scholarly journals The Naturally Evolved EPSPS From Goosegrass Confers High Glyphosate Resistance to Rice

2021 ◽  
Vol 12 ◽  
Author(s):  
Chao Ouyang ◽  
Wei Liu ◽  
Silan Chen ◽  
Huimin Zhao ◽  
Xinyan Chen ◽  
...  
Keyword(s):  
Selection Pressure ◽  
High Efficiency ◽  
Rice Genome ◽  
Selection System ◽  
Transformation System ◽  
Wild Type ◽  
Promising Alternative ◽  
Two Generations ◽  
Massive Scale ◽  
Similar Growth

Glyphosate-resistant crops developed by the CP4-EPSPS gene from Agrobacterium have been planted on a massive scale globally, which benefits from the high efficiency and broad spectrum of glyphosate in weed control. Some glyphosate-resistant (GR) genes from microbes have been reported, which might raise biosafety concerns. Most of them were obtained through a hygromycin-HPT transformation system. Here we reported the plant source with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from goosegrass endowed rice with high resistance to glyphosate. The integrations and inheritability of the transgenes in the rice genome were investigated within two generations. The EiEPSPS transgenic plants displayed similar growth and development to wild type under no glyphosate selection pressure but better reproductive performance under lower glyphosate selection pressure. Furthermore, we reconstructed a binary vector pCEiEPSPS and established the whole stage glyphosate selection using the vector. The Glyphosate-pCEiEPSPS selection system showed a significantly higher transformation efficiency compared with the hygromycin-HPT transformation system. Our results provided a promising alternative gene resource to the development of GR plants and also extended the plant transformation toolbox.

scholarly journals Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

2021 ◽  
Author(s):  
Magdy Mahfouz ◽  
Haroon Butt ◽  
Jose Luis Moreno Ramirez
Keyword(s):  
Directed Evolution ◽  
Herbicide Resistance ◽  
Selection Pressure ◽  
High Efficiency ◽  
Rice Genome ◽  
Acetolactate Synthase ◽  
Target Sequence ◽  
T7 Promoter ◽  
Simultaneous Application ◽  
Dna Base

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated into the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as a selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

scholarly journals Selection index in Drosophila melanogaster Meig. progeny after exposure to acute γ-irradiation

2019 ◽  
Vol 25 ◽  
pp. 86-91
Author(s):  
D. O. Skorobagatko ◽  
V. Yu. Strashnyuk ◽  
A. A. Mazilov
Keyword(s):  
Drosophila Melanogaster ◽  
Linear Accelerator ◽  
Selection Pressure ◽  
First Generation ◽  
Selection Index ◽  
Control Level ◽  
Wild Type ◽  
Γ Irradiation ◽  
Two Generations ◽  
Selection Indexes

Aim. The purpose of investigation was to study the intensity of selection in two generations of Drosophila melanogaster Meig. after acute g-irradiation. Methods. Experiments were conducted on a wild-type Oregon-R strain. Adult flies in the age of 3 days were irradiated with bremsstrahlung gamma quanta at doses of 8 Gy, 16 Gy and 25 Gy on a linear accelerator of electrons LAE-10. Irradiated (O) and non-irradiated (K) flies were crossed in four different combinations: K×K (control), O×K, K×O, and O×O. The selection index were calculated from the Crow formula based on fertility, mortality/survival in pre-productive period of development. Results. Selection indexes in the generation F1 after irradiation grow in proportion to the dose received: at embryonal stage 2.0–7.2 times, at post-embryonic development – in 1.3–7.6 times. In the generation F2, the indexes of selection were significantly reduced. Conclusions. The selection pressure is substantially increased in the first generation after g-irradiation and weakens, approaching the control level and lower in F2. Keywords: Drosophila melanogaster Meig., fertility, embryonic mortality, pupae mortality, ionizing radiation.

