Selectable Marker Gene Research Articles

Development of methods for regeneration and genetic transformation of the commercial plum variety Startovaya from somatic tissues

An effi cient technique for regeneration of plants from somatic tissues of domestic plum (Prunus domestica L.) varieties, as well as selecting schemes using various reporter (uidA, gfp) and selective marker genes (nptII, hpt) have been developed. During the transformation of somatic tissues of the commercial plum variety, transgenic plants containing both reporter genes and genes of economically valuable traits were obtained. For the fi rst time, the eff ect of calcium pantothenate on the adventive regeneration of plum shoots was investigated. It was shown that the selective antibiotic hygromycin is more eff ective for the selection of plum transformants than kanamycin or geneticin, which was expressed in a higher frequency of transformation — 1.2-2.2 % versus 0.2-0.5 % in selection on kanamycin. When using geneticin, transgenic plants were not obtained. Optimization of conditions made it possible to increase the percentage of regeneration of shoots to 79.4 % for the variety Startovaya and up to 39.6 % for the variety Etude. Analysis of GFP expression in transgenic lines showed that the leader sequences of herbaceous plant species encoding signal peptides can be used to eff ectively cellular compartmentalization of heterologous gene expression products in the plum. Preliminary results also show a higher level of expression of the reticular form of GFP in comparison with the vacuolar and cytosolic forms. Nine transgenic lines containing the insertion of the target bar gene, which gives resistance to phosphinothricin-containing herbicides of the “Basta” type, were obtained, which was confi rmed by the presence in the genome of the sequence of the transferred gene and the resistance of the obtained transgenic plants to 3 times the fi eld concentration of the herbicide “Basta”.

Effect of the fatty acid marker gene diacylglycerol o-acyl transferase 1 (DGAT1) on the fatty acid composition and organoleptic properties of samples of butter produced from the milk of domestic breeds of cows

In modern conditions, effective breeding work in animal husbandry is impossible without DNA technologies. The search for selective marker genes that determine the dairy productivity of animals allows us to more reliably assess the genetic potential of breeds, adjust breeding processes, and select animals of desirable genotypes. In the Republic of Belarus, almost no work was carried out to identify genetic markers that affect the fat content, fatty acid composition of milk fat, and as a result, the yield and quality of the finished product (butter). In this regard, a special interest is the diacylglycerol O-acyltransferase 1 (DGAT1) gene, which determines the fat content. The paper presents a comparative assessment of the fatty acid composition and organoleptic properties of butter samples produced from the milk of cows of natural selection according to the gene marker of fatty milk diacylglycerol o-acyl transferase 1 (DGAT1). The results of tests of the fatty acid composition of butter from cow’s milk obtained from cows of different breeds and pedigreed its balance, compliance with the established values for volatile and unsaturated fatty acids, absence of phytosterols and adulteration of the fatty phase of the oil were emphasised. The practical significance of the conducted research lies in the use of the results obtained to carry out targeted breeding of dairy cattle according to the gene marker of fat content of diacylglycerol o-acyl transferase 1 (DGAT1) in order to increase the mass fraction of fat in milk and the yield of the finished product, as well as to improve the technology of making butter from cow’s milk and the formation of an evidence base in case of disagreement when evaluating the fatty acid composition of the product.

