Transgenic Research最新文献

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

Transgene expression and genome editing can help improve cucumber varieties to better respond to climate change. This study aimed to evaluate the applicability of the CsUBL5 promoter in transgene expression and genome editing in cucumber. The CsUBL5 promoter was cloned and analyzed to identify cis-elements that respond to abiotic signals, hormones, signal molecules, and nutrient treatments. 5' deletion constructs of the promoter were tested for their ability to drive GUS reporter expression in cucumber cotyledons, Arabidopsis seedlings, and tobacco leaves, and their response to various treatments including SA, light, drought, IAA, and GA was determined. The results showed that the CsUBL5 promoter effectively drove transgene expression in these plants, and their expressions under treatments were consistent with the predicted cis-elements, with some exceptions. Furthermore, the pCsUBL5-749 deletion construct can improve genome editing efficiency in cucumber when driving Cas9 expression. The editing efficiency of two sgRNAs targeting the ATG6 gene in cucumber was up to 4.6-fold higher using pCsUBL5-749 compared to a rice UBI promoter, although the effects of changing promoter on the editing efficiency is sgRNA specific. These findings highlight the potential utility of the CsUBL5 promoter for improving cucumber varieties through genetic engineering and genome editing. It also demonstrates the importance of modulating Cas9 expression to increase genome editing efficiency in cucumbers.

The co-expression of multiple antimicrobial peptides (AMPs) in genetically modified (GM) crops can give plants a broader antibacterial spectrum and lower the pathogen risk of drug resistance. Therefore, four penaeidins (shrimp-derived AMPs) were fused and encoded in an artificial gene (PEN1234), driven by the seed-specific promoter Pzein, with the aim of co-expression in seeds of transgenic rice. The resistant rice plants, acquired via Agrobacterium-mediated transformation and glufosinate screening, were identified by PCR and the modified disk-diffusion method, and eight GM lines with high AMP content in the seeds were obtained. Among them, the PenOs017 line had the largest penaeidin content, at approximately 251-300 μg/g in seeds and 15-47 μg/g in roots and leaves. The AMPs in the seeds kept their antibacterial properties even after the seed had been boiled in hot water and could significantly inhibit the growth of methicillin-resistant Staphylococcus aureus, and AMPs in the leaves could effectively inhibit Xanthomonas oryzae pv. Oryzae. The results indicate that PenOs017 seeds containing AMPs are an ideal raw-material candidate for antibiotic-free food and feed, and may require fewer petrochemical fungicides or bactericides for disease control during cultivation than conventional rice.

n-3 polyunsaturated fatty acids (n-3 PUFAs), including α-linolenic acid and eicosapentaenoic acid (EPA), are essential nutrients for vertebrates including humans. Vertebrates are n-3 PUFA-auxotrophic; hence, dietary intake of n-3 PUFAs is required for their normal physiology and development. Although fish meal and oil have been utilized as primary sources of n-3 PUFAs by humans and aquaculture, these traditional n-3 PUFA sources are expected to be exhausted because of the increasing consumption requirements of humans. Hence, it is necessary to establish alternative n-3 PUFA sources to reduce the gap between the supply and demand of n-3 PUFAs. Here, we investigated whether insects, which are considered as a novel source of essential nutrients, could store n-3 PUFAs by the forced expression of n-3 PUFA biosynthetic enzymes. We utilized Drosophila as an insect model to generate transgenic strains expressing Caenorhabditis elegans PUFA biosynthetic enzymes and examined their effects on the proportion of fatty acids. The ubiquitous expression of methyl-end desaturase FAT-1 prominently enhanced the proportions of α-linolenic acid, indicating that FAT-1 is useful for metabolic engineering to fortify α-linolenic acid in insect. Furthermore, the ubiquitous expression of nematode front-end desaturases (FAT-3 and FAT-4), PUFA elongase (ELO-1), and FAT-1 led to EPA bioproduction. Hence, nematode PUFA biosynthetic genes may serve as powerful genetic tools for enhancing the proportion of EPA in insects. This study represents the first step toward the establishment of n-3 PUFA-producing insects.

Exhaustive analysis of genetically modified crops over multiple decades has increased societal confidence in the technology. New Plant Breeding Techniques are now emerging with improved precision and the ability to generate products containing no foreign DNA and mimic/replicate conventionally bred varieties. In the present study, metabolomic analysis was used to compare (i) tobacco genotypes with and without the CRISPR associated protein 9 (Cas9), (ii) tobacco lines with the edited and non-edited DE-ETIOLATED-1 gene without phenotype and (iii) leaf and fruit tissue from stable non-edited tomato progeny with and without the Cas9. In all cases, multivariate analysis based on the difference test using LC-HRMS/MS and GC-MS data indicated no significant difference in their metabolomes. The variations in metabolome composition that were evident could be associated with the processes of tissue culture regeneration and/or transformation (e.g. interaction with Agrobacterium). Metabolites responsible for the variance included quantitative changes of abundant, well characterised metabolites such as phenolics (e.g. chlorogenic acid) and several common sugars such as fructose. This study provides fundamental data on the characterisation of gene edited crops, that are important for the evaluation of the technology and its assessment. The approach also suggests that metabolomics could contribute to routine product-based analysis of crops/foods generated from New Plant Breeding approaches.

