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Lilium regale antifungal gene Lr14-3-3 and application thereof

An anti-fungal and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of high chemical pesticide residues, endangering human and animal health, time-consuming and labor-intensive problems, achieve broad market application prospects, shorten the breeding cycle, reduce the Effects of Environmental Pollution

Inactive Publication Date: 2013-07-10
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some results have been achieved, there are also serious disadvantages, such as time-consuming and labor-intensive use, long cycles, high chemical pesticide residues, harm to human and animal health, and easy to pollute the environment. Therefore, traditional disease control methods cannot fundamentally solve diseases. question

Method used

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  • Lilium regale antifungal gene Lr14-3-3 and application thereof
  • Lilium regale antifungal gene Lr14-3-3 and application thereof
  • Lilium regale antifungal gene Lr14-3-3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Lr14-3-3 Full-length gene cloning and sequence analysis

[0023] Lily Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 h after inoculation. The treated roots of Lily Minjiang were ground into powder with liquid nitrogen, transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. 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) and DEPC water to the reaction volume in sequence After mixing, heat and denature at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-stand buffer, 2 μL dNTP (2.5mM each), 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), 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 fir...

Embodiment 2

[0027] Embodiment 2: plant expression vector construction

[0028] Inserts were extracted from Escherichia coli using the SanPrep Column Plasmid DNA Miniprep Kit (Shanghai Sangong) Lr14-3-3 The plasmid pMD-18T- Lr14-3-3 As well as the plasmid of the plant expression vector pCAMBIA2300s, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI (TaKaRa) and Pst Ⅰ (TaKaRa) respectively for the plasmid pMD-18T- Lr14-3-3 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- Lr14-3-3 and pCAMBIA2300s plasmid, add 10 μL 10×H buffer, 5 μL Eco RI, 5 μL Pst Ⅰ. 60 μL ddH 2 O, mix well and then centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then Lr14-3-3 The fragments and pCAMBIA2300s large fragments were gel...

Embodiment 3

[0032] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0033] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.), the tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 5-8 days, transfer to light incubator after germination (25°C, 16h / d light), Subculture once a month with MS medium.

[0034] Take out the pCAMBIA2300s- containing pCAMBIA2300s- Lr14-3-3 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 and spread it on LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h. Scrape off an appropriate amount of Agrobacterium on the LB solid med...

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Abstract

The invention discloses a lilium regale gene Lr14-3-3 with antifungal activity. The gene Lr14-3-3 has the nucleotide sequence as shown in SEQ ID NO:1 and encodes protein 14-3-3. A relevant technology of functional genomics proves that the gene Lr14-3-3 has a function of improving the plant antifungal activity. The antifungal gene Lr14-3-3 is constructed to a plant expression vector and is transferred into tobacco to perform overexpression; the transgenic tobacco plant has strong in vitro antifungal activity; and the transgenic tobacco expressing the Lr14-3-3 has obvious inhibition effects on the growth of botryosphaeria dothidea, phomopsis fungi, fusarium oxysporum and alternaria.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a 14-3-3 protein gene with antifungal activity of Minjiang lily Lr14-3-3 and 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, not only causing huge losses of crop yields, but also seriously affecting the quality of grain and other foods. Methods of controlling the spread of plant fungal diseases mainly rely on the breeding of resistant varieties and chemical pesticides, or the adoption of cropping systems such as crop rotation. Although some results have been achieved, there are also serious disadvantages, such as time-consuming and labor-intensive use, long cycles, high chemical pesticide residues, harm to human and animal health, and easy to pollute the environment. Therefore,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 刘迪秋李红丽何华张南南葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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