Molecular characterization and n use efficiency of LeAlaAT ‘Mekongga’ transgenic rice lines

Genetic engineering is one of the strategies for developing nitrogen (N)-use-efficient rice (Oryza sativa) varieties. One gene that plays an indirect role in N metabolism is alanine aminotransferase (AlaAT). It can efficiently increase N content and crop yield. In a previous study, the tomato AlaAT gene (LeAlaAT) was successfully isolated and introduced into ‘Mekongga’ rice. The present research was conducted during 2018 and 2019 at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor, Indonesia. The objectives of the present study were to perform the molecular characterization of LeAlaAT ‘Mekongga’ rice lines on the basis of the hpt marker gene, the direct PCR of the LeAlaAT fragment, and the phenotypic evaluation of the selected LeAlaAT T1 ‘Mekongga’ rice lines in response to different N fertilizer rates (0 kg ha−1 [control] and 60, 90, and 120 kg ha−1). This research involved three activities, namely (1) Southern blot analysis, (2) direct PCR, and (3) N use efficiency (NUE) test of ‘Mekongga’ transgenic lines. Southern blot analysis revealed that in T0 transgenic lines, the copy number of the hpt marker gene varied from 1 to 3. Direct PCR confirmed the presence of the AlaAT fragment in the T1 generation of five ‘Mekongga’ transgenic lines. The five transgenic lines showed high panicle number, biomass weight, shoot dry weight, and total grain weight under 120 kg ha−1 nitrogen. The high agronomical NUE of transgenic lines under 120 kg ha−1 N implied that the transgenic rice lines have the potential for efficient N use at a certain minimum level of N (120 kg ha−1 of nitrogen) and should be further evaluated at high N levels.

Emergence of mcr-1-Harboring Salmonella enterica Serovar Sinstorf Type ST155 Isolated From Patients With Diarrhea in Jiangsu, China

Background: This study analyzed the antimicrobial resistance phenotypes and mechanisms of quinolone, cephalosporins, and colistin resistance in nontyphoidal Salmonella from patients with diarrhea in Jiangsu, China.Methods: A total of 741 nontyphoidal Salmonella isolates were collected from hospitals in major cities of Jiangsu Province, China between 2016 and 2017. Their susceptibility to commonly used antibiotics was evaluated by broth micro-dilution and sequencing analysis of resistance genes screened by a PCR method. For mcr-1 positive isolates, genetic relationship study was carried out by pulsed-field gel electrophoresis and multiloci sequence typing analysis. The transferability of these plasmids was measured with conjugation experiments and the genetic locations of mcr-1 were analyzed by pulsed-field gel electrophoresis profiles of S1-digested genomic DNA and subsequent Southern blot hybridization.Results: Among 741 nontyphoidal Salmonella isolates, the most common serotypes identified were S. Typhimurium (n=257, 34.7%) and S. Enteritidis (n=127, 17.1%), and the isolates showed 21.7, 20.6, and 5.0% resistance to cephalosporins, ciprofloxacin, and colistin, respectively. Among the 335 nalidixic acid-resistant Salmonella, 213 (63.6%) and 45 (13.4%) had at least one mutation in gyrA and parC. Among the plasmid-borne resistance, qnrS1 (85; 41.9%) and aac(6')-Ib-cr4 (75; 36.9%) were the most common quinolone resistance (PMQR) genes, while blaCTX-M-14 (n=35) and blaCTX-M-55 (n=46) were found to be dominant extended-spectrum beta-lactamase (ESBL) genes in nontyphoidal Salmonella. In addition, eight mcr-1-harboring strains were detected since 2016 and they were predominate in children under the age of 7years. Conjugation assays showed the donor Salmonella strain has functional and transferable colistin resistance and Southern blot hybridization revealed that mcr-1 was located in a high molecular weight plasmid.Conclusion: In nontyphoidal Salmonella, there is a rapidly increasing trend of colistin resistance and this is the first report of patients harboring mcr-1-positive Salmonella with a new ST type ST155 and new serotype S. Sinstorf. These findings demonstrate the necessity for cautious use and the continuous monitoring of colistin in clinical applications.

