• Title/Summary/Keyword: Southem hybridization

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Detection of cryIB Genes in Bacillus thuringiensis subsp. entomocidus and subsp. subtoxicus

  • CHOI, SOO KEUN;BYUNG SIK SHIN;BON TAG KOO;SEUNG HWAN PARK;AND JEONG IL KIM
    • Journal of Microbiology and Biotechnology
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    • v.4 no.3
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    • pp.171-175
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    • 1994
  • To find new crystal protein genes, we screened 42 Bacillus thuringiensis strains of serovar standards by Southern hybridization with a cryI-specific probe which was amplified from B. thuringiensis subsp. kurstaki HDl by polymerase chain reaction (PCR). Two strains, B. thuringiensis subsp. entomocidus HD9 and subsp. subtoxicus HD109, generated weak signals under the low-stringency hybridization conditions. Further analysis with Southern hybridization revealed that the two strains contained cryIB genes which are slightly different from those of B. thuringiensis subsp. thuringiensis HD2. These results were confirmed by PCR with cryIB-specific primers followed by the restriction analysis of PCR products.

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Expression of Canavalia Iineata Leghemoglobin cDNA in Transgenic Nicotiana tabacum (형질전환된 담배에서 해녀콩 Leghemoglobin cDNA의 발현)

  • 이선영
    • Journal of Plant Biology
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    • v.38 no.2
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    • pp.203-209
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    • 1995
  • Tobacco (Nicotiana tahacum L. cv. Wisconsin 38) leaf discs were cocultivated with Agrohacterium carrying a leghemoglobin (Lb) cDNA from Canavalia lineata. Seven plants were regenerated from the transformed leaf discs on MS media supplemented with 0.5 mg/L BAP, 0.1 mg/L ${\alpha}-NAA$, 200 mg/L kanamycin and 500 mg/L carbenicillin. Southern hybridization and PCR of genomic DNA from transgenic plants showed that the Lb cDNA was stably integrated into the genome of the tobacco. Total RNA from the transgenic tobacco showed northern hybridization signal at 1,000 nt and PCR of the first strand cDNA synthesized from the total RNA amplified 0.5 kb Lb cDNA. Furthermore, western hybridization using a polyclonal antibody against soybean Lb showed a 15.8 kD LB-like band on SDS-PAGE of proteins from the transformed tobacco. These results demonstrated that the Lb cDNA of C. lineata was not only incorporated into the genome of tobacco, but also transcribed into mRNA and translated into Lb protein in the transformed tabacco.

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Agrobacterium-mediated Transformation of Eleutherococcus sessiliflorus using Embryogenic Calli and the Regeneration of Plants (오갈피(Eleutherococcus sessiliflorus)의 배형성 세포를 이용한 고빈도 형질전환 및 재분화)

  • Jeong, Jae-Hun;Han, Seong-Soo;Choi, Yong-Eui
    • Journal of Plant Biotechnology
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    • v.30 no.3
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    • pp.233-239
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    • 2003
  • We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

Molecular Systematics of Rhizoctonia solani Isolates from Various Crops with RFLP and PCR-RFLP (각종 작물로부터 분리한 Rhizoctonia solani 균주의 RFLP 및 PCR-RFLP를 이용한 분자계통한 특성 구명)

  • 최혜선;신환성;김희종;김경수;우수진
    • Korean Journal of Microbiology
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    • v.35 no.3
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    • pp.173-179
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    • 1999
  • As a result of PCR-RFLP, the isolates used in this study were classified into five groups. Isolates 1 and 3 were included in AG-5 with 97% genetic similarity. Isolates 12 and 13 were included in AG-1 wilh 100% genetic similarily. Isolates 10 and AG-2-2 showed 97% similarity Isolates 7, 8, 11. 13, and 15 were included in AG-1. When isolates of 4, 5, 7 and 8 were restricted with Hae I. there was a single 700 bp fragment matched with AG-1. A 517 bp restriction fragment of isolate 9 was matched with AG-2-1. Based on the result of southem hybridization of genomic DNAs, all isolates restricted with Msp I showed more variable restriction differences than those restricted with Hae Ill. Isolates AG-2-1 and 9 showed 200 bp restriction fragment, and isolates 3 and AG-1 showed 1 kb restriction fragments.

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