The cryIB, truncated cryIB$[cryIB({\alpha})]$, cryIA(b), and cytA genes, encoding 135-, 89-, 131-, and 27-kDa proteins, respectively, from Bacillus thuringiensis were cloned into a shuttle vector pBES and expressed in E. coli and Bacillus species. The morphology and solubility in alkaline buffer of the insecticidal crystal proteins were investigated. Transformation of intact cells of E. coli and Bacillus species was achieved by electroporation. High field strength of 11.0 kV/cm and resistance of 129 ohms were required for efficient transformation of E. coli strains and 4.5 kV/cm and 48 ohms for Bacillus species. Strains of recombinant E. coli and Bacillus species produced the insecticidal crystal proteins and accumulated as the same bipyramidal and irregular structures as those of CryIB and IA(b) and CytA of B. thuringiensls, respectively. The insecticidal crystal proteins accumulated in recombinant E. coli wire smaller in size than those in recombinant Bacillus species. The solubility in alkaline buffer of the insecticidal crystal proteins of recombinant E. coli increased gradually as the pH increased, whereas in the case of Bacillus species the solubility increased gradually as the pH increased up to 9 and then the solubility increased greatly up to two times higher than that of E. coli proteins.
Khan, Abdul Latif;Hamayun, Muhammad;Ahmad, Nadeem;Hussain, Javid;Kang, Sang-Mo;Kim, Yoon-Ha;Adnan, Muhammad;Tang, Dong-Sheng;Waqas, Muhammad;Radhakrishnan, Ramalingam;Hwang, Young-Hyun;Lee, In-Jung
Journal of Microbiology and Biotechnology
/
v.21
no.9
/
pp.893-902
/
2011
Endophytic fungi are little known for their role in gibberellins (GAs) synthesis and abiotic stress resistance in crop plants. We isolated 10 endophytes from the roots of field-grown soybean and screened their culture filtrates (CF) on the GAs biosynthesis mutant rice line - Waito-C. CF bioassay showed that endophyte GMH-1B significantly promoted the growth of Waito-C compared with controls. GMH-1B was identified as Penicillium minioluteum LHL09 on the basis of ITS regions rDNA sequence homology and phylogenetic analyses. GC/MS-SIM analysis of CF of P. minioluteum revealed the presence of bioactive $GA_4$ and $GA_7$. In endophyte-soybean plant interaction, P. minioluteum association significantly promoted growth characteristics (shoot length, shoot fresh and dry biomasses, chlorophyll content, and leaf area) and nitrogen assimilation, with and without sodium chloride (NaCl)-induced salinity (70 and 140 mM) stress, as compared with control. Field-emission scanning electron microcopy showed active colonization of endophyte with host plants before and after stress treatments. In response to salinity stress, low endogenous abscisic acid and high salicylic acid accumulation in endophyte-associated plants elucidated the stress mitigation by P. minioluteum. The endophytic fungal symbiosis of P. minioluteum also increased the daidzein and genistein contents in the soybean as compared with control plants, under salt stress. Thus, P. minioluteum ameliorated the adverse effects of abiotic salinity stress and rescued soybean plant growth by influencing biosynthesis of the plant's hormones and flavonoids.
During 2008~2009 winter season, yellow patch and gray snow mold occurred on turfgrass plants in golf courses in Kangwon and Jeonbuk provinces, respectively. The fungi associated with the diseases were identified as Rhizoctonia cerealis Van der Hoeven and Typhlua incarnata Lasch ex Fr., based on the morphological characteristics of hyphae and sclerotia. R. cerealis and T. incarnata were pathogenic to most turfgrass and crop species tested. R. cerealis infected crown, stem and leaf tissue of the host plants, and the symptom was light yellow circular patch. Individual infected leaf near the margin of patch developed red color first and finally turn brown. The symptoms caused by gray snow mold pathogen are water-soaked spots, and became a watery soft rot. Infection parts became yellow and then turned brown followed by death of the whole plant. White mycelia were developed on higher petioles, leaves, and on soil where these plant parts lay, and black sclerotia of variable size and shape formed in the mycelial mass. All isolates tested were pathogenic on most turfgrass and crop plants, and significantly different in aggressiveness. Disease severity increased with longer snow cover days on target plants, suggesting that disease severity was expressed over snow cover days. There were significant differences in disease severity among the graminious species, and among cultivars within each species, indicating varying levels of susceptibility to R. cerealis and T. incarnata.