Monocyte/macrophage heterogeneity during skin wound healing in mice

Abstract Cells of the monocyte/macrophage (Mo/Ma) lineage orchestrate diverse biological processes required for efficient skin wound healing. Current understanding of Mo/Ma heterogeneity during wound healing has been established by traditional experimental approaches such as flow cytometry, qPCR, and IHC, which indicates a phenotype switch from pro-inflammatory to pro-healing cells as wound healing progresses. However, these phenotypes are limited by the relatively small number of markers assessed. Therefore, our study aimed to generate an unbiased atlas of Mo/Ma phenotypes over the course of skin wound healing by scRNAseq. Cells were dissociated from excisional skin wounds of normal healing (WT) mice on day 3, 6, and 10 post-injury and live CD11b+Ly6G− cells were isolated by FACS sorting. Using 10× Genomics Chromium system, we pooled data from a total of 2,813 high-quality cells and performed clustering using Seurat which resulted in 8 subpopulations of cells. Cluster dissimilarity and differential expression analysis categorized those clusters into three groups: early stage/pro-inflammatory, antigen-presenting, and late-stage/pro-healing phenotypes that changed over the course of healing. Signature genes and gene ontology analysis of each cluster were used to further characterize the cell populations. Moreover, Ly6C+ vs Ly6C− Mo/Ma were FACS sorted and qPCR was used to validate select marker genes from our scRNAseq clusters. Finally, cell trajectory analysis by pseudotime and RNA velocity indicated the transitions between different Mo/Ma clusters over the course of healing. In summary, our data demonstrated the complexity of Mo/Ma phenotypes and their diverse functions during skin wound healing in mice. Funded by NIH/NIGMS: R35GM136228

Development of a New Genetic Transformation System for White and Green Ash Using Embryogenic Cultures

All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Identification of new marker genes from plant single-cell RNA-seq data using interpretable machine learning methods.

Summary An essential step in the analysis of single‐cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single‐cell predictive marker (SPmarker) to identify novel cell‐type marker genes in the Arabidopsis root.Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers.Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single‐cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non‐Arabidopsis species, which expands the number of marker genes for this cell type by 35–154%.Our results represent a new approach to identifying cell‐type marker genes from scRNA‐seq data and pave the way for cross‐species mapping of scRNA‐seq data in plants.

Generation of Transfer-DNA-Free Base-Edited Citrus Plants.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and β-adrenoceptors (α-, β-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the β-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

SummaryWe have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium‐derived vir and T‐DNA‐mediated transformation. Oh H1 is a unique, non‐phytopathogenic species, categorized as a BSL‐1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1‐8 strain containing a binary vector system. Oh H1‐8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy‐. Oh H1‐8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1‐8 generated high‐quality, transgenic events with single‐copy, plasmid backbone‐free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock‐inducible excision system resulting in marker‐free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null‐segregant lines to wild‐type controls. We have demonstrated that Oh H1‐8, combined with spectinomycin selection, is an efficient, rapid, marker‐free and yield‐neutral transformation system for elite soybean.

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets

BackgroundWhile single-cell transcriptional profiling has greatly increased our capacity to interrogate biology, accurate cell classification within and between datasets is a key challenge. This is particularly so in pluripotent stem cell-derived organoids which represent a model of a developmental system. Here, clustering algorithms and selected marker genes can fail to accurately classify cellular identity while variation in analyses makes it difficult to meaningfully compare datasets. Kidney organoids provide a valuable resource to understand kidney development and disease. However, direct comparison of relative cellular composition between protocols has proved challenging. Hence, an unbiased approach for classifying cell identity is required.MethodsThe R package, scPred, was trained on multiple single cell RNA-seq datasets of human fetal kidney. A hierarchical model classified cellular subtypes into nephron, stroma and ureteric epithelial elements. This model, provided in the R package DevKidCC (github.com/KidneyRegeneration/DevKidCC), was then used to predict relative cell identity within published kidney organoid datasets generated using distinct cell lines and differentiation protocols, interrogating the impact of such variations. The package contains custom functions for the display of differential gene expression within cellular subtypes.ResultsDevKidCC was used to directly compare between distinct kidney organoid protocols, identifying differences in relative proportions of cell types at all hierarchical levels of the model and highlighting variations in stromal and unassigned cell types, nephron progenitor prevalence and relative maturation of individual epithelial segments. Of note, DevKidCC was able to distinguish distal nephron from ureteric epithelium, cell types with overlapping profiles that have previously confounded analyses. When applied to a variation in protocol via the addition of retinoic acid, DevKidCC identified a consequential depletion of nephron progenitors.ConclusionsThe application of DevKidCC to kidney organoids reproducibly classifies component cellular identity within distinct single-cell datasets. The application of the tool is summarised in an interactive Shiny application, as are examples of the utility of in-built functions for data presentation. This tool will enable the consistent and rapid comparison of kidney organoid protocols, driving improvements in patterning to kidney endpoints and validating new approaches.

