For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for trans...
For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.
For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
가설 설정
(A) Three-week-old calli co-cultivated with COR356. (B) Selection of resistant calli on medium containing 5 mg/L hygromycin; (a) brown calli represent dying cells, and (b) yellow calli represent regenerating cells. (C) Green spot calli appeared in regenerating medium after 3 weeks of the culture.
제안 방법
(A) Ethidium bromide-stained DNA analyzed by Pulsed-field gel electrophoresis. After plasmid DNA isolation from 24 candidate clones, Noil digested DNA was analyzed by Pulsed-field gel electrophoresis for 16 hrs on 1% Pulsed Field Certified Agarose gel under the conditions of 6 V/cmz initial switch time 50s, final switch time 90s, temperature 14*0, 120 degree angle, and 1 x TBE buffer. (B) Southern hybridization by ECL™ system.
To verify whether 24 colonies contain the full length of the inserted DNA (90 kb), pulsed-field gel electrophoresis analysis was conducted. After plasmid DNA isolation from 24 candidate clones’ plasmid DNA was digested with NotL Then, pulsed-field gel electrophoresis analysis was performed for 16 hrs on 1% Pulsed Field Certified Agarose gel under the con ditions of 6 V/cm, initial switch time 50s, final switch time 90s, temperature 14R 120 degree angle, and 1 x TBE buffer. From this analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb (Figure 3A).
coli DH10B competent cells by electroporator (Invitrogen, USA). Electroporation con ditions were examined by various condition of vol tages (11, 12z 13, 14, 15 and 16 kV / cm) and cuvette sizes (0.1 and 0.2 cm mouth) under the basal con ditions of capacity 50 uF, impedance 150 Q and 1800 Vmax. From the preliminary test, 11 kV/cm voltage and 0.
대상 데이터
Agrobacterium strain COR308 was provided from the Cornell University (Ithaca, NY, U.S.A.). Its geno type is UIA143, pMP90 and pCH32.
). Its geno type is UIA143, pMP90 and pCH32. UIA143 represents a recA-deficient derivative of A.
Genetic com plementation test for the thl-2Ql mutants was con ducted. The thl-201 mutant seeds were provided from AIMS iArabidopsis Information Management System, Arabidopsis Biological Resource Center, OH,U.S.A.). The thl-201 mutant plants were grown in the growth chamber under 16 hrs light condition at 21, 0, and treated with 1 mM thiamin for main taining normal growth.
성능/효과
4 mL SOC medium (Table 1) with 아taking (200 rpm) for one hour at 3713, and spread on LB medium containing 40 jUg/mL ka- namycin and 10% sucrose. As the results, 476 colo nies were selected on LB medium containing 40 jUg/ mL kanamycin and 10% sucrose. To confirm wheth er the selected colonies had the correct size of the in serted gene three analyses, such as the colony hy- bridization, the pulsed-field g이 electrophoresis, and Southern hybridization, were conducted.
Based on the hypothesis that if Thl gene in BI- BAC2 vector is transferred successively into mutant plants, mutant plants will be recovered to wild type by genetic complementation. Genetic com plementation test for the thl-2Ql mutants was con ducted.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.