The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance.
Agrobacterium tumefaciens ; Arabidopsis ; Arabidopsis Proteins ; genetics ; Disease Resistance ; Gibberella ; pathogenicity ; Hydrogen Peroxide ; Plants, Genetically Modified ; microbiology ; Recombinant Fusion Proteins ; genetics ; Tobacco ; genetics ; microbiology

0.05).2.5 mg/kg and 5.0 mg/kg avermetine groups can increased the activity of LDH,GS,CK in cerebrum,and decreased the activity of CaN in cerebrum,compared with control group(P0.05).Conclusion Avermectine may cause the toxic effect in rat nervous system through creating the nerve metabolism enzyme activity.

Objective To study the toxicity of fosthiazate feeding for 90 days in rats, and to determine the maximal non-effective dose of fosthiazate , in order to provide the reference dose for safety in production and chronic toxicity experiment.Methods A total of 80 SD rats ( half female and half male ) were randomly divided into 4 groups, respectively:0.8 mg/kg· bw· d group, 4.0 mg/kg· bw· d group, 20.0 mg/kg· bw· d group, and normal control group .The rats were sacrificed to determine the indices including serum biochemical parameters , body weight , routine urine test and organ coefficients after the end of the experiment , and the results were statistically analyzed .Results In the high dose group, the body weight gain was slowed in male and female rats .The TG and CHE in the high dose group of male rats and the TP, ALB, CREA, GLU, and CHE in the high dose group of female rats were significantly lower than those of normal control group.The ALP in the high dose group of female rats was higher than that of the normal control group .The positive rates of BIL, SG, and PRO in both male and female rats had significant differences compared with those of normal control group.The organ coefficients of brain , lung, kidney, adrenal, and testis of male rats, and the organ coefficients of brain , lung, and kidney of female rats in the high dose group were significantly higher than those of the normal control group .The ovaries and uterus in the female rats of high dose group were significantly lower than those of normal control group ( P<0.05,P<0.01).Conclusions The oral dose of fosthiazate at 4.0 mg/kg· bw· d fed for 90 days and above cause toxic effects on rats , and its maximal non-effect dose of long-term intake of low-dose fosthiazate on rats is 4 mg/kg· bw· d.

Objective To develop a convenient, rapid and specific method using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) for detection of facioscapulohumeral muscular dystrophy(FSHD). Methods Genomic DNA was extracted and digested by restricted endonuclease EcoR Ⅰ , followed by agarose electrophoresis. The DNA (< 38 kb) was retrieved from agarose electrophoretic gels. The primers and probe were designed in D4ZA gene in chromosome 4. One hundred and fifteen subjects were examined by FQ-PCR using the retrieved DNA (<38 kb) as a template and the result was analyzed by fluorescent curve comparing with positive control. Results The results by FQ-PCR showed that 13 cases were positive in 16 FSHD cases whose EcoR Ⅰ fragment sizes were known, 75 cases were negative in 78 cases of normal controls, 15 cases were positive in 16 FSHD cases diagnosed clinically whose EcoR Ⅰ fragment sizes were unknown, and 3 cases were positive in 5 cases of relatives of FSHD patients. Consistency was checked using Kappa index between the 2 gene diagnostic tests for FSHD (FQ-PCR test and the traditional Southern blotting test), and between the 2 diagnostic criterions (gene diagnosis by FQ-PCR and clinical diagnosis). The results were statistically significant (κ = 0. 765, P = 0. 002 ; κ = 0. 844, P = 0. 000). Conclusions A new genetic diagnostic method of FSHD by FQ-PCR was developed, which was more simplified and reliable compared to the time-consuming, radioactive Southern blotting. It could also detect the D4Z4 arrays in cases having deletion of p13E-11 as well as the interchromosomal exchange between 4q35 and 10q26. The new method of FQ-PCR for FSHD may be extended to utilize clinically in future.

