Ethylene responsive factor gene OsERF3 and application of promoter thereof in regulating and controlling development of rice roots

An ethylene response factor, rice root technology, applied in the field of plant genetic engineering, can solve problems such as unclear mechanism of action

Inactive Publication Date: 2014-01-22
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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  • Ethylene responsive factor gene OsERF3 and application of promoter thereof in regulating and controlling development of rice roots
  • Ethylene responsive factor gene OsERF3 and application of promoter thereof in regulating and controlling development of rice roots
  • Ethylene responsive factor gene OsERF3 and application of promoter thereof in regulating and controlling development of rice roots

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: ① Prepare mixed solution 1: 4 μg of total RNA, 2U of DNaseI, 1 μl of 10xDNaseI buffer, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase ) Treat wa...

Embodiment 2

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

[0052] 1) Using the NW55 plasmid as a template, first amplify three sets of PCR reactions with primers I miR-s and III miR*s, II miR-a and IV miR*, II miR-a and III miR*s (PCR system and program Consistent with the PCR system and procedures described in Example 1).

[0053] 2) Take 0.5 μl of PCR products from the above 3 groups of PCR reactions as a template, and amplify with primers G-11491' and G-11494' (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] I miR-s: 5'-AGTAAAGTAAGGATGCGTTGCTTCAGGAGATTCAGTTTGA-3'

[0056] II miR-a: 5'-TGAAGCAACGCATCCTTACTTTACTGCTGCTGCTACAGCC-3'

[0057] III miR*s: 5’-CTAGCATCGCTTCCTTACTTTATTCCTGCTGCTAGGCTG-3’

[0058] IV miR*a: 5'-T...

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 DNA extraction method is the CTAB method (Zhang et al., genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis , 1992, Theor Appl Genet, 83, 495-499), using the total leaf DNA as a template, and using the promoter amplification primers to amplify the ERF3 promoter sequence (the PCR system and program are consistent with the PCR system and program described in Example 1) .

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

[0067] ERF3 promoter-F1:5'-CGCGGATCCGCTAGCAGCGCCTGGTACAT-3'

[0068] ERF3 promoter-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.

[0...

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Abstract

The invention discloses application of an ethylene responsive factor gene OsERF3 in regulating and controlling the development of rice roots. By using a yeast two-hybrid method, an ethylene responsive factor OsERF3 interacted with a published OsWOX11 protein is obtained by screening. By using an overexpression and small artificial interference RNA method, transformed rice corresponding to a gene is obtained, and a situation that the length of a main root and the number of crown roots of a plant subjected to gene overexpression are obviously increased in comparison with those of wild compared plants, and the length increasing of the main root is caused by the length increasing of cells in a maturation zone of a root tip, the length of the main root and the number of crown roots of a plant subjected to small artificial interference RNA transgenosis are obviously reduced in comparison with those of wild compared plants, and the length reducing of the main root is caused by the length reducing of cells in a meristematic zone and a maturation zone. Results show that the gene has a function of promoting the development of rice roots. A result of fusing the promoter of the gene with a report gene beta-glucuronidase shows that the gene is expressed in a meristematic zone of a root tip.

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 Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 赵毓程赛凤周道绣
Owner HUAZHONG AGRI UNIV
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