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Glutathione S-transferase gene LrGSTU3 of lilium regale and application thereof

A lily glutathione and transferase technology, applied in transferase, application, genetic engineering, etc., can solve the problems of metalloenzyme activity reduction, cell membrane permeability change, etc., to shorten the breeding cycle and reduce the use and operation simple effect

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

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Problems solved by technology

However, ROS can also react with proteins, lipids, and DNA in plant cells, causing a series of adverse effects such as reduced activity of metalloenzymes in plants and changes in cell membrane permeability.

Method used

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  • Glutathione S-transferase gene LrGSTU3 of lilium regale and application thereof
  • Glutathione S-transferase gene LrGSTU3 of lilium regale and application thereof
  • Glutathione S-transferase gene LrGSTU3 of lilium regale and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: LrGSTU3 Full-length cDNA cloning and sequence analysis

[0022] Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 12 hours after inoculation. The treated root of Lilium Minjiang was ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. 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 Mix (2.5mM each), add DEPC water to make up the reaction volume to 14.5 μL; after mixing, heat and denature at 70°C for 5 min, then quickly 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. Aft...

Embodiment 2

[0025] Embodiment 2: plant overexpression vector construction

[0026] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrGSTU3 coli plasmid pMD18-T- LrGSTU3 As well as the plant expression vector pCAMBIA2300S plasmid, 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) against plasmid pMD18-T- LrGSTU3 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- LrGSTU3 and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL Eco RI, 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. All digested products were subjected to agarose gel electrophoresis, and then SanPrep Column DNA Gel Extraction Kit (Shanghai Shenggong) was used for LrGSTU3 The fragment and t...

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 ( Nicotiana tabacum L.), 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 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.

[0031] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrGSTU3 For the Agrobacterium LBA4404 strain of the plasmid, take 20 uL and inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and culture at 28°C until the medium is 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 Agrobacte...

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Abstract

The invention discloses a gene (LrGSTU3) of lilium regale with antifungal activity and an application thereof. The nucleotide sequence of the gene (LrGSTU3) is shown as SEQIDNO:1 to encode tau glutathione S-transferase. Functional genomics related technical research shows that the gene (LrGSTU3) of lilium regale has the function of improving the antifungal ability of the plant. The gene (LrGSTU3) of lilium regale disclosed by the invention is constructed to a plant expression vector and transformed to tobacco to be over-expressed, so that the transgenetic tobacco strain is strong in vitro antifungal activity, and the transgenetic tobacco sheet which over-expresses (LrGSTU3) has a remarkable inhibiting effect on growth of fusarium oxysporum and botrytis cinerea.

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 glutathione S-transferase gene with antifungal activity LrGSTU3 and applications. Background technique [0002] Plants are often subjected to various biotic and abiotic stresses during their growth, such as fungi, viruses, drought, heavy metals, extreme temperatures, chemical poisoning, and so on. Among them, fungal diseases are the most serious and have the highest incidence rate. When fungal diseases occur on a large scale, they will cause crop production reduction or even death. For the control of fungal diseases, chemical pesticides are commonly used, as well as improved farming systems and resistant plant varieties bred by traditional breeding methods. However, the traditional breeding method has a long cycle, and the extensive use of chemical pesticides will pollute the environment and even produce a larg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/84A01H5/00
Inventor 刘迪秋张南南季博韩青何华葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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