A case report of congenital myasthenic syndrome caused by a mutation in theCHRNE genein the Iranian population.

Congenital myasthenic syndrome (CMS) refers to a heterogeneous group of inherited disorders, characterized by defective transmissionat the neuromuscular junction (NMJ). Patients with CMS showed similar muscle weakness, while other clinical manifestations are mostly dependent on genetic factors. This disease,caused bydifferent DNA mutations, is genetically inherited. It is also associated with mutations of genes at NMJ, involving the acetylcholine receptor (AChR) subunits. Here, we present the case ofa five-year-old Iranian boywith CMS, undergoingtargeted sequencing of a panel of genes, associated with arthrogryposis and CMS. The patient had six affected relatives in his genetic pedigreechart. The investigations indicated a homozygous single base pair deletion at exon 12 of the CHRNE gene (chr17:4802186delC).This region was conserved across mammalian evolution and was not submitted to the 1000 Genomes Project database.Overall, the CHRNEvariant may beclassified as a significant variant in the etiology of CMS.It can besuggested thatthe Iranian CMS population carry regional pathogenic mutations, which can be detected viatargeted and whole genome sequencing.

Agrobacterium-Mediated Transformation of the Oryza sativa Thaumatin-Like Protein to Canola (R Line Hyola308) for Enhancing Resistance to Sclerotinia sclerotiorum.

Background: Canola is an agro-economically oilseed crop. Yield loss due to fungal disease of stem rot caused by Sclerotinia sclerotiorum is a serious problem in canola cultivation. Thaumatin-like proteins are large groups of the pathogenesis-related proteins which provide resistance to the fungal infection in response to invading pathogens and play a key role in plant defense system. Objectives: Transformation of the rice tlp into canola via Agrobacterium-mediated transformation and evaluation of the antifungal activity of the expressed TLP in the transgenic events on the S. sclerotiorum growth was subject to investigation. Materials and methods: The canola (R line Hyola308) was used for transformation experiment. The vector, pBITLPRA1, was used for the stable transformation. The PCR and southern blotting techniques were used to confirm transgene's presence in the transgenic canola events. Antifungal activity of transgenic plants was evaluated by the radial diffusion and spore germination assays. T2 transgenic plants were evaluated by the intact leaf inoculation method in greenhouse assay. Results: In this study, pBITLPRA1 construct containing tlp gene was introduced into canola and the transformed plants were verified by PCR. The glucanase activity of tlp gene in T0 generation was measured and transgenic plants with high activity were assessed by Southern blot analysis to confirm the copy number of the gene. Also, antifungal activity of the single copy T0 transgenic plants against Sclerotinia sclerotiorum was evaluated by radial diffusion and spore germination assays. In greenhouse assay, evaluation of T2 transgenic plants by the intact leaf inoculation method demonstrated that following the infection with S. sclerotiorum, there was a significant reduction in the lesion's diameter in transgenic lines compared to the non-transgenic ones. Conclusions: These results revealed that expression of TLP has an inhibitory effect against fungus compared to non-transgenic plants both in vitro and in vivo (i.e., greenhouse condition). These transgenic lines could be used as the additional sources of disease resistance for canola breeding program.

Co-transformation of canola by chimeric chitinase and tlp genes towards improving resistance to Sclerotinia sclerotiorum.

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.

Designing a new chitinase with more chitin binding and antifungal activity.

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.