevating ROS scavenging activities.

On the Evolution and Functional Diversity of Terpene Synthases in the Pinus Species: A Review.

In the biosynthesis of terpenoids, the ample catalytic versatility of terpene synthases (TPS) allows the formation of thousands of different molecules. A steadily increasing number of sequenced plant genomes invariably show that the TPS gene family is medium to large in size, comprising from 30 to 100 functional members. In conifers, TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, and diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. Such substances are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress. Oleoresin terpenoids can be also profitably used in a number of different fields, from traditional and modern medicine to fine chemicals, fragrances, and flavours, and, in the last years, in biorefinery too. In the present work, after summarizing the current views on the biosynthesis and biological functions of terpenoids, recent advances on the evolution and functional diversification of plant TPSs are reviewed, with a focus on gymnosperms. In such context, an extensive characterization and phylogeny of all the known TPSs from different Pinus species is reported, which, for such genus, can be seen as the first effort to explore the evolutionary history of the large family of TPS genes involved in specialized metabolism. Finally, an approach is described in which the phylogeny of TPSs in Pinus spp. has been exploited to isolate for the first time mono-TPS sequences from Pinus nigra subsp. laricio, an ecologically important endemic pine in the Mediterranean area.

Genome-wide analysis of the NAC transcription factor family in broomcorn millet (Panicum miliaceum L.) and expression analysis under drought stress

BackgroundBroomcorn millet is a drought-tolerant cereal that is widely cultivated in the semiarid regions of Asia, Europe, and other continents; however, the mechanisms underlying its drought-tolerance are poorly understood. The NAM, ATAF1/2, and CUC2 (NAC) transcription factors form a large plant-specific gene family that is involved in the regulation of tissue development and abiotic stress. To date, NAC transcription factors have not been systematically researched in broomcorn millet.ResultsIn the present study, a total of 180 NAC (PmNAC) genes were identified from the broomcorn millet genome and named uniformly according to their chromosomal distribution. Phylogenetic analysis demonstrated that the PmNACs clustered into 12 subgroups, including the broomcorn millet-specific subgroup Pm_NAC. Gene structure and protein motif analyses indicated that closely clustered PmNAC genes were relatively conserved within each subgroup, while genome mapping analysis revealed that the PmNAC genes were unevenly distributed on broomcorn millet chromosomes. Transcriptome analysis revealed that the PmNAC genes differed greatly in expression in various tissues and under different drought stress durations. The expression of 10 selected genes under drought stress was analyzed using quantitative real-time PCR.ConclusionIn this study, 180 NAC genes were identified in broomcorn millet, and their phylogenetic relationships, gene structures, protein motifs, chromosomal distribution, duplication, expression patterns in different tissues, and responses to drought stress were studied. These results will be useful for the further study of the functional characteristics of PmNAC genes, particularly with regards to drought resistance.

Analysis of the Rdr1gene family in different Rosaceae genomes reveals an origin of an R-gene cluster after the split of Rubeae within the Rosoideae subfamily.

The Rdr1 gene confers resistance to black spot in roses and belongs to a large TNL gene family, which is organized in two major clusters at the distal end of chromosome 1. We used the recently available chromosome scale assemblies for the R. chinensis 'Old Blush' genome, re-sequencing data for nine rose species and genome data for Fragaria, Rubus, Malus and Prunus to identify Rdr1 homologs from different taxa within Rosaceae. Members of the Rdr1 gene family are organized into two major clusters in R. chinensis and at a syntenic location in the Fragaria genome. Phylogenetic analysis indicates that the two clusters existed prior to the split of Rosa and Fragaria and that one cluster has a more recent origin than the other. Genes belonging to cluster 2, such as the functional Rdr1 gene muRdr1A, were subject to a faster evolution than genes from cluster 1. As no Rdr1 homologs were found in syntenic positions for Prunus persica, Malus x domestica and Rubus occidentalis, a translocation of the Rdr1 clusters to the current positions probably happened after the Rubeae split from other groups within the Rosoideae approximately 70-80 million years ago during the Cretaceous period.

An Evolutionary View of Trypanosoma Cruzi Telomeres.

