The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model ...organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.
In this study, PsMYB2 was successfully cloned using cDNA from Potentilla sericea as a template. It was used to construct the plant overexpression vector pBI121- PsMYB2-GFP, which was transferred into ...Arabidopsis thaliana wild-type plants. We studied the gene function using real-time quantitative PCR and performed a preliminary characterization and analysis of the function of PsMYB2 under abiotic stresses. This study showed that under cadmium stress, the gene expression of PsMYB2 gene in roots, stems, and leaves was up to 3–6 times higher than the control. The germination rate of transgenic A. thaliana T3 generation seeds reached more than 95%. The O
2
·
−
, H
2
O
2
, and malondialdehyde contents of the transgenic P. sericea plant lines were increased but lower than those of the wild-type strain. The superoxide dismutase, peroxidase, and hydrogen peroxidase activities were increased in both wild-type and transgenic strains, and the transgenic strains showed higher enzyme activities than the wild-type. We concluded that PsMYB2 could improve plant resistance to cadmium, which provides a theoretical basis for using transgenic plants to remediate cadmium-contaminated soil and for sustainable land use.
Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise ...genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools.
is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant ...resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the
gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.
•Apigenin and anthocyanins are products of secondary metabolism in plants.•AgFNS gene was cloned from purple celery and overexpressed in purple celery.•AgFNS overexpression increases apigenin and ...decreases anthocyanins content.•AgFNS overexpression influence on anthocyanin and apigenin metabolism.
Apigenin and anthocyanin biosyntheses share common precursors in plants. Flavone synthase (FNS) converts naringenin into apigenin in higher plants. Celery is an important edible and medical vegetable crop that contains apigenin in its tissues. However, the effect of high AgFNS gene expression on the apigenin and anthocyanins contents of purple celery remains to be elucidated. In this study, the AgFNS gene was cloned from purple celery (‘Nanxuan liuhe purple celery’) and overexpressed in this purple celery to determine its influence on anthocyanins and apigenin contents. Results showed that the AgFNS gene was 1068bp, which encodes 355 amino acid residues. Evolution analysis showed that the AgFNS protein belongs to the FSN I type. In AgFNS transgenic celery, the anthocyanins content in petioles was lower than that wild-type celery plants. Apigenin content increased in the petioles of AgFNS transgenic celery. The transcript levels of the AgPAL, AgC4H, AgCHS, and AgCHI genes were up-regulated, whereas those of the AgF3H, AgF3′H, AgDFR, AgANS, and Ag3GT genes were down-regulated in the petioles of AgFNS transgenic plants compared with wild-type celery plants. This work provides basic knowledge about the function of the AgFNS gene in the anthocyanin and apigenin biosyntheses of celery.
GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a ...deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.
Summary
Post‐transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target ...region and with de novo RNA‐directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to the transgene. Our data indicate that RdDM is correlated with the in situ generation of secondary siRNAs, occurring in P35S‐driven transgenes but not in most endogenes. We conclude that although both endogenes and transgenes are equally sensitive to transitive silencing, differences exist in their susceptibility to undergo secondary RdDM.
Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and ...introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.
Display omitted
•Obtention of kenaf transgenic plants.•Efficient protocol of kenaf seeds genetic transformation.•Fast and reliable procedure of kenaf transgenic plants obtention from seeds.•Useful ...and helpful method for genetic manipulation and gene functional analysis within Hibiscus cannabinus species.
Kenaf (Hibiscus cannabinus L.) is an economic and ecological fiber crop but suffers severe losses in fiber yield and quality under the stressful conditions of excess salinity and drought. Therefore, in order to obtain new cultivars of kenaf that could face and overcome abiotic stress, it is crucial to have a suitable protocol of genetic transformation. Therein, experiments were carried out on transformation of kenaf mature seeds using a co-culture of Agrobacterium tumefaciens. In this study, a A. tumefaciens-mediated transformation of kenaf seed have been developed by tissue-culture-independent procedure. The GV3010 Agrobacterium strain harboring the pGreenII binary vector that carries the neomymycin phosphotransferase II (npt II) gene for selection was used for transformation. The presence of the transgene and its stable expression were confirmed by PCR. In addition, the transgenic character of the selected transgenic T0 and T1 plants has been confirmed by germination test in the presence of kanamycin. Molecular analysis of F0 plants of three transgenic lines revealed the real integration of VvWRKY2 gene into the kenaf genome. Thus, our described method was an efficient, fast, and reliable procedure by which stable transgenic flowering plants were obtained within a short period of 3 months with 6% transformation efficiency.
Chorispora bungeana (C. bungeana) is a rare alpine subnival species that is highly tolerant to low temperature stress. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid ...catabolism during plant growth and the stress response. In this study, one member of CbPLD gene family, CbPLDδ, was cloned from C. bungeana and was introduced into tobacco. This gene encodes an 864-amino acid protein with two catalytic HxKxxxxD motifs which are essential for phospholipase D activity. After the CbPLDδ gene is fused with the vector containing the GFP tag, subcellular localization showed that CbPLDδ was predominately located in the cell membrane. RT-qPCR and histochemical GUS assays showed that CbPLDδ gene was induced by low temperature and expressed predominantly in leaf and root. Compared with wild-type tobacco, CbPLDδ transgenic tobacco showed higher activities of antioxidant enzymes, and lower levels of malonidiadehyde and electrolyte leakage under low temperature stress. These results reflected that CbPLDδ is involved in the response to low temperature stress, and has the potential to improve the low temperature tolerance of plants.
PDF