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

scholarly journals Yield QTL analysis of Oryza sativa x O. glumaepatula introgression lines

2013 ◽  
Vol 48 (3) ◽  
pp. 280-286 ◽  
Author(s):  
Priscila Nascimento Rangel ◽  
Rosana Pereira Vianello ◽  
Arthur Tavares Oliveira Melo ◽  
Paulo Hideo Nakano Rangel ◽  
João Antônio Mendonça ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Interspecific Cross ◽  
Rice Genome ◽  
Introgression Lines ◽  
Wild Accession ◽  
Cultivated Rice ◽  
Two Generations ◽  
Ssr Loci ◽  
Donor Parent ◽  
Effect On Yield

The objective of this work was to evaluate the yield performance of two generations (BC2F2 and BC2F9) of introgression lines developed from the interspecific cross between Oryza sativa and O. glumaepatula, and to identify the SSR markers associated to yield. The wild accession RS‑16 (O. glumaepatula) was used as donor parent in the backcross with the high yielding cultivar Cica‑8 (O. sativa). A set of 114 BC2F1 introgression lines was genotyped with 141 polymorphic SSR loci distributed across the whole rice genome. Molecular analysis showed that in average 22% of the O. glumaepatula genome was introgressed into BC2F1 generation. Nine BC2F9 introgression lines had a significantly higher yield than the genitor Cica‑8, thus showing a positive genome interaction among cultivated rice and the wild O. glumaepatula. Seven QTL were identified in the overall BC2F2, with one marker interval (4879‑EST20) of great effect on yield. The alleles with positive effect on yield came from the cultivated parent Cica‑8.

scholarly journals Identification of Selection Signatures in Commercial Asian Rice Subspecies Deciphered Selection Footprints for Four Overrepresented Biological Processes

2020 ◽  
Author(s):  
Siavash Salek Ardestani ◽  
Mahmoud Amiri Roudbar ◽  
Mohammad Hossein Banabazi ◽  
Seyedeh Fatemeh Mousavi ◽  
Madhav Bhatta ◽  
...  
Keyword(s):  
Selection Pressure ◽  
Rice Genome ◽  
Fixation Index ◽  
Biological Processes ◽  
Selection Signatures ◽  
Genomic Changes ◽  
Asian Rice ◽  
Genome Level ◽  
Window Approach ◽  
Genomic Regions

Abstract BackgroundSelective breeding pressures have led to gradual genomic changes in Asian commercial rice, which have shaped selection footprints on its genome level. Tracing genomic selection footprints might be illuminative for better understanding of recent selection breeding objectives, and how breeding strategies have formed the Asian commercial rice genome. ResultsIn this study, the genotypic information (HDRA 700K) of four Asian commercial rice subspecies including Indica (n=498), Aus (n=187), Temperate japonica (n=241), and Tropical japonica (n=361) were downloaded from Rice Diversity Project database (http://www.ricediversity.org) to detect selection signatures by employing the Z-transformed of fixation index and Tajima’s D test, based on a sliding window approach. Although we could not identify overrepresented genomic regions underlying selection pressure among all aforementioned Asian commercial rice subspecies, interestingly, our findings revealed four overrepresented biological processes underlying selection pressure including proteolysis (GO:0006508), phosphorylation (GO:0016310), protein catabolic process (GO:0030163), and transmembrane transport (GO:0055085) that might be associated with immunity, senescing leaves, transporting, and absorption of ions. ConclusionsThese results can provide knowledge on how breeding efforts shaped the Asian commercial rice subspecies genome, and which genomic regions of these subspecies have been targeted in recent decades.

Genetic rescue of muscle defects associated with a mutant Drosophila melanogaster tropomyosin allele

1987 ◽  
Vol 7 (8) ◽  
pp. 2977-2980
Author(s):  
E A Fyrberg ◽  
C C Karlik
Keyword(s):  
Germ Line ◽  
Regulatory Mechanisms ◽  
Directed Mutagenesis ◽  
Transformation System ◽  
Wild Type ◽  
Genetic Rescue ◽  
Transformation Protocol ◽  
Type Gene ◽  
Genetic Transformation System ◽  
And Function

We describe a genetic transformation system which should prove useful for investigating tropomyosin assembly and function. Muscle abnormalities associated with a defective tropomyosin allele were corrected by integrating the wild-type gene into germ line chromosomes. The transformation protocol permits application of directed mutagenesis techniques in investigations of contractile regulatory mechanisms.

scholarly journals Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system

2019 ◽  
Vol 71 (4) ◽  
pp. 1337-1349 ◽  
Author(s):  
Huiyun Liu ◽  
Ke Wang ◽  
Zimiao Jia ◽  
Qiang Gong ◽  
Zhishan Lin ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
High Efficiency ◽  
Seed Set ◽  
Large Fragment ◽  
Wild Type ◽  
Haploid Induction ◽  
Wheat Grains ◽  
Editing Efficiency ◽  
Efficient Induction ◽  
Endogenous Genes

Abstract The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.

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