Transgenic Research最新文献

Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.

Globally, nearly one million species are currently threatened with extinction, highlighting the need for more efficient solutions to biological conservation. Genome editing, which allows for faster and more precise changes in genomes, is a promising technique for boosting populations through facilitated adaptation, management of invasive or pathogenic populations, and potentially even facilitating the revival of extinct species. These approaches belong to a new field of research termed conservation biotechnology, which places a great responsibility on researchers and decision makers to ensure sustainability. In this paper, we have mapped the emerging trends in genome editing of wild animals. Current projects primarily focus on population control and de-extinction, with fewer initiatives aimed at preserving threatened species. We then explore four critical dimensions of conservation biotechnology: the technology itself, new perspectives on conservation practices, research organization, and governance and policy. Despite its potential, key questions remain-particularly whether genome editing can increase genetic diversity without causing unintended non-target impacts. Genome editing also provokes new perspectives on conservation practices where ecosystem-wide impact assessment, case-by-case evaluations, and post-release monitoring needs to be prioritized. Furthermore, conservation biotechnology is heavily funded through private funding showing varying stakeholder interest, which can lead to untraditional and less transparent research processes. Stakeholders, including local and indigenous people, are only to a certain degree involved, which may weaken inclusion of local knowledge and monitoring efforts. Finally, concerning governance and policy, there is an urgent need to develop more adequate regulation of conservation biotechnology, as environmental release of genome-edited animals challenges definitions and guidelines in current nature protection laws and GMO regulations. Based on our analysis, we outline key points for further investigation toward a more sustainable approach to conservation biotechnology.

Poplar is severely damaged by Hyphantria cunea (fall webworm), which significantly reduces tree productivity. However, conventional pest management methods are largely ineffective against fall webworm infestation. In this study, we demonstrated that the Vip3A protein possesses high insecticidal activity against H. cunea by overexpressing a synthetic THI1-Vip3A gene in poplar plants. A dicot codon-optimized Vip3A gene, fused with the THI1 chloroplast signal peptide sequence, was chemically synthesized and introduced into the poplar cv. '741' genome via Agrobacterium-mediated transformation. PCR, RT-PCR, and ELISA analyses confirmed the integration and successful expression of the transgene at both the mRNA and protein levels. The Vip3A protein concentration in chloroplasts was approximately 4.8-fold higher than in the whole leaf extract, indicating that the Vip3A protein was successfully targeted to and accumulated within the chloroplasts by the THI1 signal peptide. Subsequently, four transgenic lines with high Vip3A expression were subjected to H. cunea infestation. Compared to wild-type plants, these four transgenic lines exhibited significantly higher resistance, resulting in pest mortality rates exceeding 95% and significantly reduced leaf damage. Together, these results indicate that Vip3A possesses high insecticidal activity against H. cunea. Therefore, transgenic THI1-Vip3A poplar plants can serve as valuable germplasm for breeding poplar cultivars with high resistance to H. cunea infestation.

Sesame (Sesamum indicum), an important oilseed crop, is well known for its therapeutic values. It is one of the richest sources of polyunsaturated fatty acids and health beneficiary lignans. However, nutraceutical potential is restricted due to the presence of secoisolariciresinol diglucoside (SDG), a potent anticarcinogen, in imperceptible amount (~ 0.03 mg/25 g of seeds). Consequently, a logical strategy was implemented to augment the SDG content in sesame by heterologous expression of the pinoresinol-lariciresinol reductase (Fiplr) gene from Forsythia intermedia. Four individual transgenic lines were generated by Agrobacterium-mediated transformation of pCAMBIA/Fiplr gene cassette into sesame and was further confirmed by PCR and Southern blot analyses. Among the Individual transgenic lines, PT₁1.1 was found to demonstrate ~ 3.5-fold higher expression of plr transcript in comparison to other transformants and untransformed control. PT₁1.1 plant also showed ~ twofold increased PLR enzyme activity that led to ~ threefold increased accumulation of SDG content as compared to that in untransformed control when confirmed by the HPLC analyses. Thus, the study suggests that the presently developed transgenic sesame line may have the better dietary prospect for people suffering particularly from estrogen-induced cancers and can emerge having significant medical implications.

In 2017 and 2018, studies were conducted in Iowa, US across three environments with a history of greater than expected corn rootworm injury i.e., greater than one node of injury to (US EPA 2009) and one environment without rootworm injury, and at different growth stages, to determine the expression levels of mCry3A and eCry3.1Ab transgenes in the roots of different Bt corn hybrids namely the molecular stack (MZIR098), breeding stack (MIR604 × 5307), 5307 and MIR604. ELISA results showed that expressions of both mCry3A and eCry3.1Ab transgenes, were higher in the MZIR098 molecular stack and breeding stack (MIR604 × 5307), than the MIR604 and 5307 at V3 and VT growth stages over both years. To protect the roots from feeding damage by the corn rootworm larvae, expression of the transgenes must be high at the V3 growth stage. The expression of the transgene was significantly impacted by the stage of plant growth while the environments with greater than expected corn rootworm injury did not impact expression of the transgenes. It was found that the expression of mCry3A and eCry3.1Ab transgenes were high at the V3 plant growth stage compared to the VT growth stage. Stacking two or more genes together in the same plant such as the molecular and breeding stacks have the potential to protect roots in environments with higher-than-expected damage and slow down the evolution of resistance in field populations of rootworms.

Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.

Climate change enhances the damaging consequences of abiotic and biotic stressors, leading to severe soil fertility loss and ecosystem degradation worldwide. Leguminous have contributed significantly to replenishing soil nitrogen via symbiotic nitrogen fixation, contributing approximately 15% of nitrogen input, which is crucial for soil health and enhancing crop production. There is an increasing integration of new biotechnological interventions, such as genome editing, including the CRISPR/Cas9 system, and transgenesis, in addition to classical breeding, to make agriculture more resilient. In this review, we examine several elements that influence the genetic transformation system employing Agrobacterium tumefaciens strains in leguminous to make it an ideal vehicle for CRISPR/Cas9 component delivery. The variables investigated in our study included the incubation period, co-cultivation duration, bacterial density, selectable marker, concentration, and growth regulators used. In addition, the selection and efficiency of the explant choice for transformation should be considered in future studies. However, there have been parallel recommendations for the gradual application of selectable markers such as kanamycin.

Cold stress can greatly narrow the development and living spaces of plants. As one endangered xerophytic shrub, Reaumuria trigyna can adapt the harsh cold, but the underlying molecular mechanisms remain unknown. Here, the RtWRKY49 gene of Reaumuria trigyna was screened and encoded a nuclear WRKY transcription protein. RtWRKY49 expression mainly located in stem of Reaumuria trigyna, activated by cold, salt and ABA treatments but inhibited by heat and drought treatments. The overexpression of RtWRKY49 remarkably improved cold tolerance in Arabidopsis with the increased growth, chlorophyll content and decreased levels of malondialdehyde, oxygen free radical and hydrogen peroxide. Under cold stress, RtWRKY49 transgenic Arabidopsis had greater antioxidase activities (SOD, POD, CAT, and DHAR), AsA, and proline levels than wild-type plants, with a significant upregulation of AtCAT1, AtPOD, AtDHAR2, and AtP5CS1 genes. Our findings indicated that the RtWRKY49 protein improved the cold tolerance of plants by coordinating the antioxidant and osmoregulation pathways to maintain plant homeostasis under cold stress. In addition to offering the RtWRKY49 as a promising target gene in breeding cold stress resistant crop, our work also provides a basis for the research of Reaumuria trigyna response to cold stress.

Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.