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Application of an ethylene response factor gene oserf3 and its promoter in regulating rice root development

A rice and gene technology, applied in the field of plant genetic engineering, can solve problems such as unclear mechanism of action

Inactive Publication Date: 2016-08-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, rice root development is regulated by multiple mechanisms, such as auxin and other specific transcription factor complexes, and the mechanism of AP2 / ERF transcription factors in regulating rice root development has not yet been clarified.

Method used

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  • Application of an ethylene response factor gene oserf3 and its promoter in regulating rice root development
  • Application of an ethylene response factor gene oserf3 and its promoter in regulating rice root development
  • Application of an ethylene response factor gene oserf3 and its promoter in regulating rice root development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Construction of OsERF3 overexpression vector

[0041] For the gene OsERF3 required by the present invention, mainly by RT-PCR method (see: J. Sambrook, EF Fritsch, T Mani Artis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition) ), Beijing, Science Press, 2002 edition) was amplified to obtain a specific sequence of the OsERF3 gene. The specific operation is as follows:

[0042] 1) Extract the RNA from the seedling leaves of the rice variety "Zhonghua 11" (see Genetic Resources Source Disclosure Table-1). The RNA extraction reagent is Trizol Extraction Kit from Invitrogen Company (see the kit instruction manual for specific operation steps) ;

[0043] 2) The steps to synthesize the first strand of cDNA by reverse transcription in RT-PCR are as follows: ①Preparation of mixed solution 1: total RNA 4μg, DNaseI2U, 10xDNaseIbuffer1μl, add DEPC (diethyl pyrocarbonate, strong inhibitor of RNase) treated water ( 0.0...

Embodiment 2

[0051] Embodiment 2: Construction of OsERF3 artificial small interfering RNA vector

[0052] 1) Using the NW55 plasmid as a template, first amplify 3 groups of PCR reactions with primers ImiR-s and IIImiR*s, IImiR-a and IVmiR*, IImiR-a and IIImiR*s (PCR system and program are the same as those described in Example 1) The same PCR system and procedures as described above).

[0053] 2) Take 0.5 μl of PCR product from each of the above 3 groups of PCR reactions as a template, and amplify with primer pairs G-11491' and G-11494' (the PCR system and program are consistent with the PCR system and program described in Example 1; G-11491' and G-11494' were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.).

[0054] The primers used to construct the artificial small interfering RNA vector are:

[0055] ImiR-s: 5'-AGTAAAGTAAGGATGCGTTGCTTCAGGAGATTCAGTTTGA-3'

[0056] IImiR-a: 5'-TGAAGCAACGCATCCTTACTTTACTGCTGCTGCTACAGCC-3'

[0057] IIImiR*s: 5'-CTAGCATCGCTTCCTTACTTTATTCCTGCTGC...

Embodiment 3

[0064] Embodiment 3: The vector construction of OsERF3 promoter fusion reporter gene GUS

[0065] 1) Extract the total DNA from the leaves of the rice variety "Zhonghua 11" ((see Genetic Resources Source Disclosure Table-1). The total DNA was used as a template, and the ERF3 promoter sequence was amplified with promoter amplification primers (the PCR system and program were consistent with those described in Example 1).

[0066] The primers used to construct the vector of the promoter fusion reporter gene GUS are:

[0067] ERF3promoter-F1:5'-CGCGGATCCGCTAGCAGCGCCTGGTACAT-3'

[0068] ERF3promoter-R1:5'-ACGCGTCGACGTGTTTTGGGAGGTTGGGTT-3'

[0069] The cDNA sequence of the finally obtained OsERF3 gene promoter fusion reporter gene GUS is shown in SEQ ID NO:3.

[0070] 2) The amplified product was connected to the T / A cloning vector pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., namely Promega Company of the United States) and verified by sequencing with ...

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Abstract

The invention discloses the application of an ethylene response factor gene OsERF3 in regulating rice root development. An ethylene response factor OsERF3 that interacts with a published OsWOX11 protein is screened through a yeast two-hybrid method. Through the method of overexpression and artificial small interfering RNA, the corresponding transformed rice of the gene was obtained, and it was found that the length of the main root and the number of crown roots of the overexpressed gene were significantly increased and increased compared with the wild-type control plant, and the growth of the main root was due to the root tip The length of the main root and the number of crown roots of the artificial small interfering RNA transgenic plants were significantly shorter and less than those of the wild-type control plants, and the shortening of the main root was due to the length of the cells in the meristematic and mature zones. small cause. It shows that the gene has the function of promoting rice root development. Fusion of the promoter of this gene to the reporter gene β‑glucuronidase indicates that the gene is expressed in the root apical meristem.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the use of an ethylene response factor gene OsERF3 for regulating rice root development, and also relates to the use of an ethylene response factor gene OsERF3 promoter in regulating rice root development. Background technique [0002] Roots are important organs for plants to adapt to terrestrial life. Most of them grow in the soil and constitute the underground parts of plants. Their main functions are absorption, output, support, synthesis and storage. Therefore, the root plays an important role in the growth and development of the whole plant, and the study of the development mechanism of the plant root provides an important theoretical basis and direction for us to improve crop habits and increase crop yield in production practice. [0003] At present, the most clearly studied root structure and development among plants is the dicotyledonous plant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 赵毓程赛凤周道绣
Owner HUAZHONG AGRI UNIV
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