Hereditary Behavior of bar Gene Cassette is Complex in Rice Mediated by Particle Bombardment

Particle bombardment transformation using minimal gene cassette （containing the promoter, open reading frame and terminator） is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T1 to T3 generations of the transgenic rice （Oryza sativa L.） lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of ‘false homozygotes’, with abnormal segregation ratios ranging from 35：1 to 144：1 （Basta-resistant： sensitive plants） in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T2 progeny. The homozygotes were obtained in three transgenic lines in T1 generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expres- sion interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.

Characterization of Class 1 Integron Gene Cassettes among Clinical Bacteria Isolated from One Large Hospital in Northern China

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

Prevalence of Class 1 Integron, Resistance Gene Cassettes and Antimicrobial Susceptibility Profiles among Isolates of Pseudomonas aeruginosa in Iran

Pseudomonas aeruginosa is one of the most important opportunistic human pathogens worldwide. High prevalence of Multi Drug Resistant P. aeruginosa (MDRPa) in Iran is a serious problem for antimicrobial therapy. Several studies have reported the MDRPa in Europe and Asia. Due to the use of broad-spectrum antibiotics, bacterial resistance is increasing in Iran, located in Middle East. The present cross-sectional study was designed to investigate the prevalence of class1 integron, resistance gene cassettes and antimicrobial susceptibility profiles among isolates of P. aeruginosa in Al-Zahra Hospital, Isfahan City, central part of Iran from Jan-Sep 2014. The aim of this study was to determine the antimicrobial susceptibility, the prevalence of Class1 integron, resistance gene a measuring in Iran. A total of 231 P. aeruginosa isolates were collected from clinical specimens including urine (50.6%), tracheal tube (25.5%), wound (13.4%), blood (6.1%), catheter (2.2%), cerebrospinal fluid (1.7%) and sputum (0.4%) isolates from hospitalized patients (mean age: 50.27 ± 24.12 years).The majority of patients (68%) were male. Isolates were collected from different parts of the hospital as follows: ICU, Internal Medicine, Emergency care, Pediatrics, Nephrology, Transplant Center, General surgery and Infectious. Revealed data show a high rate of MDR P. aeruginosa isolates in the studied area;also, the result signifies the spread of aadA6 among clinical isolates in hospitalized patients.

Science Letters:Optimization of soybean (Glycine max (L.) Merrill) in planta ovary transformation using a linear minimal gus gene cassette

Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transformation reproducibility and efficiency. Using a linear minimal gus gene cassette as the foreign DNA, a maximum transformation frequency of 11% was obtained in flowers of the soybean cultivar ‘Liaodou 14’ with their styles mostly removed, whereas removal of only the stigma, partial style cutting and partial ovary cutting gave transformation frequencies of 0%, 1%, and 2%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (‘Liaodou 14’, ‘Liaodou 13’, and ‘Tiefeng 29’) were transformed by this optimized method. Southern blotting analysis showed that the gus reporter gene (encoding β-glucuronidase) was stably inherited with a simple pattern. Reverse transcription-polymerase chain reaction (RT-PCR) and GUS staining confirmed the expression of the gus gene in transgenic plants.

Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.

Class Ⅰ integron with a novel cassette array in an ESBL-producing multidrug-resistant Klebsiella pneumoniae isolate

Objective： To analyze the molecular mechanism of integron mediated mulfi-resistanc, e in an ESBL-producing K. pneumoniae NJ 12 isolate. Methods： Susceptibility test was carried out by Kirby-Bauer method. Class Ⅰ, Ⅱ and Ⅲ integrons were detected by integrase gene PCR with primers that annealed to conserved regions of integron-encoded integrase genes intll, intl2 and intl3. The variable region of integron was amplified by integron PCR with primers that targeted the conserved flanking regions, and the product was sequenced. Six aminoglycoside modifying-enzyme genes, including ant（2＂）-Ⅰ, ant（3＂）- Ⅰ, aac（3）- Ⅰ, aac（3）-Ⅱ, aac （6＇）-Ⅰ, and aac（6＇）-Ⅱ , were detected. Results： K. pneumoniae NJ 12 was resistant to nine antibiotics, including piperacillin, ampicillin, cefuroxime, ceftazidime, cefotaxime, aztreonam, streptomycin, gentamicin and amikacin. This isolate was shown that there was positive with class Ⅰ integron, ant（2＂）- Ⅰ , ant（3＂）- Ⅰ , aac（3）-Ⅱ and aac（6＇）- Ⅰ modifying-enzyme genes. Neither class Ⅱ nor Ⅲ integron was detected; DNA sequencing of the fragment amplified by integron PCR revealed a novel cassette array aadR-cat-blaoxa-10/ aadA1. Conclusion： Class I integron with a novel cassette array in an ESBL-producing multidrug-resistant K. pneumon/ae NJ 12 isolate was reported from Nanjing area of China, with the GenBank accession number DQ141319.

Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.

Survey of Multidrug Resistance and Class I Integron Prevalence in Chicken-derived Salmonella

[Objective] This study aimed to investigate the multidrug resistance and prevalence of class I integrons in Salmonel a. [Method] Salmonel a strains were isolated from chicken farms in Shandong Province. Kauffmann-White classification method was employed to analyze the serotypes of Salmonel a strains. Minimum in-hibition concentration （MIC） of Salmonel a strains against 19 common antimicrobial drugs was analyzed determined with microdilution method. The class I integrons and carried drug resistance gene cassettes were detected by PCR. [Result] A total of 311 Salmonel a strains were isolated and classified into two serotypes, including 133 Salmonel a Indiana strains and 178 Salmonel a Enteritidis strains. Drug sensitivity test showed that the isolated Salmonel a strains were general y resistant to sulfadiazine, sulfamethoxazole, nalidixic acid, ampicil in, tetracycline, doxycycline and trimethoprim, with a multidrug resistance rate of 91.0% （283/311）; 99% strains were sensitive to amikacin and colistin. PCR assay indicated that the detection rate of class I integrons was 65.0% （202/311）; the positive rate of class I integrons in Salmonel a strains with multidrug resistance was 92.6%; among 202 positive strains, six strains carried gene cassette dfr17-aadA5. [Conclusion] According to the above results, class I integrons exist general y in Salmonel a and are closely associated with the multidrug resistance of Salmonel a strains.

Multi-drug Resistance and Characteristic of Integrons in Shigella spp.Isolated from China

Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains （83 S. flexneri and 7 S. sonnei） were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. Results High prevalence of multi‐drug resistance （95.6%） was identified. Of the isolates 79 （87.8%） carried integrase genes of class 1 integron （3.3%）, class 2 integron （10.0%） or both （74.4%）. No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi‐drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics （P0.05）. Gene cassettes dfrA17‐aadA5, dfrA12‐orfF‐aadA2 of class 1 integron and dfrA1‐sat1‐aadA1 of class 2 integron were identified. Conclusion The class 2 integron may play a role in the emergence of multi‐drug resistance in Shigella spp.

Prevalence of Carbapenem-Resistant Klebsiella Pneumoniae （CRKP） and the Distribution of Class 1 Integron in Their Strains Isolated from a Hospital in Central China

Objective The aim of our study is to investigate the prevalence of Carbapenem-resistant Klebsiella pneumoniae （CRKP） and the genetic characteristics of the class 1 integron in CRKP on multi-drug resistance. Methods Clinical Klebsiella pneumoniae strains were collected from multiple departments of a hospital in central China. CRKP strains were identified among the isolates, and antibiotics susceptibility of CRKP strains was analyzed. The polymerase chain reaction （PCR） was adopted to amplify the class 1 integron variable area. The integron genetic structure was analyzed with enzyme digestion and DNA sequencing technology. The relation between class 1 integron and drug resistance was analyzed statistically. Results Totally 955 strains of Klebsiella pneumoniae were isolated from varied sites of the hospital, and 117（12.3%） of them were identified as CRKP, with a separation rate of 8.9% （26/292） in 2013, 11.3% （38/336） in 2014 and 16.2% （53/327） in 2015, which shows an increasing trend by year. 44.4% （52/117） of CRKP strains were separated from specimen of ICU, and 61.5% （72/117） were from sputum. Over 95% CRKP strains were resistant to ampicillin/sulbactam, aztreonam, imipenem, meropenem Ceftazidme,Cefotaxime,Cefepime,and Piperacillin, while relatively low resistant rates were found in Tigecycline （12.8%） and colistin （35.9%）. The class 1 integron was detected in 77.8% （91/117） of CRKP strains. Class 1 integron of CRKP was significantly correlated with the antibiotic resistance to the tobramycin, gentamicin and amikacin （all P〈0.01）. The gene cassette analysis of variable area of class 1 integron showed that aadA2 accounts for 64.8% （59/91）, aacA4-catB8-aadA1 23.1% （21/91）, and aadA2-dfrA25 12.1% （11/91）. Conclusions CRKP has an increasing trend in a clinical setting in China, and most of them were resistant to multiple antibiotics. Class 1 integron in CRKP has strong ability to capture the genes resistant to aminoglycosides antibiotics from environment, with the aadA2 gene as the most popular one.