Like in most eukaryotes, the linear chromosomes of Trypanosoma cruzi end in a nucleoprotein structure called the telomere, which is preceded by regions of variable length called subtelomeres. Together telomeres and subtelomeres are dynamic sites where DNA sequence rearrangements can occur without compromising essential interstitial genes or chromosomal synteny. Good examples of subtelomeres involvement are the expansion of human olfactory receptors genes, variant surface antigens in Trypanosoma brucei, and Saccharomyces cerevisiae mating types. T. cruzi telomeres are made of long stretches of the hexameric repeat 5′-TTAGGG-OH-3′, and its subtelomeres are enriched in genes and pseudogenes from the large gene families RHS, TS and DGF1, DEAD/H-RNA helicase and N-acetyltransferase, intermingled with sequences of retrotransposons elements. In particular, members of the Trans-sialidase type II family appear to have played a role in shaping the current T. cruzi telomere structure. Although the structure and function of T. cruzi telomeric and subtelomeric regions have been documented, recent experiments are providing new insights into T. cruzi's telomere-subtelomere dynamics. In this review, I discuss the co-evolution of telomere, subtelomeres and the TS gene family, and the role that these regions may have played in shaping T. cruzi's genome.

Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat

Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients.

Towards coeliac-safe bread.

SummaryGluten‐free foods cannot substitute for products made from wheat flour. When wheat products are digested, the remaining peptides can trigger an autoimmune disease in 1% of the North American and European population, called coeliac disease. Because wheat proteins are encoded by a large gene family, it has been impossible to use conventional breeding to select wheat varieties that are coeliac‐safe. However, one can test the properties of protein variants by expressing single genes in coeliac‐safe cereals like maize. One source of protein that can be considered as coeliac‐safe and has bread‐making properties is teff (Eragrostis tef), a grain consumed in Ethiopia. Here, we show that teff α‐globulin3 (Etglo3) forms storage vacuoles in maize that are morphologically similar to those of wheat. Using transmission electron microscopy, immunogold labelling shows that Etglo3 is almost exclusively deposited in the storage vacuole as electron‐dense aggregates. Of maize seed storage proteins, 27‐kDa γ‐zein is co‐deposited with Etglo3. Etglo3 polymerizes via intermolecular disulphide bonds in maize, similar to wheat HMW glutenins under non‐reducing conditions. Crossing maize Etglo3 transgenic lines with α‐, β‐ and γ‐zein RNA interference (RNAi) lines reveals that Etglo3 accumulation is only dramatically reduced in γ‐zein RNAi background. This suggests that Etglo3 and 27‐kDa γ‐zein together cause storage vacuole formation and behave similar to the interactions of glutenins and gliadins in wheat. Therefore, expression of teff α‐globulins in maize presents a major step in the development of a coeliac‐safe grain with bread‐making properties.

Evolutionary Analysis of Calcium-Dependent Protein Kinase in Five Asteraceae Species.

Calcium-dependent protein kinase (CPK) is crucial in Ca2+ signal transduction, and is a large gene family in plants. In our previous work, we reported Hevea brasiliensis CPKs were important for natural rubber biosynthesis. However, this CPK gene family in other rubber producing plants has not been investigated. Here, we report the CPKs in five representative Asteraceae species, including three rubber-producing and two non-rubber species. A total of 34, 34, 40, 34 and 30 CPKs were identified from Taraxacum koksaghyz, Lactuca sativa, Helianthus annuus, Chrysanthemum nankingense and Cynara cardunculus, respectively. All CPKs were classified into four individual groups (group I to IV). In addition, 10 TkCPKs, 11 LsCPKs, 20 HaCPKs, 13 CnCPKs and 7 CcCPKs duplicated paralogs were identified. Further evolutionary analysis showed that, compared to other subfamilies, the group III had been expanded in the Asteraceae species, especially in the rubber-producing species. Meanwhile, the CPKs in group III from Asteraceae species tend to expand with low calcium binding capacity. This study provides a systematical evolutionary investigation of the CPKs in five representative Asteraceae species, suggesting that the sub-family specific expansion of CPKs might be related to natural rubber producing.

Predicting transfer RNA gene activity from sequence and genome context.

Transfer RNA (tRNA) genes are among the most highly transcribed genes in the genome owing to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with difficulty in measuring transcript abundance and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish sequence-based correlates to tRNA gene expression and develop a tRNA gene classification method that does not require, but benefits from, comparative genomic information and achieves accuracy comparable to molecular assays. We observe that guanine + cytosine (G + C) content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our tRNA gene activity predictions in conjunction with a comprehensive tRNA gene ortholog set spanning 29 placental mammals to estimate the evolutionary rate of functional changes among orthologs. Our method adds a new dimension to large-scale tRNA functional prediction and will help prioritize characterization of functional tRNA variants. Its simplicity and robustness should enable development of similar approaches for other clades, as well as exploration of functional diversification of members of large gene families.

