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Glutathione S-transferase gene LrGSTL1 of lilium regale and application thereof

A technology of lily glutathione and transferase, applied in the direction of transferase, application, genetic engineering, etc., can solve the problems that cannot be fundamentally solved, such as plant diseases, less beneficial variation of plant resources, and adverse effects on human and animal health, etc. Market application prospects, short breeding cycle, and cost-saving effects

Inactive Publication Date: 2014-07-23
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional methods of preventing and controlling plant diseases are mainly the use of chemical pesticides and the cultivation of resistant varieties. Although both methods have achieved certain results, they also have serious drawbacks.
Extensive use of chemical pesticides has caused serious environmental pollution and has had adverse effects on human and animal health; however, conventional breeding has disadvantages such as long cycle, time-consuming and labor-intensive, and less beneficial variation of plant resources, making them unable to fundamentally solve the problem of plant diseases

Method used

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  • Glutathione S-transferase gene LrGSTL1 of lilium regale and application thereof
  • Glutathione S-transferase gene LrGSTL1 of lilium regale and application thereof
  • Glutathione S-transferase gene LrGSTL1 of lilium regale and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: LrGSTL1 Cloning and sequence analysis of full-length genes

[0022] Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 hours after inoculation. Grind the treated root of Lilium Minjiang into powder with liquid nitrogen, transfer it to a centrifuge tube, extract total RNA by guanidine isothiocyanate method, and use reverse transcriptase M-MLV (promega) to synthesize cDNA using total RNA as a template The first strand, the reaction system and the operation process are as follows: take 5 μg Total RNA, add 50 ng oligo(dT) 15, 2 μL dNTP (2.5mM each), DEPC water in sequence until the reaction volume is 13.5 μL; after mixing, 70 Heat and denature at ℃ for 5 minutes, then cool quickly on ice for 5 minutes, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U), 1 μL M-MLV (200U) in sequence, mix well and centrifuge for a short time, and incubate at 42℃ After 1.5 h, take it out and heat at 70°C for 10 minutes to t...

Embodiment 2

[0025] Embodiment 2: plant expression vector construction

[0026] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrGSTL1 Escherichia coli plasmid pMD-18T- LrGSTL1 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. use Eco RI (TaKaRa) and Pst I (TaKaRa) for plasmid pMD-18T- LrGSTL1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- LrGSTL1 or pCAMBIA2300S plasmid, add 10 μL 10×H buffer, 5 μL EcoR I, 5 μL Pst I. 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. Spot all digested products on agarose gel for electrophoresis, and then LrGSTL1 The fragment and the pCAMBIA2300S large fragment were gel-recovered separately, and the SanPrep column DNA gel-rec...

Embodiment 3

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

[0030] 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 Disinfect the surface 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, and transfer to light incubator after germination (25°C, 16h / d light) , subculture once a month with 1 / 2 MS medium.

[0031] Take out the stored pCAMBIA2300S containing pCAMBIA2300S from the -80℃ refrigerator -LrGSTL1 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 cloudy. Pipette 1 mL of turbid bacterial solution onto 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 sol...

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Abstract

The invention discloses an anti-disease gene (LrGSTL1) of lilium regale and an application thereof. The nucleotide sequence of the gene (LrGSTL1) is shown as SEQIDNO:1 to encode Lambda glutathione S-transferase. Functional genomics related technical research shows that the gene (LrGSTL1) of lilium regale has the function of improving the antifungal ability of the plant. The glutathione S-transferase gene (LrGSTL1) of lilium regale disclosed by the invention is constructed to a plant expression vector and transformed to tobacco to be over-expressed. The transgenetic tobacco has strong antifungal activity, and the transgenetic tobacco sheet which expresses (LrGSTL1) has remarkable resistance to infection by fusarium oxysporum and botrytis cinerea.

Description

technical field [0001] The invention relates to a Minjiang lily glutathione S-transferase gene with antifungal activity LrGSTL1 The invention and its application belong to the research fields of molecular biology and genetic engineering related technologies. technical background [0002] With the continuous increase of world population, the demand for food is increasing day by day, so improving food production is an urgent problem to be solved. Crops are constantly attacked by various pathogenic microorganisms during their growth and development. The diseases caused by fungi account for more than 80% of the total plant diseases, and have seriously affected the yield of food. In addition, fungal diseases are the most common types of diseases, and fungi can invade any organ and tissue of plants and cause them to cause disease. The traditional methods of preventing and treating plant diseases are mainly the use of chemical pesticides and the cultivation of resistant varieties...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/84A01H5/00
Inventor 刘迪秋何华韩青季博张南南葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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