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Anti-plant-bacterial-wilt gene, preparation method and application thereof

A bacterial wilt and bacterial wilt resistance technology, applied in the field of plant disease resistance, can solve the problems of bacterial wilt disease encroaching on the environment, pollution, narrow genetic basis, etc. Effect

Inactive Publication Date: 2012-01-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a gene with anti-bacterial wilt function according to the problems of narrow genetic basis, serious bacterial wilt damage and environmental pollution caused by the use of pesticides in existing plant breeding.

Method used

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  • Anti-plant-bacterial-wilt gene, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of disease resistance gene

[0023] 1. For the materials used (E-31, E-32 and their F1 and F2 generation plants), carry out artificial anti-bacterial wilt resistance identification by root irrigation method, and determine the disease-resistant materials and disease-susceptible materials.

[0024] 2. The total RNA of the disease-resistant material E-31, the disease-resistant material E-32 and their F1 and F2 generations of disease-resistant plants and susceptible plants was isolated and extracted by the Trizol method.

[0025] 3. Synthesis of cDNA

[0026] Double-stranded cDNA synthesis protocol refers to SMART of CLONTECH company TM cDNA Library construction kit Manual. Among them, Powerscript Reverse Transcriptase was purchased from Invitrogen Company, and 50×advantage 2 polymerase Mix was purchased from CLONTECH Company. cDNA Synthesis (CDS), SMARTП primer and 5′-PCR primer were synthesized by Shanghai Sangong. The SMARTП sequence is shown in...

Embodiment 2

[0151] Example 2 Method for introducing genes into plants

[0152] Digest pMD19-T containing the target gene with sma I and sac I to cut out the target gene, then also use sma I and sac I to double digest the GUS gene of pBI121, and use T4 ligase to digest the disease resistance gene with the restriction enzyme After connecting pBI121 at 16°C for 12 hours, the sense plant expression vector pBI121-RE-bw of the disease resistance gene can be obtained, and the constructed plant expression vector can be transformed into Agrobacterium EHA105 or LBA4404 by freeze-thaw method, and genetic transformation can be carried out .

[0153] Taking the susceptible eggplant inbred line "E-32" as the transformation material, the specific genetic transformation method mediated by Agrobacterium was introduced. The specific operation method is: cultivate aseptic seedlings of eggplant, cut off the epicotyl of 7d seedling age, soak in 600 0.5 Agrobacterium bacteria liquid for 13 minutes, dry the e...

Embodiment 3

[0155] Example 3 Evaluation of the disease resistance of the transgenic plants obtained

[0156] The transgenic contemporary plants are planted in the field, and the incidence rate is less than 20%. The symptoms of the diseased plants are 1-2 (that is, the whole plant has 1-3 withers), while the incidence rate of the control (transformed plants) is more than 80%. , the symptoms of the diseased plants showed 4-5 grades (that is, the leaves of the whole plant were withered or even died); the transgenic plants were artificially inoculated for bacterial wilt identification, and the onset time was delayed by 15 days compared with the control plants, while the control plants showed symptoms 7 days after inoculation. Symptoms of infection. The disease index of the transgenic plants was 8.74, and the group resistance was resistant, while the disease index of the control plants was 76.68, and the group resistance was high.

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Abstract

The invention discloses an anti-plant-bacterial-wilt gene, a preparation method and an application thereof. The anti-plant-bacterial-wilt gene is obtained from a semi-natural eggplant E-31, the DNA sequence thereof is shown in SEQ ID NO:1, and protein encoded by the gene is shown in SEQIDNO:2. The anti-plant-bacterial-wilt gene is transferred into a plant, a transgenic plant with the anti-bacterial-wilt capability is obtained by screening, and the disease resistance of the plant is enhanced; the plant variety is improved; therefore, the invention can be widely applied in the agricultural production.

Description

technical field [0001] The invention relates to the field of plant disease resistance, in particular to a plant bacterial wilt resistance gene and its preparation method and application. Background technique [0002] As one of the main vegetables cultivated in China, eggplant has a national planting area of ​​702,700 hm in 2006 2 , with a total output of 22.47 million tons. However, in the production process of eggplant, the occurrence of diseases often causes serious economic losses to the vegetable farmers. The main diseases that harm eggplant include Verticillium wilt, bacterial wilt, cotton blight, brown spot disease, etc. For example, the occurrence of eggplant bacterial wilt is generally It will reduce production by 20-30%, and when it is serious, the loss will reach 50-60%, which has become the main factor affecting the high yield of eggplant. Moreover, this disease is difficult to eradicate, and comprehensive control measures based on chemical control can only rece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/82C07K14/415A01H5/00
Inventor 曹必好雷建军陈国菊曾国平王勇柯剑陈清华
Owner SOUTH CHINA AGRI UNIV
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