Towards a plant model for enigmatic U-to-C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis.

Hornworts are crucial to understand the phylogeny of early land plants. The emergence of 'reverse' U-to-C RNA editing accompanying the widespread C-to-U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort-tracheophyte clade. C-to-U RNA editing is well understood after identification of many editing factors in models like Arabidopsis thaliana and Physcomitrella patens, but there is no plant model yet to investigate U-to-C RNA editing. The hornwort Anthoceros agrestis is now emerging as such a model system. We report on the assembly and analyses of the A.agrestis chloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors. Both organelles in A.agrestis feature high amounts of RNA editing, with altogether >1100 sites of C-to-U and 1300 sites of U-to-C editing. The nuclear genome reveals >1400 genes for PPR proteins with variable carboxyterminal DYW domains. We observe significant variants of the 'classic' DYW domain, in the meantime confirmed as the cytidine deaminase for C-to-U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U-to-C editing targets according to the PPR-RNA binding code.

Programmed Cell Death in Neurospora crassa Is Controlled by the Allorecognition Determinant rcd-1.

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.

Genome-wide identification, characterisation and functional evaluation of WRKY genes in the sweet potato wild ancestor Ipomoea trifida (H.B.K.) G. Don. under abiotic stresses

BackgroundWRKY DNA-binding protein (WRKY) is a large gene family involved in plant responses and adaptation to salt, drought, cold and heat stresses. Sweet potato from the genus Ipomoea is a staple food crop, but the WRKY genes in Ipomoea species remain unknown to date. Hence, we carried out a genome-wide analysis of WRKYs in Ipomoea trifida (H.B.K.) G. Don., the wild ancestor of sweet potato.ResultsA total of 83 WRKY genes encoding 96 proteins were identified in I. trifida, and their gene distribution, duplication, structure, phylogeny and expression patterns were studied. ItfWRKYs were distributed on 15 chromosomes of I. trifida. Gene duplication analysis showed that segmental duplication played an important role in the WRKY gene family expansion in I. trifida. Gene structure analysis showed that the intron-exon model of the ItfWRKY gene was highly conserved. Meanwhile, the ItfWRKYs were divided into five groups (I, IIa + IIb, IIc, IId + IIe and III) on the basis of the phylogenetic analysis on I. trifida and Arabidopsis thaliana WRKY proteins. In addition, gene expression profiles confirmed by quantitative polymerase chain reaction showed that ItfWRKYs were highly up-regulated or down-regulated under salt, drought, cold and heat stress conditions, implying that these genes play important roles in response and adaptation to abiotic stresses.ConclusionsIn summary, genome-wide identification, gene structure, phylogeny and expression analysis of WRKY gene in I. trifida provide basic information for further functional studies of ItfWRKYs and for the molecular breeding of sweet potato.

Silencing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem

Both suberin and its associated waxes contribute to the formation of apoplastic barriers that protect plants from the environment. Some transcription factors have emerged as regulators of the suberization process. The potato StNAC103 gene was reported as a repressor of suberin polyester and suberin-associated waxes deposition because its RNAi-mediated downregulation (StNAC103-RNAi) over-accumulated suberin and associated waxes in the tuber phellem concomitantly with the induction of representative biosynthetic genes. Here, to explore if other genes of the large NAC gene family participate to this repressive function, we extended the silencing to other NAC members by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c). Transcript profile of the StNAC103-RNAi-c phellem indicated that StNAC101 gene was an additional potential target. In comparison with StNAC103-RNAi, the silencing with StNAC103-RNAi-c construct resulted in a similar effect in suberin but yielded an increased load of associated waxes in tuber phellem, mainly alkanes and feruloyl esters. Globally, the chemical effects in both silenced lines are supported by the transcript accumulation profile of genes involved in the biosynthesis, transport and regulation of apoplastic lipids. In contrast, the genes of polyamine biosynthesis were downregulated. Altogether these results point out to StNAC101 as a candidate to repress the suberin-associated waxes.

