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Lilium regle bZIP transcription factor LrbZIP1 and application

A technology of transcription factors and genes, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of high chemical pesticide residues, long breeding cycle, environmental pollution, etc., achieve broad market application prospects, shorten the breeding cycle, reduce Effects of Environmental Pollution

Inactive Publication Date: 2013-09-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although traditional disease control methods have achieved certain results, they mainly rely on traditional breeding methods to cultivate resistant varieties and use chemical pesticides, or adopt farming systems such as crop rotation. These methods have more or less disadvantages, such as time-consuming and labor-intensive use. , high chemical pesticide residues and easy to cause pollution to the environment, long breeding cycle, etc., so the traditional method of controlling plant diseases cannot completely solve the problem

Method used

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  • Lilium regle bZIP transcription factor LrbZIP1 and application
  • Lilium regle bZIP transcription factor LrbZIP1 and application
  • Lilium regle bZIP transcription factor LrbZIP1 and application

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: LrbZIP1 Full-length gene cloning and sequence analysis

[0022] 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, reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP ( 2.5mM each), DEPC water to a reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 minutes, and then rapidly cool on ice for 5 minutes, 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. After th...

Embodiment 2

[0025] Embodiment 2: plant overexpression vector construction

[0026] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrbZIP1 Escherichia coli plasmid pMD-18T- LrbZIP1 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) respectively on the plasmid pMD-18T- LrbZIP1 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- LrbZIP1 and pCAMBIA2300s plasmid, add 10 μL 10×K buffer, 5 μL Eco RI, 5 μL Bam HI, 60 μL ddH 2 O, mix well and then 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 LrbZIP1The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separat...

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 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.

[0031] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrbZIP1 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 the Agrobacterium on LB solid medium and inoculate it in an appropriate amount of acetyl c...

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Abstract

The inventing discloses a Lilium regle bZIP transcription factor LrbZIP1. According to the invention, the nucleotide sequence of the gene LrbZIP1 is as shown in SEQ ID No:1, and the gene LrbZIP1 encodes a protein of a nucleotide sequence as shown in SEQ ID No:2. Functional genomics related technical research proves that LrbZIP1 has a function for improving the antifungal property of plants, after the antifungal LrbZIP1 gene is constructed in a plant expression vector and transferred to tobacco for overexpression, the result shows that the transgenic tobacco plant has strong in-vitro antifungal activity, so the experiment shows that the LrbZIP1-overexpressed transgenic tobacco has an obvious inhibiting effect on growth of various fungus such as ascomycetes 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 bZIP transcription factor gene of Minjiang lily with antifungal activity LrbZIP1 and 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. Although traditional disease control methods have achieved certain results, they mainly rely on traditional breeding methods to cultivate resistant varieties and use chemical pesticides, or adopt farming systems such as crop rotation. These methods have more or less disadvantages, such as time-consuming and labor-intensive use. , high chemical pesticide residues and easy to cause pollution to the environment, long breeding cycle, etc., so the traditional method of controlling plant diseases can...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 刘迪秋李红丽张南南饶健陈朝银葛锋
Owner KUNMING UNIV OF SCI & TECH
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