Orange canker resistant pthA-nls gene and its construction method and application
A ptha-nls, citrus canker technology, applied in the field of citrus canker resistance gene and its construction, can solve the problem that citrus variety resources have not been found to be completely resistant to the disease, there is no specific method for eradicating citrus canker, and no substantial progress has been made. And other issues
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Embodiment 1
[0024] (1) Using the citrus canker bacteria isolated from the citrus canker epidemic area of Nanyue District, Hengyang City, Hunan Province as a template, and using P1 / P3 as a template, the NLS sequence was amplified by PCR. The PCR reaction conditions were: 94°C for 4min; 94°C 30sec, 58°C for 30sec, 72°C for 1.5min, 35 cycles, and finally 72°C for 10min; (2) PCR products were electrophoresed on 1%-1.5% agarose gel and then recovered with Qiaquick Gel Extraction Ket DNA gel recovery kit ( (purchased from Qiagen Company) was recovered and purified, and the purified product was dissolved in 15-25 μl of sterile water; the recovered fragment was double-digested with Xba I and Sac I; the enzyme digestion reaction system was 20 μl: 15 μl of the purified product, 1.5 μl of each of the two enzymes, 10 ×Buffer 2μl; (3) Double enzyme digestion of the plant expression vector pBI121, the double enzyme digestion reaction system and conditions of the pBI121 plasmid with Xba I and Sac I are...
Embodiment 2
[0027] Using the citrus canker isolated from the citrus canker epidemic area of Nanyue District, Hengyang City, Hunan Province as a template, using P1 / P4 as a template, the NLS sequence was amplified by PCR, and the PCR product was electrophoresed on 1% to 1.5% agarose gel For recovery and purification, the purified product was dissolved in 15-25 μl sterile water; the recovered fragment was double digested with Xba I and Sma I; the enzyme digestion reaction system was 20 μl: 15 μl of the purified product, 1.5 μl of each of the two enzymes, and 2 μl of 10×Buffer; The pBI121 plasmid was double digested with Xba I and Sma I. The digested product was recovered and purified by 1%-1.5% agarose gel electrophoresis. Ligate the pBI121 digestion product and the NLS sequence PCR digestion product with T4 DNA ligase. The ligation reaction system is 10 μl: 4 μl of pBI121 digestion product, 4 μl of NLS sequence PCR digestion product, 1 μl of T4 DNA ligase, and 1 μl of 10×Buffer. React at...
Embodiment 3
[0030]Using the citrus canker pathogen isolated from the citrus canker epidemic area of Nanyue District, Hengyang City, Hunan Province as a template, using P2 / P3 as a template, the NLS sequence was amplified by PCR method, and the PCR product was electrophoresed on 1% to 1.5% agarose gel Recovery and purification, the purified product was dissolved in 15-25 μl sterile water; the recovered fragment was double-digested with BamHI and Sac I; the enzyme digestion reaction system was 20 μl: 15 μl of purified product, 1.5 μl of each of the two enzymes, 2 μl of 10×Buffer; pBI121 The plasmid was double digested with BamHI and Sac I. The digested product was recovered and purified by 1%-1.5% agarose gel electrophoresis; the pBI121 digested product was ligated with the PCR digested product of NLS sequence with T4 DNA ligase, and the ligation reaction system was 10 μl: 4 μl of pBI121 digested product, NLS sequence PCR digestion product 4 μl, T4 DNA ligase 1 μl, 10×Buffer 1 μl. React a...
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