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Application of OsMYB91 transcription factor in rice growth and stress-tolerance

A transcription factor, rice technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, some R2R3-type MYB genes have also been found to be involved in growth and development in rice, such as the establishment of root structure, pollen development, resistance to abiotic stress, efficient use of nitrogen and phosphorus, etc., but they are not the same as those in Arabidopsis. In comparison, there are relatively few studies (Zhang et al., 2010. Plant Biologists Carbon Starved Anther Encodes a MYB Domain Protein That Regulates Sugar Partitioning Required for Rice Pollen Development. The Plant Cell, Vol.22: 672-689; Dai et al., 2013.OsMYB2P-1, an R2R3 MYB Transcription Factor, Is Involved in the Regulation of Phosphate-Starvation Responses and Root Architecture in Rice. Plant Physiology 159:169-183; Yang et al, 2012.A R2R3- type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J Exp Bot63: 2541-2556; Ashraf et al., The Rice R2R3-MYB Transcription Factor OsMYB55 Is Involved in the Tolerance to High Temperature and Modulates Amino Acid Metabolism)

Method used

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  • Application of OsMYB91 transcription factor in rice growth and stress-tolerance
  • Application of OsMYB91 transcription factor in rice growth and stress-tolerance
  • Application of OsMYB91 transcription factor in rice growth and stress-tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Cloning of the OsMYB91 gene

[0047] The cloning (LOC_Os12g38400) of the gene OsMYB91 of the present invention is mainly obtained by the method of RT-PCR (methods are referred to: J. Sam Brook, EF Fritsch, T Mani Artis, translated by Huang Peitang, Wang Jiaxi, etc., molecular cloning Experiment Guide (Third Edition), Beijing, Science Press, 2002 edition). The specific operation steps are as follows:

[0048] 1) RNA was extracted from the seedling leaves of the rice variety "Zhonghua 11" (Institute of Crop Science, Chinese Academy of Agricultural Sciences), and the RNA was extracted using the Trizol extraction kit from Invitrogen (see the kit's instruction manual for specific steps);

[0049] 2) The steps of RT-PCR reverse transcription to synthesize the first strand of cDNA are as follows: ① Preparation of mixed solution 1: 4 μg of total RNA, DNaseI2U, 1 μl of 10xDNaseI buffer, and DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase) treated water (0.0...

Embodiment 2

[0056] Embodiment 2: Construction of OsMYB91 overexpression vector

[0057] 1) Design primers suitable for constructing overexpression vectors and add KpnI and BamH I enzyme-cut adapters at both ends of the primers, and use the full-length cDNA obtained in Example 1 as a template to amplify the OsMYB91 gene by PCR sequence.

[0058] 2) Digest the PCR product and the overexpression vector plasmid pU1301 with Kpn I and BamH I respectively (see figure 1 ), the target fragment is recovered and purified and connected with ligase, and the connected product is introduced into Escherichia coli DH10B (purchased from Promer Grid (Beijing) Biotechnology Co., Ltd., that is, Promega Company of the United States), in LA containing 250ppm kanamycin (purchased from Roche Biological Company products) (LA formulation sees J. Sambrook, EF Fritsch, T Manny Worked by Artis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 edi...

Embodiment 3

[0062] Embodiment 3: Construction of double-strand suppression dsRNAi vector

[0063] According to the full-length cDNA sequence of the gene of OsMYB91 obtained in Example 1, design double-stranded suppression dsRNAi primers, and add Spe I and Kpn I restriction endonuclease sites at the 5' end of the upstream primer, and add Spe I and Kpn I restriction endonuclease sites at the 5' end of the downstream primer. Sac I and BamH I restriction endonuclease sites were added sequentially. After the target sequence was amplified by PCR, the amplified product was connected to pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., the United States Promega Company) by T / A cloning for sequencing to verify the amplified DNA The fragment is shown in SEQ ID NO: 2 in the sequence listing. Specific steps are as follows:

[0064] 1) The T / A clone with the RNAi fragment of OsMYB91 was digested with Kpn I and BamH I restriction endonucleases, and the target band was recovered,...

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Abstract

The invention belongs to the technical field of rice genetic engineering, and in particular relates to functions and application of a transcription factor gene OsMYB91 in an MYB family relevant to stress-tolerance. The gene has the nucleotide sequence length of 990 bp, and is induced by various stress conditions such as drought simulation, cold, salt, heat shock, and plant hormones including ABA, ACC, NAA, 6-BA, JA, SA and the like; under the natural growing condition, the plant height of an over-expression pant of the gene is obviously lowered than that of a wild plant and an inhibited plant; when a rooting culture medium containing mannitol and ABA is used for treating the seedlings of over-expression, RNAi and wild plants, the over-expression of the gene is discovered to be capable of increasing the resistance to mannitol, and meanwhile, the growth and the germination rate of the transgenic plant are both influenced by an exogenous ABA; measurement of physiological indexes relevant to stress-tolerance of different transgenic plants after the NaCl treatment shows that the gene has the function of reinforcing the resistance of the rice.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the functional verification of a rice MYB family transcription factor gene OsMYB91 and its application in rice development and stress resistance. Background technique [0002] Transcription factors, also known as trans-acting factors, are DNA-binding proteins that specifically interact with cis-acting elements in gene promoter regions. Many inducible genes in plants can be expressed regularly, quantitatively and at specific positions, all of which are related to the mutual regulation of specific transcription factors and specific cis-acting elements in related genes. [0003] MYB family transcription factors refer to a class of transcription factors containing MYB domains. The MYB domain is a peptide segment of about 51-52 amino acids, which contains 3 tryptophan residues, these 3 residues are separated by 18-19 amino acid residues, forming a hydroph...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00
Inventor 赵毓朱宁周道绣
Owner HUAZHONG AGRI UNIV
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