Ginkgo long-chain non-coding RNA Lnc2L and Lnc2S as well as vector and application thereof

A long-chain non-coding, Ginkgo biloba technology, applied in the field of plant genetic engineering, can solve problems such as few research reports and no relevant reports on lncRNAs.

Pending Publication Date: 2021-12-03
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many research reports on plant lncRNA, but most of them focus on model plants such as Arabidopsis and rice, which participate in the regulation of various life processes such as plant growth, development, signal transduction, morphogenesis, and stress response. , especially in gymnosperms, there are relatively few research reports, for example, there are few reports on Ginkgo biloba, and there are no relevant reports on the lncRNA that regulates the synthesis of flavonoids in Ginkgo biloba.

Method used

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  • Ginkgo long-chain non-coding RNA Lnc2L and Lnc2S as well as vector and application thereof
  • Ginkgo long-chain non-coding RNA Lnc2L and Lnc2S as well as vector and application thereof
  • Ginkgo long-chain non-coding RNA Lnc2L and Lnc2S as well as vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning of Lnc2L and Lnc2S

[0030] (1) Based on the Ginkgo lncRNA-seq data, an lncRNA was screened, and primers were artificially designed for this lncRNA using Primer Premier 5.0 software. Wherein the forward primer (F primer) is: 5'-GTATTCGTTTCCTCATAAACCAGG-3', and the reverse primer (R primer) is: 5'-TTTCAATTGGCAGGGATAATATA-3'. Two bands appeared during PCR amplification, which may be caused by variable splicing. The long sequence was named Lnc2L, and the short sequence was named Lnc2S( figure 1 ).

[0031] (2) Using the high-fidelity enzyme PrimeSTAR Max (Takara, Japan) for PCR amplification, the PCR system is as follows:

[0032]

[0033] Gently mix the above mixture, centrifuge briefly at low speed and place it in an ordinary PCR reaction instrument, set the following program:

[0034]

[0035] Gel running: Take out the gene amplification product in the PCR instrument, use the electrophoresis instrument to spot an appropriate amount of the product on a 1...

Embodiment 2

[0050] Construction of plant expression vectors for Lnc2L and Lnc2S

[0051] (1) This experiment uses TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) to pRI 101-AN vector (TaKaRa, Japan, with promoter CAMV35S, strong terminator NOS-ter, NPTⅡ gene expression cassette, LB and RB Sequence) and the sequences of Lnc2L and Lnc2S were subjected to enzyme digestion reaction experiments, the specific reaction system is as follows:

[0052]

[0053] After the solutions in the system were mixed, they were centrifuged instantaneously, incubated in a 37°C water bath for 30 minutes, and then the enzyme digestion reaction was completed. The enzyme-cut bands were observed by agarose gel electrophoresis, and then the target gene and carrier fragments were cut and recovered for subsequent use. The carrier ligation reaction.

[0054] (2) Referring to the operation manual of TaKaRa T4 DNA Ligase (TaKaRa, Japan), connect the expression vector recovered after the double enzyme digestion reac...

Embodiment 3

[0061] Genetic transformation of Lnc2L and Lnc2S

[0062] 1. Ginkgo callus transformation

[0063] (1) Agrobacteria containing 35S::Lnc2L and Lnc2S vectors obtained in Example 2 were coated on LB plates respectively. After culturing, pick the single clone of Agrobacterium on the LB plate, inoculate it into LB liquid medium, and culture it at 28°C for 16h to OD 600 0.5-0.6;

[0064] (2) Put the bacterial solution into a centrifuge tube, centrifuge at 18°C, 3500rpm for 15min, and remove the supernatant;

[0065] (3) Add resuspension solution (100 mL MS liquid medium containing 100 μM acetosyringone) to the centrifuge tube to resuspend the bottom bacteria, and place it at room temperature for 2 h;

[0066] (4) Put the ginkgo callus fragments of the same size into the Agrobacterium resuspension, let it stand at room temperature and soak for 15min, then gently clip it out with tweezers, and suck off the resuspension liquid on the surface with sterile filter paper;

[0067] (5) ...

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PUM

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Abstract

The invention discloses ginkgo long-chain non-coding RNA (Ribonucleic Acid) Lnc2L and Lnc2S as well as a carrier and application thereof, and the nucleotide sequences of the ginkgo long-chain non-coding RNA Lnc2L or Lnc2S are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2. The non-coding RNA provided by the invention can be used for regulating and controlling the synthesis of gingko flavonoids after overexpression in gingko, and the content of flavonoids is obviously increased after overexpression of long-chain non-coding RNA Lnc2L; and after the long-chain non-coding RNA Lnc2S is over-expressed, the flavonoid content is obviously reduced. The Lnc2L and the Lnc2S are key lncRNA for regulating and controlling the synthesis of the gingko flavonoids, the content of the gingko flavonoids is regulated and controlled through overexpression or knockout of the Lnc2L and the Lnc2S, and gingko with high flavonoid content can be cultivated according to requirements, so that the Lnc2L and the Lnc2S have important application value in the gingko molecular breeding process.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to two new ginkgo long-chain non-coding RNAs Lnc2L and Lnc2S, their carriers and applications. Background technique [0002] Ginkgo biloba (Gingo biloba L.) is an important economic tree species. Its leaves, kernels and outer testa contain medicinal ingredients, and it is called "a living fossil full of treasures all over the body". Ginkgo biloba has been used as medicine for more than 600 years, and its efficacy was first recorded in "Shen Nong's Herbal Classic". Ginkgo biloba leaves are rich in secondary metabolites, such as flavonoids, terpene lactones, polyprenol and other active substances, which are widely used in medicines, health products and food. Ginkgo biloba Extract (GbE) is the raw material of many medicines, and it has certain effects on the prevention and treatment of early Alzheimer's disease and cardiovascular diseases. At present, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84A01H7/00
CPCC12N15/113C12N15/8243
Inventor 刘思安操萌孟钊龙王莉
Owner YANGZHOU UNIV
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