Fusion protein and application thereof

A fusion protein and protein technology, applied in fusion protein and its application field, can solve problems affecting protein-DNA interaction, gene silencing, etc.

Pending Publication Date: 2021-08-27
SHANDONG SHUNFENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, DNA methylation can lead to changes in the conformation of DNA in certain region

Method used

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  • Fusion protein and application thereof
  • Fusion protein and application thereof
  • Fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0183] Example 1: Construction of demethylation gene editing tool vector

[0184] 1.1dCas9-TET1cd demethylation tool

[0185] (1) Use the existing dCas9 and TET1 sequences in the laboratory, and use high-fidelity enzyme Q5 to amplify the sequence fragments of dCas9 and TET1cd. Gel recovered fragments for future use.

[0186] (2) Using the p1300-UBQ-CAS9 vector cut with Nco I and BamH I, the p1300-UBQ fragment was recovered by cutting the gel and used for future use.

[0187] (3) Using recombinase to recombine the dCas9 fragment into the p1300-UBQ fragment to obtain p1300-UBQ-dCas9, which is ready for use. Sanger sequencing was used to prove that the fragment recombination was successful.

[0188] (4) Then use BamHI to cut the p1300-UBQ-dCas9 vector obtained above.

[0189] (5) Using recombinase to recombine the TET1cd fragment into the p1300-UBQ-dCas9 fragment to obtain the p1300-UBQ-dCas9-TET1cd vector, which is the final vector for targeted editing of DNA methylation. S...

Embodiment 2

[0193] Example 2: Regulation of ROS1 expression by MEMS demethylation in the ROS1 promoter region

[0194] 2.1 Target design and construction

[0195] (1) Five sgRNAs targeting MEMS regions were designed according to the rules of sgRNA design, and the sequences of corresponding sgRNAs are shown in Table 1. In addition to the 20 bp targeting the MEMS region, the sgRNA also has a sticky end for ligation.

[0196] Table 1 sgRNA sequences targeting MEMS regions

[0197]

[0198]

[0199] (2) Change the F and R sequences of sgRNA into double-stranded DNA fragments with cohesive ends through annealling program. The process is as follows: dilute the forward and reverse primers to 100 μM, mix 1 μL each with 1 μL T4 DNA ligase buffer, 0.5 μL T4 polynucleotide kinase, and 6.5 μL ddH2O, keep the mixture at 37 °C for 30 min, 95 °C for 5 min, and then The speed of 0.2°C / s is reduced to 25°C, and finally diluted 250 times with water.

[0200] (3) U6, U3b, and 7SL vectors were dige...

Embodiment 3

[0251] Example 3: Demethylation experiment in RdDM mutant (nrpd1)

[0252] 3.1 Target design

[0253] The target design is consistent with the previous ones. sgRNAs 1, 2, and 3 are connected to the upstream of the fusion protein, while sgRNAs 4, 5, and 6 are connected to the downstream of the fusion protein. The sequence of the sgRNA is shown in Table 3. The composition of the sgRNA sequence is the same as that of the previous one. sgRNAs are consistent.

[0254] Table 3 sgRNA sequences targeting multiple regions

[0255]

[0256]

[0257] 3.2 Genetic Transformation

[0258] See step 2 in the experimental example for the genetic transformation process.

[0259] 3.3 Screening of positive seedlings

[0260] Refer to step 3 positive seedling screening in the experimental example.

[0261] 3.4 Detection of DNA methylation level and editing efficiency

[0262] (1) When the positive seedlings grow to an appropriate size, the leaves of the positive seedlings are taken to ...

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Abstract

The invention provides a fusion protein and an application thereof. Specifically, the invention provides a fusion protein, which comprises or consists of the following components: (1) a positioning functional element D1 with functions of targeting and combining DNA; and (2) a demethylated functional element D2 having a function of converting methylated nucleotides into non-methylated nucleotides. The demethylation method provided by the invention has accurate and efficient demethylation modification efficiency in plants, and has important scientific values for researching plant epigenetics and regulating plant traits through demethylation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein and its application. Background technique [0002] DNA methylation is a form of chemical modification of DNA, which can change genetic expression without changing the DNA sequence, and is a common modification method in eukaryotic cells. The establishment of DNA methylation is formed by the reaction catalyzed by DNA methyltransferase with S-adenosylmethionine (SAM) as the methyl donor. [0003] There are many ways of methylation modification. The bases at the modified site can be the N-6 position of adenine (6mA), the N-4 position of cytosine, the N-7 position of guanine (7mG) and cytosine The C-5 position (5mC). They are respectively catalyzed by different DNA methylases. However, the most well-studied and most common is the methylation of 5mC, the C-5 position of cytosine. [0004] Methylated DNA can undergo demethylation. There are two mechanisms ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/22C12N9/12C12N9/48C12N15/62C12N15/82A01H5/00A01H6/20
CPCC07K2319/00C07K2319/09C07K2319/80C12N9/12C12N9/22C12N9/485C12N15/8218C12Y207/10001C12Y304/11A01H5/00A01H6/20C07K19/00C12N5/10C12N9/48C12N15/62C12N15/63C12N15/82
Inventor 牛小牧李彦莎梁亚峰
Owner SHANDONG SHUNFENG BIOTECH CO LTD
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