查看更多>>摘要：The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.
查看更多>>摘要：A molecular description of lignan biosynthesis in Isatis indigotica displaying its synthetic characteristics and regulatory mechanism is of great importance for the improvement of the production of this class of active compounds. To discover the potential key catalytic steps and regulatory genes, I.indigotica hairy roots elicited by methyl jasmonate (MeJA) were used as a source of systematic variation for exploring the metabolic/transcriptional changes and candidate genes that might play key roles in lignan biosynthesis. The reprogramming modulated by MeJA was classified into three distinct phases, referred to as signal responding, transcriptional activation of metabolic pathways and accumulation of metabolites. Candidate genes were pooled according to the three phases and applied to co-expression network analysis. In total, 17 genes were identified as hub genes. 4CL3 was selected to validate its impact on lignan biosynthesis. RNAi of 4CL3 resulted in a significant reduction in lignan production. Taken together with its catalytic property, a major route of lignan biosynthesis in I.indigotica was highlighted, which was catalysed by 4CL3 via the esterization of caffeic acid. In conclusion, this study provides new insights into lignan biosynthesis as well as potential targets for metabolic engineering in I.indigotica.
Bonneau, JulienBeasley, JesseLi, YuanJohnson, Alexander A. T....
12页
查看更多>>摘要：Nicotianamine (NA) is a non-protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S-adenosyl-L-methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up-regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.
Prentis, Peter J.Campbell, Bradley C.Gilding, Edward K.Mace, Emma S....
14页
查看更多>>摘要：Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either Landraces' or Wild and Weedy' genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.
查看更多>>摘要：The developmental variation in stem height with respect to stem diameter is related to a broad range of ecological and evolutionary phenomena in trees, but the underlying genetic basis of this variation remains elusive. We implement a dynamic statistical model, functional mapping, to formulate a general procedure for the computational identification of quantitative trait loci (QTLs) that control stem height-diameter allometry during development. Functional mapping integrates the biological principles underlying trait formation and development into the association analysis of DNA genotype and endpoint phenotype, thus providing an incentive for understanding the mechanistic interplay between genes and development. Built on the basic tenet of functional mapping, we explore two core ecological scenarios of how stem height and stem diameter covary in response to environmental stimuli: (i) trees pioneer sunlit space by allocating more growth to stem height than diameter and (ii) trees maintain their competitive advantage through an inverse pattern. The model is equipped to characterize pioneering' QTLs (piQTLs) and maintaining' QTLs (miQTLs) which modulate these two ecological scenarios, respectively. In a practical application to a mapping population of full-sib hybrids derived from two Populus species, the model has well proven its versatility by identifying several piQTLs that promote height growth at a cost of diameter growth and several miQTLs that benefit radial growth at a cost of height growth. Judicious application of functional mapping may lead to improved strategies for studying the genetic control of the formation mechanisms underlying trade-offs among quantities of assimilates allocated to different growth parts.
查看更多>>摘要：Plant-based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C-terminal fused to the heavy chain of 14D9 (vac-Abs) and compared with secreted and ER-retained variants (sec-Ab, ER-Ab, respectively). Accumulation of ER- and vac-Abs was 10- to 15-fold higher than sec-Ab. N-glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec-Ab while vac-Abs carried mainly oligomannosidic (Man 7-9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec-Ab-RFP localized in the apoplast while vac-Abs-RFP were exclusively detected in the central vacuole. The data suggest that vac-Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N-glycans). Importantly, vac-Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post-translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.
查看更多>>摘要：Flowerless trait is highly desirable for poplar because it can prevent pollen- and seed-mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the -glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non-floral-specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT-A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel-less) and carpel-less. Further, the vegetative growth of these transgenic lines was similar to that of the wild-type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild-type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT-A transgenic poplar plants was similar to that of the wild-type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT-A gene may be useful for producing flowerless poplar that retains normal vegetative growth.
查看更多>>摘要：Virus-like particles (VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N- or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.
查看更多>>摘要：Targeted manipulation of phenylalanine (Phe) synthesis is a potentially powerful strategy to boost biologically and economically important metabolites, including phenylpropanoids, aromatic volatiles and other specialized plant metabolites. Here, we use two transgenes to significantly increase the levels of aromatic amino acids, tomato flavour-associated volatiles and antioxidant phenylpropanoids. Overexpression of the petunia MYB transcript factor, ODORANT1 (ODO1), combined with expression of a feedback-insensitive E.coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (AroG), altered the levels of multiple primary and secondary metabolites in tomato fruit, boosting levels of multiple secondary metabolites. Our results indicate that coexpression of AroG and ODO1 has a dual effect on Phe and related biosynthetic pathways: (i) positively impacting tyrosine (Tyr) and antioxidant related metabolites, including ones derived from coumaric acid and ferulic acid; (ii) negatively impacting other downstream secondary metabolites of the Phe pathway, including kaempferol-, naringenin- and quercetin-derived metabolites, as well as aromatic volatiles. The metabolite profiles were distinct from those obtained with either single transgene. In addition to providing fruits that are increased in flavour and nutritional chemicals, coexpression of the two genes provides insights into regulation of branches of phenylpropanoid metabolic pathways.
查看更多>>摘要：Flowering of higher plants is orchestrated by complex regulatory networks through integration of various environmental signals such as photoperiod, temperature, light quality and developmental cues. In Arabidopsis, transcription of the flowering integrator gene FLOWERING LOCUS T (FT) that several flowering pathways converge to is directly regulated by more than ten transcription factors. However, very little is known about the transcriptional regulation of the FT homolog SINGLE FLOWER TRUESS (SFT) in the day-neutral plant tomato (Solanum lycopersicum). Previously, we showed that the zinc finger transcription factor SlZFP2 plays important roles in regulation of seed germination and fruit ripening in tomato and also found that overexpression of SlZFP2 impacted flowering and branching. Here, we characterized in detail the early flowering and high branching phenotypes by overexpression of this transcription factor. Our data showed that overexpression of SlZFP2 accelerated flowering in an SFT-dependent manner as demonstrated by elevated SFT expression in the leaves and the transcription factor's binding ability to SFT promoter invitro and invivo. Furthermore, overexpression of the SlZFP2 gene in the sft plants failed to rescue the mutant's late flowering. Through analysis of grafting phenotype, growth response of branches to auxin application and transcriptome profiling by RNA sequencing, we also showed that overexpression of SlZFP2 affected shoot apical dominance through multiple regulatory pathways. Our results suggest that the transcription factor SlZFP2 has potential applications in genetic modification of plant architecture and flowering time for tomato production and other crops as well.
NSTL
Wiley