Oryza sativa histone methyltransferase as well as encoding gene and application thereof

A technology of methyltransferase and histone, which is applied in the fields of transferase, application, genetic engineering, etc., can solve the problems of research lag and few, and achieve the effects of delayed flowering time, short growth and broad application prospects

Inactive Publication Date: 2016-07-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long life cycle of rice and other reasons, the related research is still quite lagging behind that of Arabidopsis, and the research on the methylation modification of histone H3K4 in rice is still very little.

Method used

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  • Oryza sativa histone methyltransferase as well as encoding gene and application thereof
  • Oryza sativa histone methyltransferase as well as encoding gene and application thereof
  • Oryza sativa histone methyltransferase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Rice histone methyltransferase gene SDG701 the acquisition

[0027] The SDG2 gene sequence of Arabidopsis thaliana published in GenBank (GenBank number: NC_003075.7) was compared with the homologous sequence, and the homologous sequence was obtained from the rice genome, and according to the 5' and 3' end sequences of the homologous sequence A pair of primers were designed, and the primer sequences were: 5'-ATGCCGGACAAGGGGGAGA-3' (SEQ ID No: 3) and 5'-CTAGCCTAGGAAGACGTTGGATCTTG-3' (SEQ ID No: 4).

[0028] The total RNA of etiolated rice seedlings was extracted (Promega, SVtotalRNAisolationsystem), and the total RNA of rice was used as a template to synthesize cDNA with AMV reverse transcriptase (TaKaRa) (according to the user manual of PlantRT-PCRKit2.01 (TaKaRa)). PCR Amplification of Rice Histone Methyltransferase Gene Using cDNA as Template SDG701 full-length cDNA sequence. The 50 μl PCR reaction system contains: 2 μl template, 1 μl high-fidelity enzy...

Embodiment 2

[0029] Example 2. Rice histone methyltransferase gene SDG701 protein activity assay

[0030] Using the pUC19-SDG701 obtained in Example 1 as a template, PCR amplification SDG701From the nucleotide sequence at position 4035-6648 at the 5' end (including the catalytic active center of histone methyltransferase - the SETdomain domain), the 5' and 3' primers are: 5'-cccgggCCAAGGTTGATTTCTGATAG-3'( SEQ ID No: 5), and 5'-gcggccgcCTAGCCTAGGAAGAC-3' (SEQ ID No: 6). The 50 μl PCR amplification system contained template pUC19-SDG701200ng, 20 pmol of 5' and 3' primers, 5 μl of 10×buffer, 20 μM of dNTP, and 1 μl of high-fidelity KODplus (TOYOBO). Amplification was performed under the following conditions: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 3 minutes, a total of 30 cycles. The PCR product was Sma I and not The restriction site I was cloned into the Escherichia coli expression vecto...

Embodiment 3

[0034] Example 3. Detection SDG701 Regulatory effect on rice growth and development

[0035] one, SDG701 Construction of antisense expression vector

[0036] Using RNA interference (RNAi) technology to SDG701 The gene is the target gene, and the RNA interference vector is constructed. Using the pUC19-SDG701 obtained in Example 1 as a template, PCR amplification was performed with two pairs of different primers SDG701 The nucleotide sequence from the 98th to the 463rd position of the 5' end is a complementary DNA duplex as a hairpin structure. The primer pair 1 used was: 5'-ccatggggatccGGGAGCTGCTGCTCAATGGGGAG-3' (SEQ ID No: 7) and 5'-gaattcCCGACGAGCCGAGCTTCTT-3' (SEQ ID No: 8), and the obtained PCR product was fSDG701i-1 (forward). Primer pair 2 is 5'-tctagaGGGAGCTGCTCAATGGGGAG-3' (SEQ ID No: 9) and 5'-aagcttCCGACGAGCCGAGCTTCTT-3' (SEQ ID No: 10), and the obtained PCR product is rSDG701i-1 (reversed). In order to facilitate subsequent vector construction, the 5' and 3...

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Abstract

The invention belongs to the technical field of biological genetic engineering and particularly discloses an oryza sativa histone methyltransferase as well as an encoding gene and an application thereof. The methyltransferase adopts one of the following amino acid residue sequences: (1) SEQ ID NO.1 in a sequence table; (2) substitution and / or deletion and / or addition of 1-50 amino acid residues performed on the amino acid residue sequence shown in the SEQ ID NO.1 in the sequence table, the methyltransferase is a protein adopting the sequence and having a regulation effect on growth and development of plants. The invention further discloses a recombinant expression vector, a transgenic cell line and engineering bacteria containing the gene as well as a primer pair for amplifying any fragment of the gene. Antisense transgenic plants of the encoding gene of the oryza sativa histone lysine methyltransferase grow short and small, and the flowering time is delayed. One excellent gene is provided for improvement of plant varieties and has higher actual application value and broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a histone methyltransferase related to rice epigenetic regulation, its coding gene and its application. Background technique [0002] Different from animals that can move, the growth, development and environmental response of sessile plants are closely related to the regulation of gene transcription and expression. In addition to the influence of genetic factors such as transcription factors, the regulation of gene transcription and expression is also regulated by epigenetic factors such as genetic cofactors. In recent years, epigenetics has become a hot field of life science research. In addition to the DNA sequence in the genome, there is also a lot of information that regulates genes. They do not change the sequence of the gene itself, but can affect and regulate the function and characteristics of the gene through genetic modification, and inherit i...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N5/10C12N15/11C12N15/82A01H5/00C12R1/91
CPCC12N9/1007C12N15/11C12N15/8205C12N15/8222C12N15/827C12N2310/10C12N2810/10C12Y201/01043
Inventor 沈文辉刘坤朋董爱武俞瑜
Owner FUDAN UNIV
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