Kim, Soo-Yong;Choi, Myung-Sik;Chang, Woo-Hyun;Cha, Chang-Yong
The Journal of the Korean Society for Microbiology
/
v.22
no.3
/
pp.233-240
/
1987
The amniotic fluid provides a medium in which the fetus can readily move, cushions him against possible injury and helps him maintain an even temperature. Besides above mentioned functions, investigators reported that human amniotic fluid contains host-resistance factors which prevent bacteria from producing infectious disease and this activity shows difference among human racial groups or bacterial genera, species and strains. 40 amniotic fluid specimens from Korean women in their second and third trimesters of pregnancy were examined for inhibiting the growth of Escherichia coli. And various factors which might affect bacterial growth inhibiting activity such as pH, initial inoculum size, concentration of amniotic fluid, and heat resistance, were also tested using a strongly inhibitory amniotic fluid specimen. Finally plate diffusion tests were carried out using other strongly inhibitory amniotic fluid. The following results were obtained: 1. Of the 40 fluid samples examined, 18 specimens(45%) had inhibitory activity and samples from women in their second trimester of pregnanancy showed non-inhibitory activity(2 specimens). 2. The pH of the fluids varied between 7.43 and 8.33. There was no correlation between pH and inhibitory activity. 3. No. 19 amniotic fluid showed bacteriostatic activity after 24 hours incubation when an inoculum of $10^2$ organisms per milliliter was used, but non-inhibitory with an inoculum of $10^3$ and $10^4$ bacteria per milliliter. 4. The content of amniotic fluid in culture media influenced E. coli growth. At 90 percent, E. coli was inhibited growth but at 10 percent and 50 percent. 5. Inhibitory activity of No. 19 amniotic fluid was retained after heating to $50^{\circ}C$ for 30 minutes or 100^{\circ}C$ for 30 minutes. 6. Plate diffusion tests with No. 27 amniotic fluid showed that 0.7ml amniotic fluid gave clear zone of growth inhibition around the central well but 0.2ml and 0.1ml amniotic fluids were not.
This study was performed to evaluate the resistance of three different rice cultivars to the BPH biotypes 1, 2, 3 and their hybrid progenies, Nilaparvata lugens STAL. The rice cultivars tested were Chucheongbyeo, Cheongcheongbyeo and Milyang 63. The following characteristics of hybrid progenies were compared with these of their parents: Seedling response, Host preference, feeding amount, Esterase polymorphism of the brown rice. According to the responses of rice seedlings to the three BPH biotypes and their crosses progenies nymph, Chucheongbyeo was shown highly susceptible to their nymphs. The Cheongcheongbyeo was susceptible to the biotypes 2 and the Milyang 63 was susceptible to the biotype 3. The Cheongchengbyeo and the Milyang 63 were moderate to the biotypes 2(female)${\times}$3(male) and 3(female)${\times}$2(male). The preference in feeding and oviposition of three BPH biotypes and their hybrid progenies to Chucheongbyeo were shown higher than to another cultivars. The two preference were not only shown highly in Cheongcheongbyeo by the biotypes 2,2(female)${\times}$3(male) and 3(female)${\times}$2(male) but also in Milyang 63 by the biotypes 3,2(female)${\times}$3(male) and 3(femlae)${\times}$2(male). The feeding amount of female adult was much on three BPH biotypes and their hybrid progenies in Chucheongbyeo and on the biotypes 2 and 3(female)${\times}$2(male) in Cheongcheongbyeo, On the biotypes 3 and 3(female)${\times}$2(male) in Milyang 63 were as same as the above. The esterase bands of Est ${\alpha}-1,\;{\beta}-4,\;{\beta}-5\;and\;{\beta}-I$ were detected on brown rice of all the tested cultivars. The bands of Est ${\beta}-2\;and\;{\alpha}-I$ were detected on Cheongcheongbyeo (Bph 1 gene for resistance) and Milyang 63 (bph 2 gene for resistance). The Est ${\beta}-3\;and\;{\beta}-1$ of esterase bands were detected on Chucheongbyeo.
This experiment was attempted to investigate a cause of the scion death in green pepper grafting system. A tobamovirus particle examined in the rootstock of the sample but not in the scion showing necrosis. The virus isolated from the rootstock was identified as Pepper mild mottle virus (PMMoV), pepper tobamovirus pathotype P1.2. (PMMoV-2), by nucleotide sequence analysis and host plant reaction. The virus isolate infected systematically in 6 commercial rootstock varieties using for green pepper grafting seedling production. Green pepper varieties 'Long green mart' and 'Daechan' represented resistance to the virus showing local lesions only on the inoculated leaves and 'Manitda' was systematically infected. In the experiment with grafting 'Long green mart' or 'Daechan' onto the those rootstocks, the upper leaves of the scions first showed vein necrosis and wilt symptoms 7 days after inoculation with PMMoV-2 on the cotyledon of the rootstock, following to the scion stem necrosis and then only the scion death. The virus was detected in the rootstock but not in the scion. However, 'Manitda' of susceptible variety in the grafting system showed mottle symptom on the leaves of the scion but not necrosis on the plant. PMMoV-3 isolate, pepper tobamovirus pathotype P1.2.3, did not cause the scion death in the grafting system. All of the varieties were susceptible to PMMoV-3. These results suggest that the scion death is caused by infecting with pepper tobamovirus pathotype P1.2. in the green pepper grafting system combined with the susceptible rootstock and the resistance scion to the virus pathotype.
