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Tomato long non-coding RNA-lncrna23468 and its cloning method and application method

A technology of long-chain non-coding and application methods, which is applied in the cloning and application field of tomato long-chain non-coding RNA-lncRNA23468, which can solve the problems of drug resistance of environmental pollution pathogens and problems of agricultural production, and achieve enhanced resistance, excellent effect, The effect of decreased expression

Active Publication Date: 2021-05-18
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although chemical pesticides have a certain control effect on the occurrence and spread of late blight, the resulting environmental pollution, pathogenic bacteria resistance and other problems still bring great troubles to agricultural production

Method used

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  • Tomato long non-coding RNA-lncrna23468 and its cloning method and application method
  • Tomato long non-coding RNA-lncrna23468 and its cloning method and application method
  • Tomato long non-coding RNA-lncrna23468 and its cloning method and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning of tomato long non-coding RNA-lncRNA23468

[0032] 1. Extraction of Tomato Total RNA

[0033] (1) Put the sample into a mortar, add liquid nitrogen and grind it fully to powder.

[0034] (2) Take an appropriate amount of powder, put it in a 1.5mL RNase / DNase Free centrifuge tube, and quickly add 1mL pre-cooled Trizol at the same time, shake well, and let stand at room temperature for 5min.

[0035] (3) Add 200 μL of chloroform to the centrifuge tube, shake well, let stand at room temperature for 5 minutes, and centrifuge at 12000 r / min at 4°C for 15 minutes.

[0036] (4) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix gently by inversion, let stand at -20°C for 20min, then centrifuge at 12000r / min at 4°C for 10min.

[0037] (5) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, wash the precipitate, and centrifuge at 12000 r / min at 4°C for 5 min.

[0038] (6) Discard the supernatant carefully, op...

Embodiment 2

[0065] Example 2: Construction of tomato long-chain non-coding RNA-lncRNA23468 expression vector

[0066] 1. Enzyme digestion of pMD-19T-lncRNA23468 plasmid

[0067] The pMD-19T-lncRNA23468 plasmid was double digested with BamHI and SacI (purchased from Takara), and the target fragment, namely a small fragment, was recovered. The enzyme digestion reaction system and method are as follows:

[0068]

[0069] Digested at 37°C for 6 hours, and detected the digested products by 1% agarose gel electrophoresis.

[0070] 2. Double digestion of pBI121 plasmid

[0071] The pBI121 plasmid was double digested with BamHI and SacI, the GUS gene was excised, and the pBI121 fragment, which is a large fragment, was recovered. The reaction system is as follows:

[0072]

[0073] Digested at 37°C for 6 hours, and detected the digested products by 1% agarose gel electrophoresis.

[0074] 3. The target fragment is connected to the pBI121 vector

[0075] Using T4 DNA ligase (purchased f...

Embodiment 3

[0078] Example 3: Application of tomato long non-coding RNA-lncRNA23468

[0079] 1. Preparation of pBI121-lncRNA23468 Agrobacterium engineering bacteria

[0080] (1) Add 2 μL of the pBI121-lncRNA23468 plasmid into 100 μL of Agrobacterium competent cells, mix well, bathe in ice water for 10 minutes, and quickly transfer to liquid nitrogen to freeze for 5 minutes;

[0081] (2) Place the frozen mixture in a water bath at 37°C for 5 minutes, add 1 mL of fresh YEB medium, and culture at 28°C for 3 hours with constant temperature shaking (180 rpm);

[0082] (3) After centrifugation, suck off 1 mL of the supernatant, fully mix and suspend the remaining 200 μL of bacterial liquid, and spread evenly on the YEB solid medium (containing 100 mg / L streptomycin, 100 mg / L rifampicin and 50 mg / L kanamycin) Mycin), cultured at 28°C for 36h;

[0083] (4) PCR detection of Agrobacterium engineering bacteria

[0084] The colony obtained by antibiotic selection from the above plate was picked, p...

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Abstract

The present invention provides a long-chain non-coding RNA-lncRNA23468 that improves resistance to tomato late blight by silencing miRNA482b, its DNA molecular sequence is shown in SEQ ID NO.1; a cloning method for the long-chain non-coding RNA is provided: Using the cDNA of wild-type late blight-resistant tomato L3708 as a template for PCR amplification, the obtained PCR product was connected to the pMD-19T cloning vector, transformed into Escherichia coli DH5α, and a single colony was picked for sequencing; the long-chain non-coding Methods of RNA application. After the long-chain non-coding RNA obtained by the method of the present invention is transiently overexpressed in tomato, the expression level of tomato miR482b is significantly reduced, and the resistance to late blight is significantly improved; it is used for silencing miR482b, which is important for cultivating tomato varieties resistant to late blight significance.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the cloning and application of a tomato long-chain non-coding RNA-lncRNA23468. Background technique [0002] Long noncoding RNAs (lncRNAs) are a class of RNAs with a length of more than 200nt that do not contain a coding sequence (CDS), which are abundant in plants and have various biological functions. In recent years, with the rapid development of omics technology, lncRNAs have been identified in Arabidopsis, corn, rice, wheat, cotton, tomato, cucumber, poplar and other plants. Studies have found that plant lncRNAs play an important regulatory role in flowering regulation, photomorphogenesis and other developmental processes, as well as biotic and abiotic stress responses. During the interaction between plants and pathogens, four wheat lncRNAs respond to the infection of stripe rust; some Arabidopsis lncRNAs are induced by Fusarium fungus; some tom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/12A01H6/82
CPCC12N15/113C12N15/8218C12N15/8282C12N2310/11C12Q1/686C12Q2531/113
Inventor 栾雨时姜宁崔军
Owner DALIAN UNIV OF TECH
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