Occurrence and Molecular Characteristics of Extended-Spectrum Beta-Lactamase-Producing Enterobacterales Recovered From Chicken, Chicken Meat, and Human Infections in Sao Paulo State, Brazil

This study aimed to investigate the phylogenetic diversity and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from chicken, chicken meat, and human clinical isolates in Sao Paolo, Brazil, and characterize their respective ESBL-encoding plasmids. Three hundred samples from chicken cloaca, chicken meat, and clinical isolates were phenotypically and genotypically assessed for ESBL resistance. Isolates were identified by MALDI TOF-MS and further characterized by MLST analysis and phylogenetic grouping. ESBL genes were characterized and their location was determined by I-Ceu-I-PFGE and Southern blot, conjugation, transformation, and PCR-based replicon typing experiments. Thirty-seven ESBL-producing isolates (28 E. coli and 9 K. pneumoniae) that were positive for the blaCTX–M–1 or blaCTX–M–2 gene groups were obtained. Two isolates were negative in the transformation assay, and the chromosomal location of the genes was deduced by Southern blot. The blaCTX–M genes identified were carried on plasmid replicon-types X1, HI2, N, FII-variants, I1 and R. The E. coli isolates belonged to nine sequence types, while the K. pneumoniae isolates belonged to four sequence types. The E. coli isolates belonged to phylotype classification groups A, B1, D, and F. This study demonstrated that isolates from cloacal swabs, chicken meat, and human feces had genetic diversity, with a high frequency of blaCTX–M–15 among chickens, chicken meat, and human feces. Thus, this reinforces the hypothesis that chickens, as well as their by-products, could be an important source of transmission for ESBL-producing pathogens to humans in South America.

Detection of Cryptic Fragile X Full Mutation Alleles by Southern Blot in a Female and Her Foetal DNA via Chorionic Villus Sampling, Complicated by Mosaicism for 45,X0/46,XX/47,XXX

We describe a female with a 72 CGG FMR1 premutation (PM) (CGG 55–199) and family history of fragile X syndrome (FXS), referred for prenatal testing. The proband had a high risk of having an affected pregnancy with a full mutation allele (FM) (CGG > 200), that causes FXS through hypermethylation of the FMR1 promoter. The CGG sizing analysis in this study used AmplideX triplet repeat primed polymerase chain reaction (TP-PCR) and long-range methylation sensitive PCR (mPCR). These methods detected a 73 CGG PM allele in the proband’s blood, and a 164 CGG PM allele in her male cultured chorionic villus sample (CVS). In contrast, the Southern blot analysis showed mosaicism for: (i) a PM (71 CGG) and an FM (285–768 CGG) in the proband’s blood, and (ii) a PM (165 CGG) and an FM (408–625 CGG) in the male CVS. The FMR1 methylation analysis, using an EpiTYPER system in the proband, showed levels in the range observed for mosaic Turner syndrome. This was confirmed by molecular and cytogenetic karyotyping, identifying 45,X0/46,XX/47,XXX lines. In conclusion, this case highlights the importance of Southern blot in pre- and postnatal testing for presence of an FM, which was not detected using AmplideX TP-PCR or mPCR in the proband and her CVS.

Construction and identification of Rab7-ROSA26 knock-in mice by CRISPR/Cas9 technology

Background: CRISPR/Cas9 system, a Genome editing technique, can achieve the purpose of gene knockout and gene insertion. Because we want to study the relationship between Rab7 and renal fibrosis in vivo, we should construct Rab7-knock-in mice. Results: We constructed Rab7-knock-in mice by CRISPR/Cas9 technology. Using the identification of PCR, southern blot and sequencing, the detected results were in line with our expectation. Conclusions: We constructed Rab7-knock-in mice successful, which can be provided for studying the relationship between Rab7 and renal fibrosis.

HIV-1 P24 EXPRESSION IN TRANSPLASTOMIC TOBACCO PLANTS

Expression of HIV-1 p24 gene in chloroplasts was achieved in a tobacco variety V2 (Virginia TBE2). Through PCR and Southern blot analyses, it was demonstrated that the transgene integrated into the target site in the chloroplasts, between trnfM and trnG. Western blot results showed that HIV-1 p24 gene expressed in transplastomic tobacco plants. p24 protein accumulations were detected by ELISA in the range from 1.7% to 6.3% TSP and the high concentrations in the leaves near the top. p24 protein was purified by gel filtration chromatography demonstrated that the purification is 9.694 folds and the performance is 31.94%, however, protein p24 largely was inactive after purification.