This study aimed to assess the disease incidence and distribution of toxigenic in Korean triticale. The pathogen of triticale that cause Fusarium head blight were isolated from five different triticale cultivars that cultivated in Suwon Korea at 2021 year. The 72 candidate were classified as a Fusarium asiaticum by morphology analysis and by ITS1, TEF-1α gene sequence analysis. And the results of pathogenicity with 72 isolates on seedling triticale, 71 isolates were showed disease symptom. Also, seven out of 71 Fusarium isolates were inoculated on the wheat, to test the pathogenicity on the different host. The results showed more low pathogenicity on the wheat than triticale. The results of analysis of toxin type with 72 isolates, 64.6% isolates were produced nivalenol type toxin and other 4.6% and 30.8% isolates were produce 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, respectively. To select fungicide for control, the 72 Fusarium isolates were cultivated on the media that containing four kinds fungicide. The captan, hexaconazole, and difenoconazole·propiconazole treated Fusarium isolates were not showed resistance response against each fungicide. However, six isolates out of 72 isolates, showed resistance response to fludioxonil. This study is first report that F. asiaticum causes Fusarium head blight disease of triticale in Korea.
Purpose : Infection with Shiga-like toxin (SLT)-producing Escherichia coli, an emerging human pathogen found particularly in young children under 5 years of age, causes a spectrum of illnesses with high morbidity and mortality, ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome. Host mediators play an important role in the pathogenesis of SLT-I toxicity. The experiments described here were designed to investigate the effect of SLT-I on TNF-${\alpha}$ production and to understand the effect of TNF-${\alpha}$ on GB3 expression. We also further examine the relationship between the Gb3 level and the differential susceptibility of cells to the cytotoxic action of SLT-I. Methods : The effect of purified SLT-1 from E. coli O157 : H7 (ATCC 43890) on tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) production in Raw264.7 cells was investigated. Many mediators regulate endothelial cell membrane expression of the glycolipid globotriaosyleramide (Gb3), which serves as the toxin receptor, suggesting that the host response to the toxin or other bacterial products may contribute to pathogenesis by regulating target cell sensitivity to the toxins. Therefore, the relationships between Gb3 expression and cytotoxicity against SLT-I on three types of cells were evaluated. Results : Detectable levels of TNF-${\alpha}$ were produced as early as six hours after induction and continued to increase during 48 hours by SLT-I. It was also found that Vero cells and dendritic cells (DC2.4 cells) expressed high levels of Gb3, 83% and 68%, respectively, and that Raw264.7 cells had a low level of Gb3 (29%) and appeared refractory to cytotoxicity against SLT-I. Vero cells and DC2.4 cells expressing high levels of Gb3 were highly susceptible to SLT-I. Furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. Conclusion : These results strongly suggest that the expression of Gb3 is necessary but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its Gb3 receptors in the pathogenesis of E. coli O157 infection.
Lee, Sang Mi;Kim, Ji Woo;Jeong, Young-Hee;Kim, Se Eun;Kim, Yeong Ji;Moon, Seung Ju;Lee, Ji-Hye;Kim, Keun-Jung;Kim, Min-Kyu;Kang, Man-Jong
Asian-Australasian Journal of Animal Sciences
/
v.27
no.11
/
pp.1644-1651
/
2014
Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ${\beta}$-casein gene locus using a knock-in vector for the ${\beta}$-casein gene locus. We developed the knock-in vector on the porcine ${\beta}$-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ${\beta}$-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ${\beta}$-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ${\beta}$-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
Background: Host genetic polymorphisms in the HIV-1 co-receptor CCR5 and CCR2b and SDF-1, ligand for co-receptor CXCR4, have been known to be associated with the resistance of HIV infection and/or the delayed disease progression in HIV-infected patients. Methods: We examined the frequencies of SDF1-3'A and CCR2b-64I alleles of 354 Koreans including 100 HIV-uninfected persons, 13 discordant spouses of HIV-infected persons, and 241 HIV-infected persons. The genotyping assays of SDF1 and CCR2b genes were carried out by polymerase chain reaction-restriction fragment length polymorphism. Results: The frequencies of CCR2b-64I and SDF1-3'A alleles in Koreans were very high compared with Caucasians and blacks. Observed frequencies of CCR2b-64I and SDF1-3'A allelic variants were 25.1% and 28.7%, respectively. The frequency of the CCR2b-64I allele in Koreans was 2~4 times higher than those of other ethnic groups with the exception of Asian. The frequencies of CCR2b-64I and SDF1-3'A genotypes did not show the significant difference between HIV-infected and uninfected Koreans. However, the prevalence of CCR2b-64I genotype of the LTNP group was about two times higher than that of the remainder group (P< 0.05). Four (45%) out of 9 LTNPs (long-term nonprogressors) showed having the SDF1-3'A allele and 7 (78%) out of 9 LTNPs carried the CCR2b-64I allele. 3 (33%) out of 9 LTNPs had both SDF1-3'A and CCR2b-64I alleles. But none of 5 RPs (rapid progressors) appeared to have both SDF1-3'A and CCR2b-64I alleles. Conclusion: The different genetic backgrounds in study populations may affect the disease progression and the AIDS epidemic in each country. Further studies need to define whether high frequencies of CCR2b-64I and SDF1-3'A allelic variants may affect the HIV disease progression.
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