Detection of class 1 integron in Acinetobacter baumannii isolates collected from nine hospitals in Turkey

Objective:To investigate the antibiotic resistance genes inserted into class I and class 2integrons in Acinetobacler baumannii[A.baumannii)isolates obtained from nine different cities in Turkey.Methods:A collection of 281 A.baumannii clinical isolates were collected from nine diferenl state hospitals in Turkey and were confirmed as A.baumannU by conventional biochemical,API testing and bla_(-OXA-51)specific PCR.The isolates were examined by PCR for existence of class I and2 integron gene cassettes.Results:They were characterized by antimicrobial susceptibility testing and the highest resistance rates were determined for piperacillin(90.03%),ciprofloxacin(87.54%),cefepime and trimethoprim/sulfamethoxazole(81.13%).The lowest resistance rates was for cefotaxime(3.55%).class 1 integrons were detected in 6.4%(18/281)of A.baumannii strains and no class 2 integron was detected.The gene casselles of class 1 inlegrons AacCI-AAC(3)l-aadAI,AacCI-aadA1,AAC(3)-I,AAC(3)-I-AAC(3)-I-aadA1,TEM-1.AAC(3)-I-aadA1-AAC(3)-I-AAC(3)-I,AAC(3)-I-AAC(3)-I-AAC(3)-I-aadA1,AAC(3)-I-aadA1,AAC(3)-I-AAC(3)-I,AAC(3)-I-aadA1-AAC(3)-I-aadA1,AAC(3)-I-AAC(3)-I-aadA1-AAC(3)-I-aadA1 were detected in eighteen strains.The aac genes family were most frequently found integrated into the class 1 inlegrons and it was followed by aadA genes and TEM-1 genes.Conclusions:This is an extensive study on the distribulion of class 1 integron among A.baumannii in Turkey.In addition to these,two new alleles were observed.Their percentage rates of similarity to other cassettes are 95%aadA1(TK A18)and 89%,aadA 1(ANKA3).

发夹式siRNA对hDNMT1基因表达的抑制作用

目的 研究发夹式siRNA对hDNMT1基因表达的抑制作用。方法 根据hDNMT1全长cDNA序列设计和合成与其互补的发夹式siRNA序列 ,并以发夹式siRNA序列为模板经体外合成siRNA表达盒 (SECs)。SECs通过脂质体载体转染结直肠癌细胞株HCT1 1 6后 ,经半定量RT PCR法检测hDNMT1mRNA的表达水平。结果 发夹式siRNA可以特异性地抑制hDN MT1基因。结论 通过SECs产生的发夹式siRNAs可以特异性抑制hDNMT1基因的表达 ;本研究为通过构建表达发夹式siR

Identification and characterization of integron mediated antibiotic resistance in pentachlorophenol degrading bacterium isolated from the chemostat

A bacterial consortium was developed by continuous enrichment of microbial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium（MSM） supplemented with pentachlorophenol（PCP） as sole source of carbon and energy in the chemostat.The consortia contained three bacterial strains.They were identified as Escherichia coli,Pseudomonas aeruginosa and Acinetobacter sp.by 16S rRNA gene sequence analysis.Acinetobacter sp.readily degraded PCP through the formation of tetrachloro-p-hydroquinone（TecH）,2-chloro-1,4-benzenediol and products of ortho ring cleavage detected by gas chromatograph/mass spectrometer（GC-MS）.Out of the three acclimated PCP degrading bacterial strains only one strain,Acinetobacter sp.showed the presence of integron gene cassette as a marker of its stability and antibiotic resistance.The strain possessed a 4.17 kb amplicon with 22 ORF＇s.The plasmid isolated from the Acinetobacter sp.was subjected to shotgun cloning through restriction digestion by BamHI,HindIII and SalI,ligated to pUC19 vector and transformed into E.coli XLBlue1α,and finally selected on MSM containing PCP as sole source of carbon and energy with ampicillin as antibiotic marker.DNA sequence analysis of recombinant clones indicated homology with integron gene cassette and multiple antibiotic resistance genes.