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Application of lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5

A technology related to proteins and genes, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear disease resistance mechanism, achieve the effects of reducing the use of chemical pesticides, reducing environmental pollution, and shortening the breeding cycle

Inactive Publication Date: 2014-11-05
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] PR-10 plays an important role in the invasion of pathogenic bacteria, and PR-10 protein has antibacterial function in vitro, but its anti-disease mechanism is still unclear

Method used

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  • Application of lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5
  • Application of lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5
  • Application of lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: LrPR10-5 Full-length gene cloning and sequence analysis

[0023] Lilium Minjiang was inoculated with Fusarium oxysporum, total RNA was extracted from the roots 24 hours after inoculation, the treated roots of Lily Minjiang were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and extracted by guanidine isothiocyanate method For total RNA, use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5mM each), DEPC water to a reaction volume of 14.5 μL; after mixing, heat denaturation at 70 °C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U), 1 μL M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction. After the first strand of...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrPR10-5 Escherichia coli plasmid pMD-18T- LrPR10-5 As well as the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Eco RI (TaKaRa) and Bam HI (TaKaRa) for plasmid pMD-18T- LrPR10-5 and pCAMBIA2300s for double enzyme digestion (100μL system), the reaction system and operation process are as follows: Take 20μL pMD-18T- LrPR10-5 and pCAMBIA2300s plasmid, add 10μL 10×K buffer, 5μL EcoRI , 5μL Bam HI, 60 μL ddH 2 O, mix well and then centrifuge for a short time, and put it at 37 ℃ for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then LrPR10-5The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately, and the ...

Embodiment 3

[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0031] The transgenic recipient in this experiment was tobacco. The tobacco seeds were soaked in 75% alcohol for 30 s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 min, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 6 days, transfer to light incubator (25°C, 16 h / d light) after germination, and then Subculture once a month with MS medium.

[0032] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrPR10-5 The plasmid Agrobacterium LBA4404 strain was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultured at 28 °C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and incubate at 28 °C for 48 h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it with 20 mg / ...

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Abstract

The invention discloses an application of a lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5. A nucleotide sequence of the LrPR10-5 is as shown in SEQIDNO:1, and the pathogenesis-related protein 10 gene is encoded. The research proves that the LrPR10-5 gene has the function of improving the plant antifungal capability by functional genomics-related technologies, the antifungal LrPR10-5 gene is built on a plant expression vector and is switched into tobacco to perform overexpression, the transgenic tobacco plant has strong in-vitro antifungal activity in the result, and the experiment result shows that the transgenic tobacco for overexpression of the LrPR10-5 has an obvious inhibiting effect on growth of a plurality of fungi, such as botrytis cinerea, rhizoctonia solani and sclerotinia sclerotiorum.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a Minjiang lily disease course-related protein 10 gene with antifungal activity LrPR10-5 Applications. Background technique [0002] Plant diseases are a very difficult problem in agricultural production, especially fungal diseases, which account for about 80% of the total plant diseases and seriously affect the yield and quality of crops. Traditional disease control methods such as relying on plant breeding to breed resistant varieties, using chemical pesticides, or adopting crop rotation and other farming systems have achieved certain results. However, these methods have more or less disadvantages, such as long traditional breeding cycles, high chemical pesticide residues and It is easy to cause pollution to the environment and is time-consuming and labor-intensive, so the traditional method of controlling plant diseases cann...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C12Q1/68A01H5/00
Inventor 刘迪秋何华季博韩青葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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