Selectable Markers to Marker-Free Selection in Rice.

Inadequate rice production worldwide is largely attributed to abiotic and biotic stresses, along with high sensitivity of cultivable plant germplasm. In the field of cereal biotechnology, rice engineering plays an important role in achieving tolerance to such stresses. Plant transformation and selection play crucial role in rice engineering. This review summarized the antibiotic, herbicide and metabolic selection marker genes (SMG) employed in diverse rice engineering studies. These SMGs are no longer required after the transformation has been achieved, hence undesirable at the commercial level. This study also included several strategies employed in rice engineering to eliminate such foreign DNA elements. These include co-transformation, site-specific recombination, transposon and CRISPR base approaches. CRISPR/Cas9 being simple and efficient, is considered a crucial step toward clean gene technology. Further ease and applicability of CRISPR/Cas9 in the embryos directly can help us to modify target genes with efficient marker-free selection in minimum time. Overall, this review summarizes and analyse the recent advances that have enormous potential in rice improvement.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Herbicide tolerance and gene silencing stability over generations in the ricin bio-detoxicated castor bean

Castor bean (Ricinus communis L.) is an important cultivated oilseed. Seeds contain ricinoleic acid, a valuable product for a variety of industries. Castor cake is a residue of ricinoleic manufacture and could be used as animal feed due to its high amount of protein. However, castor cake contains ricin and RCA120, both highly toxic and allergenic proteins. In 2017, we reported the development of a transgenic event (named TB14S-5D) with an undetectable amount of ricin/RCA120. In the present work, we evaluate TB14S-5D for tolerance to the herbicide imazapyr, as it contains the selectable marker gene, ahas, which was previously isolated from Arabidopsis thaliana and contains a mutation at position 653 bp. In addition, we demonstrated that the ricin coding genes are stably silenced over three generations.

An Efficient Agrobacterium-mediated Genetic Transformation Using cry1F gene in Castor (Ricinus communis L.) for protection Against lepidopteran Insects

to foliage feeders like Spodoptera litura which resulted in a loss of production. This report focuses on the development of an optimized protocol for the transformation of castor shoot apices by Agrobacterium tumefaciens strain LBA4404 containing plasmid construct pBIN1F harboring neomycin phosphotransferase (nptII), as selectable marker gene and Bacillus thuringiensis var. aizawai (Bt) cry1F gene controlled by cauliflower mosaic virus 35S promoter. Several parameters like O.D., concentration of kanamycin and acetosyringone were optimized and produced a significant difference in the transformation efficiency. Co-cultivation time and seedling age were factors, with overall transformation efficiency of 2.0% in 15 days-old seedlings and co-cultivated for 3 days. The surviving and actively developing shoot apices were validated for gene integration by molecular analysis after being preliminarily screened on kanamycin. Furthermore, PCR, qRT-PCR and insect bioassay were used to confirm the putative primary transformants. When bioassayed against newly hatched Spodoptera litura hatchlings, these putative transgenics with cry1F gene caused significant (≤93%) insect mortality. Cry1F gene expressing transgenic plants had adequate defence against Spodoptera litura when exposed to castor.

Genotype-independent plant transformation.

Plant transformation and regeneration remain highly species- and genotype-dependent. Conventional hormone-based plant regeneration via somatic embryogenesis or organogenesis is tedious, time-consuming, and requires specialized skills and experience. Over the last 40 years, significant advances have been made to elucidate the molecular mechanisms underlying embryogenesis and organogenesis. These pioneering studies have led to a better understanding of the key steps and factors involved in plant regeneration, resulting in the identification of crucial growth and developmental regulatory genes that can dramatically improve regeneration efficiency, shorten transformation time, and make transformation of recalcitrant genotypes possible. Co-opting these regulatory genes offers great potential to develop innovative genotype-independent genetic transformation methods for various plant species, including specialty crops. Further developing these approaches has the potential to result in plant transformation without the use of hormones, antibiotics, selectable marker genes, or tissue culture. As an enabling technology, the use of these regulatory genes has great potential to enable the application of advanced breeding technologies such as genetic engineering and gene editing for crop improvement in transformation-recalcitrant crops and cultivars. This review will discuss the recent advances in the use of regulatory genes in plant transformation and regeneration, and their potential to facilitate genotype-independent plant transformation and regeneration.

