Application of a Minjiang lily disease course related protein 10 gene lrpr10-5

A related protein and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of unclear disease resistance mechanism, reduce the use of chemical pesticides, have broad market application prospects, and save costs.

Inactive Publication Date: 2016-05-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] PR-10 plays an important role in the invasion of pathogenic bacteria, and PR-10 protein has antibacterial function in vitro, but its anti-disease mechanism is still unclear

Method used

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  • Application of a Minjiang lily disease course related protein 10 gene lrpr10-5
  • Application of a Minjiang lily disease course related protein 10 gene lrpr10-5
  • Application of a Minjiang lily disease course related protein 10 gene lrpr10-5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: LrPR10-5 full-length gene cloning and sequence analysis

[0023] Lily Minjiang was inoculated with Fusarium oxysporum, and 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 total RNA was extracted by guanidine isothiocyanate method. For 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 TotalRNA, add 50 ngoligo (dT), 2 μL dNTP (2.5 mMeach), DEPC water to the reaction The volume is 14.5 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-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix and centrifuge for a short time , in a warm bath at 42°C for 1.5h, and after taking it out, heat it at 70°C for 10min to termina...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] Use the SanPrep Column Plasmid DNA Small Extraction Kit (Shanghai Sangong) to extract the E. coli plasmid pMD-18T-LrPR10-5 inserted into LrPR10-5 and the plasmid of the plant expression vector pCAMBIA2300s, and take 1 μL for agarose gel electrophoresis To detect the integrity and concentration of the extracted plasmids; use restriction endonucleases EcoRI (TaKaRa) and BamHI (TaKaRa) to double-enzyme digest the plasmids pMD-18T-LrPR10-5 and pCAMBIA2300s (100 μL system), and the reaction system And the operation process is: take 20 μL pMD-18T-LrPR10-5 and pCAMBIA2300s plasmid, add 10 μL 10×Kbuffer, 5 μL CoRI, 5 μL BamHI, 60 μL ddH in sequence 2 O, after mixing, centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then perform gel recovery on the LrPR10-5 fragment and the large fragment of the pCAMBIA2300s vector, and u...

Embodiment 3

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

[0031] 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 minutes, 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 use MS every month thereafter The medium was subcultured once.

[0032] Take out the stored Agrobacterium LBA4404 strain containing the pCAMBIA2300s-LrPR10-5 plasmid from the -80°C refrigerator, inoculate it in 5 mL of LB liquid medium containing 50 mg / LKm and 20 mg / L rifampicin, and culture it at 28°C until the culture medium turbid. Pipette 1mL of turbid bacteria solution onto LB solid medium containing 50mg / LKm, incubate at 28°C for 48h; then scrape off an appropriate amount of Agrobacterium on LB solid medi...

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Abstract

The invention discloses an application of a lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5. A nucleotide sequence of the LrPR10-5 is as shown in SEQIDNO:1, and the pathogenesis-related protein 10 gene is encoded. The research proves that the LrPR10-5 gene has the function of improving the plant antifungal capability by functional genomics-related technologies, the antifungal LrPR10-5 gene is built on a plant expression vector and is switched into tobacco to perform overexpression, the transgenic tobacco plant has strong in-vitro antifungal activity in the result, and the experiment result shows that the transgenic tobacco for overexpression of the LrPR10-5 has an obvious inhibiting effect on growth of a plurality of fungi, such as botrytis cinerea, rhizoctonia solani and sclerotinia sclerotiorum.

Description

technical field [0001] The invention relates to the research fields of molecular biology and genetic engineering related technologies, in particular to the application of a Minjiang lily disease process-related protein 10 gene LrPR10-5 with antifungal activity. 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 such as relying on plant breeding to breed resistant varieties, using chemical pesticides, or adopting crop rotation and other farming systems have achieved certain results. However, these methods have more or less disadvantages, such as long traditional breeding cycles, high chemical pesticide residues and It is easy to cause pollution to the environment and is time-consuming and labor-intensive, so the traditional method of controlling plant dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/84C12Q1/68A01H5/00
Inventor 刘迪秋何华季博韩青葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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