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Lilium regale glutathione S-transferase gene LrGSTU5 and application thereof

A lily glutathione and glutathione technology, which is applied in the fields of molecular biology and genetic engineering, can solve the problems of inability to fundamentally solve the problem of plant diseases, adverse effects on human and animal health and ecological balance, and less favorable variation. Achieve broad market application prospects, reduce the use of pesticides, and shorten the breeding cycle

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

AI Technical Summary

Problems solved by technology

Traditional disease control mainly relies on chemical pesticides and the cultivation of resistant varieties. Although some results have been achieved, the large amount and long-term use of pesticides has caused adverse effects on human and animal health and ecological balance. Disadvantages such as less variation make them unable to fundamentally solve the problem of plant diseases

Method used

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  • Lilium regale glutathione S-transferase gene LrGSTU5 and application thereof
  • Lilium regale glutathione S-transferase gene LrGSTU5 and application thereof
  • Lilium regale glutathione S-transferase gene LrGSTU5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: LrGSTU5 Cloning and sequence analysis of full-length genes

[0024] Inoculate Minjiang Lily with Fusarium oxysporum, extract total RNA from the root 24 hours after inoculation, grind the treated root of Minjiang Lily into powder with liquid nitrogen, transfer it to a centrifuge tube, and extract with 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) 15, DEPC water to The reaction volume is 12.5 μL; 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, 0.5 μL RNasin (200U), 2 μL dNTP (2.5mM each) , 1 μL M-MLV (200U), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min to terminate the reaction. After the first strand of cDN...

Embodiment 2

[0027] Embodiment 2: plant expression vector construction

[0028] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrGSTU5 Escherichia coli plasmid pMD-18T- LrGSTU5 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 Bam HI (TaKaRa) and Pst I (TaKaRa) for plasmid pMD-18T- LrGSTU5 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- LrGSTU5 or pCAMBIA2300S plasmid, add 10 μL 10×H buffer, 5 μL Bam HI, 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 LrGSTU5The fragment and the pCAMBIA2300S large fragment were gel-recovered separately, and the SanPrep column DNA gel...

Embodiment 3

[0031] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0032] 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 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) , and then subculture once a month with MS medium.

[0033] Take out the stored pCAMBIA2300S containing pCAMBIA2300S from the -80℃ refrigerator -LrGSTU5 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, incubate at 28°C for 48 hours, scrape off the Agrobacterium on LB solid medium and inoculate ...

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Abstract

The invention discloses a glutathione S-transferase gene LrGSTU5, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1; and the gene encodes the glutathione S-transferase. According to the invention, related technology of functional genomics is adopted to prove that the lilium regale LrGSTU5 gene has the function of improving the antifungal ability of plants; the lilium regale glutathione S-transferase gene LrGSTU5 provided by the invention is built on plant expression carriers and then transferred into tobacco for overexpression; the transgenic tobacco has a high in-vitro antifungal activity; and the transgenic tobacco protein expressing LrGSTU5 has an obvious inhibition effect on the growth of various pathogenic fungi such as botryosphaeria dothidea, fusarium oxysporum, alternaria sp and the like.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering, especially a glutathione S-transferase gene with antifungal activity of Minjiang lily LrGSTU5 and applications. technical background [0002] With the gradual increase of population, the problem of food shortage is increasing day by day, so increasing food production is an urgent problem to be solved. Crop disease is one of the main agricultural disasters in my country, and it is also one of the important factors affecting grain production. In agricultural production, plant diseases are becoming more and more serious, especially fungal diseases, which account for more than 80% of the total plant diseases. There are many types of fungal diseases, and the symptoms of the diseases caused by them are also ever-changing, and can appear in any part of the plant. Traditional disease control mainly relies on chemical pesticides and the cultivation of resistant varieties. Althou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/00
Inventor 刘迪秋刘亚龙张南南何华陈朝银葛锋
Owner KUNMING UNIV OF SCI & TECH
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