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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

Inactive Publication Date: 2008-09-10
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no specific method that can eradicate citrus canker, and no completely disease-resistant varieties have been found in citrus varieties. Citrus has been bred for many years to resist canker, but no substantial progress has been made.

Method used

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  • Orange canker resistant pthA-nls gene and its construction method and application
  • Orange canker resistant pthA-nls gene and its construction method and application
  • Orange canker resistant pthA-nls gene and its construction method and application

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Effect test

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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Abstract

The invention provides an anti-citrus bacterial canker disease pthA-nls gene, a construction method and application thereof. The invention uses the pathogenic gene pthA-mediated broad-spectrum disease resistance theory of the citrus bacterial canker disease to construct a new anti-citrus bacterial canker disease gene (pthA-nls). The construction method comprises the following steps of: (1) primer design; (2) clone of NLS sequence; (3) recovery and enzyme digestion of PCR product; (4) double enzyme digestion of a plant expression vector; (5) connection between product of enzyme digestion of pBI121 and corresponding product of PCR enzyme digestion of the NLS sequence by T4 DNA ligase; (6) acquisition of positive cloned gene pthA-nls by converting ligation product into colon bacillus DH5alpha. The gene pthA-nls can further perform genetic transformation on susceptible varieties of orange so as to acquire new anti-citrus bacterial canker disease germ plasm. The anti-citrus bacterial canker disease pthA-nls gene provides an effective tool for breeding of anti-citrus bacterial canker disease.

Description

technical field [0001] The invention relates to a citrus canker resistance gene and a construction method thereof, in particular to a citrus canker resistance gene constructed by using the citrus canker pathogenic gene pthA and a construction method thereof. Background technique [0002] Citrus canker disease is a bacterial disease caused by Xanthomonas axonopodis pv.citri. It has a great impact on citrus seedlings, yield and fruit quality, and seriously endangers the healthy development of citrus industry. So far, there is no specific method that can eradicate citrus canker, and no completely disease-resistant varieties have been found in citrus variety resources. Citrus canker-resistant breeding has been breeding for many years, but no substantial progress has been made. Contents of the invention [0003] The purpose of the present invention is to provide a citrus canker resistance gene and its construction method. [0004] The purpose of the present invention is achie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10A01H1/00A01H5/00C12N15/31C12N15/84
Inventor 邓子牛胡春华张家银徐磊杨莉李娜
Owner HUNAN AGRICULTURAL UNIV
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