Objective:To analyze the gene variants of a patient affected with Duchenne/Becker muscular dystrophy in a pedigree and further explore the genotype-phenotype correlation for providing basis for family genetic counseling.Methods:The clinical features and family history of family members were collected.Multiplex ligation-dependent probe amplification(MLPA)was utilized to detect copy number variation of target genes.The pathogenic variations were ana-lyzed by whole exome sequencing(WES).The suspected gene variations were verified by Sanger sequencing.For the splice site mutations,mini-gene was constructed and expressed in vitro to detect the number of transcript and cDNA se-quence.Results:The proband of this family is a male,with no obvious involvement of the lower limbs.Laboratory tests showed an elevated level of creatine kinase(CK)in peripheral blood(700-1600 U/L),and electromyography showed myogenic damage.MLPA did not detect pathogenic exon copy number variation in dystrophin(DMD)gene.Genetic testing showed the proband carried a maternal hemizygotic splicing variation of DMD gene(NM_004006.2):c.2622+2T＞C.An in vitro mini-gene splicing assay confirmed that this splicing mutation could affect RNA splicing.According to clinical features and genetic testing results,the proband was speculated first proof of Duchenne/Becker muscular dys-trophy(DMD/BMD)caused by DMD gene mutation.Conclusion:This study identified the pathogenic variation of a proband with DMD/BMD of DMD gene,which enriched the variation spectrum of DMD/BMD in China.It was con-firmed that the splicing variation of the DMD gene c.2622+2T＞C can produce multiple transcripts leading to different functional impairments,and based on the specificity of temporal and spatial expression,it corresponded to the mild clin-ical manifestations of the patient,providing some reference value for the correlation between genotype and phenotype.

Objective:To explore the clinical phenotype and genetic variant in a Chinese pedigree affected with Hunter syndrome and create immortalized cell lines for the affected pedigree members.Methods:A pedigree of six members who had visited Xi′an Children′s Hospital in July 2022 was selected as the study subject. Clinical data was collected. Whole exome sequencing was carried out for the pedigree members. Candidate variant was verified by Sanger sequencing. In addition, peripheral B lymphocytes were transfected with Epstein-Barr virus to create immortalized cell lines, which were then subjected to enzyme activity analysis.Results:The patient, a five-year-and-seven-month-old boy, had exhibited stiff limbs and enlarged joints. He had developed hernia, scaphocephaly, and barrel chest from 3 months of age. His uncle also had stiff limbs, poor hearing, blindness, and right oblique inguinal hernia. Above features had resembled those of Hunter syndrome. Genetic testing revealed that both the child and his uncle had harbored an IDS (NM_000202.8): c. 823G>A (p.D275N) variant, which was unreported previously. Bioinformatic analysis indicated that the D275 to be a highly conserved site, and the D275N variant may affect the stability of the protein′s spatial conformation, thereby decrease the catalytic activity of the enzyme. The successfully constructed immortalized lymphoblastoid cell lines for the child and his parents showed increased volume, irregular shape, burr structure and cluster growth. And the value of IDS activity of the patient′s immortalized lymphoblastoid cells was below the limit of detection. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PS3+ PM2_Supporting+ PM5+ PP1+ PP3). Conclusion:Above finding has enriched the phenotypic and mutational spectra of Hunter syndrome, and provided a basis for the genetic counseling for this pedigree. The creation of immortalized cell lines has offered a model for further investigation of the impact of variant on the function of IDS and development of targeted drugs.

Objective:To analyze the clinical characteristics of a child with Congenital disorder of glycosylation due to compound heterozygous variants of COG6 gene ( COG6-CDG). Methods:A child who was admitted to Xi′an Children′s Hospital on January 10, 2023 was selected as the study subject. Clinical data were collected. Pathogenic variants were analyzed by whole exome sequencing, and candidate variants were verified by Sanger sequencing, in vitro experiments and bioinformatic analysis. This study was approved by the Medical Ethics Committee of Xi′an Children′s Hospital (No. 20230101). Results:The child, a 1-month-8-day-old male, was admitted for diarrhea and weight loss for one month. He had presented with cholestasis, diarrhea, facial dysmorphism, poor response, bilateral Simian crease, and brain atrophy. After discharge, he had continued to have high fever, feeding difficulty, and deceased finally. Whole exome sequencing results showed that he had harbored compound heterozygous variants of the COG6 gene, namely c. 807delT (p.F269Lfs*37) and c. 1746+ 1G>C (p.Gly565_Met582del). Sanger sequencing verified that the variants were inherited from his father and mother, respectively. In vitro experiments verified that the c. 1746+ 1G>C variant could affect the mRNA splicing and produce a truncated protein, whilst the c. 807delT variant could significantly reduce gene expression at both mRNA and protein levels. Based on the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP), the variants were classified as pathogenic (PVS1+ PM3+ PM2_Supporting) and likely pathogenic (PVS1+ PM2_Supporting), respectively. Conclusion:The c. 807delT (p.F269Lfs*37) and c. 1746+ 1G>C (p.Gly565_Met582del) compound heterozygous variants of the COG6 gene probably underlay the pathogenesis of this child. Above finding has enriched the mutational spectrum of COG6-CDG and provided a basis for the genetic counseling for this family.