Molecular switch from MYC to MYCN expression in MYC protein negative Burkitt lymphoma cases

MYC is the most altered oncogene in human cancer, and belongs to a large family of genes, including MYCN and MYCL. Recently, while assessing the degree of correlation between MYC gene rearrangement and MYC protein expression in aggressive B-cell lymphomas, we observed few Burkitt lymphoma (BL) cases lacking MYC protein expression despite the translocation involving the MYC gene. Therefore, in the present study we aimed to better characterize such cases. Our results identified two sub-groups of MYC protein negative BL: one lacking detectable MYC protein expression but presenting MYCN mRNA and protein expression; the second characterized by the lack of both MYC and MYCN proteins but showing MYC mRNA. Interestingly, the two sub-groups presented a different pattern of SNVs affecting MYC gene family members that may induce the switch from MYC to MYCN. Particulary, MYCN-expressing cases show MYCN SNVs at interaction interface that stabilize the protein associated with loss-of-function of MYC. This finding highlights MYCN as a reliable diagnostic marker in such cases. Nevertheless, due to the overlapping clinic, morphology and immunohistochemistry (apart for MYC versus MYCN protein expression) of both sub-groups, the described cases represent bona fide BL according to the current criteria of the World Health Organization.

The poplar R2R3 MYB transcription factor PtrMYB94 coordinates with abscisic acid signaling to improve drought tolerance in plants.

In plants, R2R3 MYB transcription factors (TFs) consist of one large gene family and are involved in the regulation of many developmental processes and various stresses. However, the functions of most of MYB TFs in woody plants remain unknown. Here, PtrMYB94, an R2R3 MYB TF from Populus trichocarpa, is characterized to be involved in the regulation of drought responses and abscisic acid (ABA) signaling. PtrMYB94 encodes a nuclear-localized R2R3 MYB TF. RT-PCR results showed that the PtrMYB94 transcripts were relatively abundant in leaves and stems, and were induced rapidly in response to dehydration stress. Overexpression of PtrMYB94 improved plant drought responses, suggesting that this MYB TF may functionally regulate poplar adaptability to drought stress. Furthermore, the analysis of transcriptional expression and PtrMYB94 promoter: GUS activity showed that PtrMYB94 responded to ABA induction. PtrMYB94-overexpressing plants exhibited the inhibition of seed germination compared with the wild-type (WT) control under ABA exposure condition. The ABA content was evidently increased in the PtrMYB94-overexpressing plants relative to the WT plants. In addition, transcript levels of several ABA- and drought-responsive genes, such as ABA1 and DREB2B, were up-regulated. Taken together, our results suggest that PtrMYB94 is involved in an ABA-dependent drought stress regulation in Populus.

Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala

BackgroundLinoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members.ResultsFull-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid.ConclusionsThis research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

The Basis for Strain-Dependent Rat Aldehyde Dehydrogenase 1A7 (ALDH1A7) Gene Expression.

Aldehyde hydrogenases (ALDHs) belong to a large gene family involved in oxidation of both endogenous and exogenous compounds in mammalian tissues. Among ALDHs, the rat ALDH1A7 gene displays a curious strain dependence in phenobarbital (PB)-induced hepatic expression: the responsive RR strains exhibit induction of both ALDH1A7 and CYP2B mRNAs and activities, whereas the nonresponsive rr strains show induction of CYP2B only. Here, we investigated the responsiveness of ALDH1A1, ALDH1A7, CYP2B1, and CYP3A23 genes to prototypical P450 inducers, expression of nuclear receptors CAR and pregnane X receptor, and structure of the ALDH1A7 promoter in both rat strains. ALDH1A7 mRNA, associated protein and activity were strongly induced by PB and modestly induced by pregnenolone 16α-carbonitrile in the RR strain but negligibly in the rr strain, whereas induction of ALDH1A1 and P450 mRNAs was similar between the strains. Reporter gene and chromatin immunoprecipitation assays indicated that the loss of ALDH1A7 inducibility in the rr strain is profoundly linked with a 16-base pair deletion in the proximal promoter and inability of the upstream DNA sequences to recruit constitutive androstane receptor-retinoid X receptor heterodimers. SIGNIFICANCE STATEMENT: Genetic variation in rat ALDH1A7 promoter sequences underlie the large strain-dependent differences in expression and inducibility by phenobarbital of the aldehyde dehydrogenase activity. This finding has implications for the design and interpretation of pharmacological and toxicological studies on the effects and disposition of aldehydes.

Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi

BackgroundCRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan–chitin crosslinking enzymes involved in cell wall biosynthesis.ResultsHere, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9–sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9–sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members.ConclusionsOur findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.