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Application of reducing expression of OsSAMS1 protein and encoding gene thereof in improving plant resistance to rice dwarf virus

A transgenic plant, dwarf disease technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve problems such as plant death

Active Publication Date: 2019-02-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the expression of three genes is reduced at the same time, it will lead to lethality of the plant

Method used

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  • Application of reducing expression of OsSAMS1 protein and encoding gene thereof in improving plant resistance to rice dwarf virus
  • Application of reducing expression of OsSAMS1 protein and encoding gene thereof in improving plant resistance to rice dwarf virus
  • Application of reducing expression of OsSAMS1 protein and encoding gene thereof in improving plant resistance to rice dwarf virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Obtaining OsSAMS1 protein and its coding gene

[0047] 1. Acquisition of OsSAMS1 protein and its coding genes

[0048] The kit used for the construction of the rice library is HybridZAP-2.1Two-HybridPredigested Vector / Gigapack Cloning Kit (Stratagene), and the library construction and screening methods are carried out according to the instructions. The S11 sequence in the bait clone pDHB1-S11- used was the same as in the literature (Xu et al., 1998. Rice Dwarf Phytoreovirus segment S11 encodes a nucleic acid binding protein. Virology. 240(2): 267-272.). The yeast strain used was NMY51. The positive clones obtained by the screening were sequenced and analyzed, and it was determined that the protein fragments encoded by them had the amino acid sequence shown in sequence 2 in the sequence table. The full length of the protein (ie, the protein corresponding to sequence 2) was named OsSAMS1, and its coding gene was sequence The nucleotide shown in sequence 1 in the l...

Embodiment 2

[0054] Example 2. Demonstration of the interaction between OsSAMS1 protein and RDV Pns11 in vitro

[0055] 1. Yeast two-hybrid experiment proved the interaction between OsSAMS1 and RDV Pns11

[0056] The recombinant vector pRR3-N-SAMS1 obtained in Example 1 and pDHB1-S11 (S11 is the encoding gene of RDV Pns11 protein, and the encoding gene of RDV Pns11 protein was introduced into pDHB1.) co-transformed yeast strain NMY51 (purchased from Dualsystems Biotech) Company, the product catalog number is P01601-P01629), the yeast transformation method adopts the well-known PEG / LiAc method; the transformed yeast cells are spread on the auxotrophic plate SD / -Trp-Leu and cultured at 30°C for 2 days. The transformants were streaked onto SD / -Trp-Leu-His-Ade auxotrophic plates, cultured at 30°C for 4 days, and the growth of the transformants was observed. In this experiment, a negative control pPR3-N (purchased from Dualsystems Biotech company, product catalog number: P01601-P01629) and pDHB1-S1...

Embodiment 3

[0070] Example 3. Pns11 can activate the enzyme activity of OsSAMS1

[0071] 1. Expression clone construction and protein expression

[0072] Using pDHB1-S11 plasmid as a template, PCR amplification was carried out with S11-5'SalI (sequence: 5'-CCCGGGTTACGCTTTGATTTGCGAGTATTGG-3' and S11-3'PstI (sequence: 5'-CTGCAGTTACTTACGCTTTGATTTGCGAGTATTGG-3'), and the result was 570bp After the 570bp PCR product was digested with SalI and PstI, the 570bp digested product was recovered by gel electrophoresis; the pMAL-p2x plasmid (New England Biolabs, E8000S, which expresses MBP protein) was used with SalI and PstI Double enzyme digestion and gel electrophoresis to recover the 6703bp vector backbone; the 570bp digestion product and the 6713bp vector backbone were ligated with T4DNA ligase to obtain the recombinant vector pMAL-p2x-S11. After sequencing, the recombinant vector is the S11 sequence The vector obtained by inserting DNA molecules between SalI and PstI restriction sites of pMAL-p2x wa...

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Abstract

The invention provides application of reducing expression of OsSAMS1 protein and an encoding gene thereof in improving plant resistance to rice dwarf virus. The amino acid sequence of the protein OsSAMS1 is a sequence 2 in a sequence table. The experiments prove that RDV coat protein Pns11 can interact with the rice OsSAMS1 protein, so that the OsSAMS1 enzyme activity is increased, the OsSAMS1 protein excessively-accumulated rice is more susceptible to diseases, and the transgenic rice with the OsSAMS1 eliminated by a CRISPR / Cas9 pathway has enhanced resistance to the RDV.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to the application of reducing the expression of the OsSAMS1 protein and its encoding gene in improving the resistance of plants to rice dwarf virus. Background technique [0002] Ethylene is one of the classic plant hormones and the only gas hormone. Ethylene plays an important role in all stages of plant growth and development, including seed germination, root hair development, stem and root elongation inhibition, plant flowering, fruit maturation, and organ senescence and shedding. In addition, because ethylene has the characteristics of quickly spreading to the entire plant of the plant and quickly responding to the external environment, it is also an important hormone that mediates plants to respond to various abiotic and biotic stresses. [0003] Almost all plants can synthesize ethylene, but in most cases the concentration is very low. After years of research, scientists have ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N9/22
CPCC12N9/1085C12N9/22C12N15/8213C12N15/8283C12Y205/01006
Inventor 李毅赵珊珊洪薇魏春红
Owner PEKING UNIV
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