Application of Lilium regale pathogenesis-related protein 10 gene LrPR10-6

A technology related to proteins and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear disease resistance mechanisms, achieve the effects of reducing the use of chemical pesticides, shortening the breeding cycle, and broad market application prospects

Inactive Publication Date: 2014-11-19
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] PR10 plays an important role in the invasion of pathogenic bacteria, and PR10 has antibacterial activity in vitro, but its anti-disease mechanism is not clear

Method used

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  • Application of Lilium regale pathogenesis-related protein 10 gene LrPR10-6
  • Application of Lilium regale pathogenesis-related protein 10 gene LrPR10-6
  • Application of Lilium regale pathogenesis-related protein 10 gene LrPR10-6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: LrPR10-6 Full-length gene cloning and sequence analysis

[0023] Lilium Minjiang was inoculated with Fusarium oxysporum, total RNA was extracted from the roots 24 hours after inoculation, the treated roots of Lily Minjiang were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and extracted by 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), 2 μL dNTP (2.5mM each), DEPC water until the reaction volume was 14.5 μL; after mixing, heated and denatured at 70 °C for 5 min, then rapidly cooled on ice for 5 min, then added 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 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

[0026] Embodiment 2: plant overexpression vector construction

[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrPR10-6 Escherichia coli plasmid pMD-18T- LrPR10-6 As well as the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Eco RI (TaKaRa) and Bam HI (TaKaRa) for plasmid pMD-18T- LrPR10-6 and pCAMBIA2300s for double enzyme digestion (100μL system), the reaction system and operation process are as follows: Take 20μL pMD-18T- LrPR10-6 and pCAMBIA2300s plasmid, add 10μL 10×K buffer, 5μL Eco RI, 5 μL Bam HI, 60 μL ddH 2 O, mix well and then centrifuge for a short time, and put it at 37 ℃ for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then LrPR10-6 The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately. The Sa...

Embodiment 3

[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0031] 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 Soak for 8 min, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 6 days, transfer to light incubator (25°C, 16h / d light) after germination, and then Subculture once a month with MS medium.

[0032] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrPR10-6 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 onto LB solid medium containing 50 mg / L Km, and incubate at 28 °C for 48 h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it with 20 mg / L...

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Abstract

The present invention discloses application of a Lilium regale pathogenesis-related protein 10 gene LrPR10-6. LrPR10-6 has nucleotide sequence shown as SEQ ID NO:1 and encodes pathogenesis-related protein 10. Through research on functional genomics related technology, the invention has confirmed that LrPR10-6 gene has the function of improving fungi resistance of plants. The anti-fungal LrPR10-6 gene provided by the invention can be constructed into a plant expression vector and transferred to tobacco for over expression, so thaytthe transgenic tobacco plants gain strong in vitro antifungal activity. The experimental results show that the transgenic tobacco with overexpression of LrPR10-6 has significant inhibitory effect on Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium oxysporum.

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 disease course-related protein 10 gene with antifungal activity LrPR10-6 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 and seriously affect the yield and quality of crops. Traditional disease control methods have achieved some success, mainly relying on breeding resistant varieties and using chemical pesticides, or adopting farming systems such as crop rotation. These methods have more or less disadvantages, such as long traditional breeding cycle, high chemical pesticide residues, easy to pollute the environment, time-consuming and laborious to use, etc. Therefore, the above-mentioned traditional methods of controlling plant diseases cannot completely solve th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/84A01H5/00
Inventor 刘迪秋何华韩青季博葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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