Research Progress on Edible Fungi Genetic System.

In order to obtain strains with targeted changes in genetic characteristics, molecular biology and genetic engineering techniques are used to integrate target gene fragments into the vector and transform them into recipient cells. Due to the different target genes and functional elements on the transformation plasmids, gene silencing, gene knockout, and gene overexpression can be carried out, which provides a new way to study the gene function of edible fungi. At present, the cloning vectors used in the transformation of edible fungi are modified by bacterial plasmids, among which pCAMBIA-1300 plasmid and pAN7 plasmid are the two most commonly used basic vectors. On this basis, some basic elements such as promoters, selective marker genes, and reporter genes were added to construct silencing vectors, knockout vectors, and overexpression vectors. At the same time, different expression vector systems are needed for different transformation methods. In this chapter, the main elements of the genetic system (promoters, screening markers), the current main genetic transformation methods (Agrobacterium-mediated transformation, liposome transformation, electroporation method), and the specific application of transformation were systematically summarized, which provides a reference for the study of the genetic system of edible fungi.

An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae.

For genetic manipulation of yeast, numerous selection marker genes have been employed. These include prototrophic markers, markers conferring drug resistance, autoselection markers, and counterselectable markers. This chapter describes the different classes of selection markers and provides a number of examples for different applications.

Advances and outlook of horticultural bioengineering

A Branch of modern biotechnology for creating unique relevant genotypes is bioengineering that harnesses a spectrum of plant genome modification technologies. The study aimed to analyse the current state of the art in genome modification of fruit and berry crops for more significant (vs. premium pure breeding varieties) deviations of norm in the traits and properties of biotic and abiotic resistance, productivity, fruit quality, etc. First horticultural crop transformation studies aimed at developing protocols based on selectable enzyme marker genes of phosphorylationmediated aminoglycoside antibiotics detoxification. Neomycin phosphotransferase nptII constitutes the most common system of transgenic fruit and berry crop selection. In pome crops, the transgenic selection priorities were resistance to scab (Venturia inaequalis (Wint.) Cke), rust (Gymnosporangium juniper-virginianae Schwein.) and bacterial blight (Erwinia amylovora Burrill, Winslow et al.), higher fruit quality, including bright colouring, and reduced enzymatic browning. In stone crops, it was tolerance to plum pox (PPV), papaya ringspot (PRSV) and Prunus necrotic ringspot (PNRSV) viruses. In berry crops — resistance to Sphaerotheca humuli (DC.) Burrill fungus, grey mould (Botrytis cinerea Pers.), root rot (Phytophthora cactorum (Lebert & Cohn) J.Schrot.) and powdery mildew (Oidium tuckeri Berkeley), as well as higher fruit quality. In citruses — resistance to bacterial canker (Xanthomonas citri sub sp.), citrus ulcer (Xanthomonas axonopodis pv citri), greening disease (Huanglongbing (HLB)) and fungi (Trichoderma harzianum Rifai). In tropical crops — resistance to papaya ringspot (PRSV) and banana streak (eBSV) viruses. Unique FT-phenotype transgenic fruit lines are leveraged in the new FasTrack breeding strategy. Nine fruit and berry transgenic crop lines have now been registered worldwide. Transgenic Arctic apples (Golden, Granny, Fuji), plums (Honey Sweet) and papaya (Rainbow, SunUp, Laie Gold) are industry-approved in fresh and processed form. The transgenic list regulated in the Russian Federation does not include fruit or berry crops.

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae.

We evaluated the Cre-lox and CRISPR-Cas9 systems as marker-recycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4-based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.