Method for regulating growth of plants and application thereof

A plant growth and plant technology, applied in the application field of rice histone lysine methyltransferase gene in regulating plant growth and development, can solve the problems of rare functional reports, achieve high practical application value and broad application prospects Effect

Inactive Publication Date: 2010-09-22
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, there are very few reports ...

Method used

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  • Method for regulating growth of plants and application thereof
  • Method for regulating growth of plants and application thereof
  • Method for regulating growth of plants and application thereof

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Embodiment 1

[0016] Embodiment 1. Construction of rice histone lysine methyltransferase gene SDG714 plant expression vector

[0017] A pair of primers were designed according to the gene sequence of SDG714 published in GenBank (GenBank number: NM_001051801). The primer sequences were: 5'-actcgagaattcggatccATGGAGGTGATGGATTCGGTG-3', and 5'-accatgggATAAAGTCGCTTCCGGCAGTACG-3'.

[0018] Extract the total RNA of rice yellow seedlings (Promega, SV total RNA isolation system), use the total RNA of rice as a template, and use AMV reverse transcriptase (TaKaRa) to synthesize cDNA (according to the user manual of Plant RT-PCR Kit 2.01 (TaKaRa) conduct). Using cDNA as a template, the full-length cDNA sequence of SDG714 was amplified by PCR. The 50ul PCR reaction system contains: template 2ul, high-fidelity enzyme KOD plus (TOYOBO) 1ul, 10× buffer 5ul, 2.5uM dNTP 8ul, 20uM 5' and 3' primers 1ul each, water 32ul. The reaction conditions were: pre-denaturation at 94°C for 2 minutes; denaturation at 94°...

Embodiment 2

[0019] Example 2. Transformation of Arabidopsis thaliana with plant expression vector of rice histone lysine methyltransferase gene SDG714

[0020] The pER8-SDG714-YFP obtained in Example 1 was transformed into Arabidopsis wild type (Columbia) through Agrobacterium GV3101 according to conventional methods. As a control, pER8-YFP ( Plant J .2004, 40:699-711) also transformed Arabidopsis at the same time. In order to further confirm the stable inheritance of the transgenic traits, T2 transgenic Arabidopsis seeds and wild-type seeds were germinated on MS medium without (-) or with (+) inducer 4 μM estrogen (Estradiol), and the results Such as figure 1 Shown: SDG714 transgenic plants can severely inhibit the growth of Arabidopsis thaliana after being induced by estrogen, while YFP transgenic plants are not significantly different from wild-type plants (WT) after induction (the photo was taken on the 9th day after seed germination) .

Embodiment 3

[0021] Example 3. Detection of the effect of SDG714 on histone lysine modification in transgenic plants

[0022] Referring to Yu et al. ( Plant J .2004, 40:699-711) to extract plant histones. All steps were performed at 4°C. Take 2 g of Arabidopsis plant material ground in liquid nitrogen, add 10 ml of histone extractbuffer A (0.4M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl2, 5mMβ-mercaptoethanol, 1mM PMSF), and pass through 100nm Filter with nylon membrane, discard the filter residue, centrifuge the filtrate at 2,000g for 20min, discard the supernatant, add 2ml of histone extract buffer B (0.25Msucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl2, 1% Triton X to the centrifugal pellet -100, 5mMβ-mercaptoethanol, 1mM PMSF), mix well and centrifuge at 12,000g for 10 minutes, discard the supernatant, add 1ml histone extract buffer C (1.7M sucrose, 10mM Tris-HCl (pH8.0) to the pellet , 2mM MgCl2, 0.15% Triton X-100, 5mM β-mercaptoethanol, 1mM PMSF), centrifuged at 16,000g for 60min, dis...

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Abstract

The invention belongs to the technical field of the biology, and relates to a method for regulating the growth of plants and an application thereof, in particular to an application of paddy rice Histone-lysine methyltransferase gene which is associated to the Epigenetics in the regulation of the plant growth. The name of the gene is SDG714, and the accession number is NM_001051801. Through a designed primer, a target gene entering plant expression carrier is obtained, and the plant Arabidopsis is transformed in an Agrobacterium rhizogenes medium transformation mode. Compared with a contrast group, in the plant of the over-expression SDG714, the methylation level of the histone H3K9 is obviously improved to cause the decrease of a great amount of genes which are related to the growth of the plant and to seriously inhibit the growth of the Arabidopsis; and once the SDG714 which is over-expressed is removed in the plant with the growth being inhibited, the plant can continue to grow. The method for regulating the growth of the plant has high real application value and promising application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for regulating plant growth and development and its application, in particular to a method for regulating plant growth and development of a rice histone lysine methyltransferase gene related to epigenetic regulation. application. Background technique [0002] The prior art discloses that processes such as plant growth and development and stress response are related to the regulation of gene transcription and expression. The regulation of gene transcription and expression is not only affected by genetic factors but also regulated by epigenetics. 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,...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00C12N1/00
Inventor 俞瑜董爱武丁波朱炎
Owner FUDAN UNIV
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