Lilium regale germin-like protein gene LrGLP2 and application thereof

A technology of germin and protein, applied in the field of molecular biology and genetic engineering, can solve problems such as fungal diseases that cannot be fundamentally solved, achieve broad market application prospects, save costs, and improve management levels

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 these methods have achieved certain results, they

Method used

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  • Lilium regale germin-like protein gene LrGLP2 and application thereof
  • Lilium regale germin-like protein gene LrGLP2 and application thereof
  • Lilium regale germin-like protein gene LrGLP2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: LrGLP2 Full-length gene cloning and sequence analysis

[0022] Lilium Minjiang was inoculated with Fusarium oxysporum, 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. The reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg Total RNA, add 50 ng oligo (dT), DEPC water in sequence to a reaction volume of 12.5 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, 2 μL dNTP (2.5mM each), 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...

Embodiment 2

[0025]Embodiment 2: plant expression vector construction

[0026] Inserts were extracted from Escherichia coli using the SanPrep Column Plasmid DNA Miniprep Kit (Shanghai Sangong) LrGLP2 The plasmid pMD-18T- LrGLP2 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 Bam HI (TaKaRa) respectively on the plasmid pMD-18T- LrGLP2 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- LrGLP2 and pCAMBIA2300s plasmid, add 10 μL 10×K buffer, 5 μL EcoRI , 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37 ℃ for overnight reaction. Spot all digested products on agarose gel for electrophoresis, and then LrGLP2 The gene fragment and the large fragment of the pCAMBIA2300s vector were ge...

Embodiment 3

[0029] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0030] The transgenic recipient in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30s, wash them with sterile water and wash them 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 5-8 days, transfer to light incubator after germination (25°C, 16 h / d light), Subculture once a month with MS medium.

[0031] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrGLP2 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. A proper amount of Agrobacterium on LB solid medium was scraped off and inoculated into MGL liquid me...

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Abstract

The invention discloses a lilium regale germin-like protein gene LrGLP2 with antifungal activity. The gene LrGLP2 has the nucleotide sequence as shown in SEQ ID:1 and encodes germin-like protein. A relevant technology of functional genomics proves that the gene LrGLP2 has a function of improving the plant antifungal activity. The antifungal gene LrGLP2 is constructed to a plant expression vector and is transferred into tobacco to perform overexpression; and the transgenic tobacco plant has strong in vitro antifungal activity. The LrGLP2 transgenic tobacco protein has different degrees of inhibition effects on the growth of botryosphaeria dothidea, phomopsis fungi, fusarium oxysporum and botrytis cinerea.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, in particular to the germin-like protein gene with antifungal activity of Minjiang lily LrGLP2 and applications. Background technique [0002] Fungal diseases are the largest category of plant diseases, accounting for about 70-80% of plant diseases. Many plant diseases with serious damage and wide distribution are caused by fungi, and cause huge losses in crop yields. There are three main conventional methods to control plant fungal diseases: one is to use chemical fungicides, the other is to breed new resistant varieties, and the third is to adopt a reasonable farming system to avoid the spread of infected soil and pathogenic plant materials. Although these methods have achieved certain results, they cannot fundamentally solve the problem of fungal diseases. With the emergence and development of recombinant DNA technology, transgenic technology has opened up ...

Claims

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Application Information

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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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