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Tomato late blight resistant long-chain non-coding RNA-lncRNA40787 as well as cloning method and application method thereof

An rna-lncRNA40787, long-chain non-coding technology, applied in the field of plant genetic engineering, can solve problems such as specific relationships and regulatory methods that have not been reported, and achieve the effects of enhanced resistance, decreased expression, and excellent effect

Pending Publication Date: 2021-01-08
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It shows that lncRNA is also involved in the process of miR394 mediating the response of fungal pathogens, but the specific relationship and regulation mode between the two have not been reported yet.

Method used

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  • Tomato late blight resistant long-chain non-coding RNA-lncRNA40787 as well as cloning method and application method thereof
  • Tomato late blight resistant long-chain non-coding RNA-lncRNA40787 as well as cloning method and application method thereof
  • Tomato late blight resistant long-chain non-coding RNA-lncRNA40787 as well as cloning method and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of tomato anti-late blight long non-coding RNA-lncRNA40787

[0030] 1. Extraction of Tomato Total RNA

[0031] (1) Put the sample into a mortar, add liquid nitrogen and grind it fully to powder.

[0032] (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.

[0033] (3) Add 200 μL of chloroform to the centrifuge tube, shake well, let stand at room temperature for 5 minutes, and centrifuge at 12,000 r / min at 4°C for 15 minutes.

[0034] (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.

[0035] (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.

[0036] (6) Carefully discard t...

Embodiment 2

[0063] Example 2: Construction of recombinant expression vector for tomato late blight resistance long non-coding RNA-lncRNA40787

[0064] 1. Enzyme digestion of pMD-18T-lncRNA40787 plasmid

[0065] The pMD-18T-lncRNA40787 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:

[0066]

[0067] Digested at 37°C for 6 hours, and detected the digested products by 1% agarose gel electrophoresis.

[0068] 2. Double digestion of pBI121 plasmid

[0069] The pBI121 plasmid was double digested with BamHI and SacI, and the pBI121 fragment, that is, the large fragment, was recovered. The reaction system is as follows:

[0070]

[0071] Digest at 37°C for 6 hours, and detect the digested product by 1% agarose gel electrophoresis.

[0072] 3. The target fragment is connected to the pBI121 vector

[0073] T4 DNA ligase (purchased fro...

Embodiment 3

[0076] Example 3: Application of tomato long-chain non-coding RNA-lncRNA40787 against late blight

[0077] l. Preparation of pBI121-lncRNA40787 Agrobacterium engineering bacteria

[0078] (1) Add 2.0 μL of the pBI121-lncRNA40787 plasmid to 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;

[0079] (2) Place the frozen mixture in a water bath at 37°C for 5 minutes, add 1 mL of fresh YEB medium, and place it in a constant temperature shaker at 28°C (180 rpm) for 3 hours;

[0080] (3) After centrifuging the above bacterial liquid, suck off 1 mL of the supernatant, blow and mix the remaining 100 μL bacterial liquid, and evenly spread it on the YEB solid medium (containing 100 mg / L streptomycin, 100 mg / L rifampicin and 50 mg / L kanamycin), cultured at 28°C for 36h;

[0081] (4) PCR detection of Agrobacterium engineering bacteria

[0082] The colony obtained by antibiotic selectio...

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Abstract

The invention provides a long-chain non-coding RNAlncRNA40787 for improving tomato late blight resistance by silencing miR394. The DNA molecule has a nucleotide sequence as shown in SEQ ID NO. 1; Thecloning method of the long-chain non-coding RNAlncRNA40787 comprises the following steps: taking cDNA of wild late blight affected tomato Zaofen No.2 as a template for PCR amplification, connecting the obtained PCR product with a pMD-18T cloning vector, converting escherichia coli DH5alpha, and selecting a single colony for sequencing. The invention further provides an application method of the long-chain non-coding RNA for tomatoes resisting infection of pathogenic bacteria, namely the tomato late blight resistant long-chain non-coding RNA-IncRNA40787 is transiently overexpressed in tomatoes.After the long-chain non-coding RNA is transiently overexpressed in tomatoes, the expression quantity of the tomato miR394 is remarkably reduced, the late blight resistance of the long-chain non-coding RNA has a better effect, namely the long-chain non-coding RNA can be used in silence miR394 for enhancing the resistance of tomatoes on late blight and used for culturing late blight resistant tomato varieties, so that a significant meaning is achieved on increasing the yield of tomatoes.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to tomato late blight resistant long-chain non-coding RNA-lncRNA40787 and its cloning method and application method. Background technique [0002] Tomato is a herbaceous plant belonging to the tomato genus, and it is an important economic horticultural crop widely grown in the world. It is rich in lycopene, vitamins, etc., and has extremely high nutritional value. However, during its growth, it is often attacked by a variety of pathogens, causing huge economic losses to tomato production. Among them, the late blight caused by Phytophthora infestans is a devastating disease that has adverse effects on tomatoes at all stages. When the disease is severe, it will cause tomato stems to rot and plants to wilt, eventually leading to a serious reduction in tomato production. The current control methods have the problems of environmental pollution, pesticide r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/82A01H5/00A01H6/82C12R1/19
CPCC12N15/113C12N15/70C12N15/8282C12N2310/14
Inventor 栾雨时张媛媛洪雨慧
Owner DALIAN UNIV OF TECH
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