Interleukin-37 is a newly discovered cytokine that plays a pivotal role in suppressing innate inflammation and acquired immunity. We have recently expressed both the mature(mat-) and pro-forms of human IL-37b in plants and demonstrated that while both forms of the plant-made hIL-37b are functional, pmat-hIL37b exhibited significantly greater activity than ppro-IL-37b. Compared to ppro-hIL-37b, on the other hand, the expression level of pmat-hIL-37b was substantially lower (100.5 µg versus 1.05 µg/g fresh leaf mass or 1% versus 0.01% TSP). Since the difference between ppro-hIL-37b and pmat-hIL-37b is that ppro-hIL-37b contains a signal sequence not cleavable by plant cells, we reasoned that this signal sequence would play a key role in stabilizing the ppro-hIL-37b protein. Here, we describe a novel approach to enhancing pmat-hIL-37b production in plants based on incorporation of a gene sequence encoding tobacco etch virus (TEV) protease between the signal peptide and the mature hIL-37b, including a TEV cleavage site at the C-termini of TEV protease. The rationale is that when expressed as a sp-TEV-matIL-37b fusion protein, the stabilizing properties of the signal peptide of pro-hIL-37b will be awarded to its fusion partners, resulting in increased yield of target proteins. The fusion protein is then expected to cleave itself in vivo to yield a mature pmat-hIL-37b. Indeed, when a sp-TEV-matIL-37b fusion gene was expressed in stable-transformed plants, a prominent band corresponding to dimeric pmat-hIL-37b was detected, with expression yields reaching 42.5 µg/g fresh leaf mass in the best expression lines. Bioassays demonstrated that plant-made mature pmat-hIL-37b is functional.

The initial compositional analysis of plants plays an important role within the internationally harmonized comparative safety assessment approach for genetically modified plants. Current EFSA guidance prescribes two types of comparison, namely difference tests with regard to a conventional comparator or control, and equivalence tests with regard to a collection of commercial reference varieties. The experience gained so far shows that most of the statistically significant differences between the test and control can be discounted based on the fact that they are still within equivalence limits of reference varieties with a presumed history of safe use. Inclusion of a test variety and reference varieties into field trial design, and of the statistical equivalence test would already suffice for the purpose of finding relevant parameters that warrant further assessment, hence both the inclusion of a conventional counterpart and the performance of difference testing can be omitted. This would also allow for the inclusion of safety testing regimes into plant variety testing VCU (value for cultivation and use) or other, independent variety trials.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

JC polyoma virus (JCPyV), a ubiquitous polyoma virus that commonly infects people, is identified as the etiologic factor for progressive multifocal leukoencephalopathy and has been closely linked to various human cancers. Transgenic mice of CAG-loxp-Laz-loxp T antigen were established. T-antigen expression was specifically activated in gastroenterological target cells with a LacZ deletion using a cre-loxp system. Gastric poorly-differentiated carcinoma was observed in T antigen-activated mice using K19-cre (stem-like cells) and PGC-cre (chief cells), but not Atp4b-cre (parietal cells) or Capn8-cre (pit cells) mice. Spontaneous hepatocellular and colorectal cancers developed in Alb-cre (hepatocytes)/T antigen and villin-cre (intestinal cells)/T antigen transgenic mice respectively. Gastric, colorectal, and breast cancers were observed in PGC-cre/T antigen mice. Pancreatic insulinoma and ductal adenocarcinoma, gastric adenoma, and duodenal cancer were detected in Pdx1-cre/T antigen mice. Alternative splicing of T antigen mRNA occurred in all target organs of these transgenic mice. Our findings suggest that JCPyV T antigen might contribute to gastroenterological carcinogenesis with respect to cell specificity. Such spontaneous tumor models provide good tools for investigating the oncogenic roles of T antigen in cancers of the digestive system.

Confined field trials (CFT) of genetically engineered (GE) crops are used to generate data to inform environmental risk assessments (ERA). ERAs are required by regulatory authorities before novel GE crops can be released for cultivation. The transportability of CFT data to inform risk assessment in countries other than those where the CFT was conducted has been discussed previously in an analysis showing that the primary difference between CFT locations potentially impacting trial outcomes is the physical environment, particularly the agroclimate. This means that data from trials carried out in similar agroclimates could be considered relevant and sufficient to satisfy regulatory requirements for CFT data, irrespective of the country where the CFTs are conducted. This paper describes the development of an open-source tool to assist in determining the transportability of CFT data. This tool provides agroclimate together with overall crop production information to assist regulators and applicants in making informed choices on whether data from previous CFTs can inform an environmental risk assessment in a new country, as well as help developers determine optimal locations for planning future CFTs. The GEnZ Explorer is a freely available, thoroughly documented, and open-source tool that allows users to identify the agroclimate zones that are relevant for the production of 21 major crops and crop categories or to determine the agroclimatic zone at a specific location. This tool will help provide additional scientific justification for CFT data transportability, along with spatial visualization, to help ensure regulatory transparency.