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Establishment of method for directly reducing protein levels in plants

A plant protein and plant technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, plant peptides, etc., can solve problems such as the SMASh system that has not been successfully reported, and achieve fine regulation, reversible degradation, and avoidance of lethal appearance. type effect

Pending Publication Date: 2022-03-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is currently mainly used in mammalian cells and yeast cells, and there is no successful reported SMASh system suitable for plants.

Method used

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  • Establishment of method for directly reducing protein levels in plants
  • Establishment of method for directly reducing protein levels in plants
  • Establishment of method for directly reducing protein levels in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Use of vegetative SMASh tags to regulate the level of GFP in Arabidopsis

[0065] 1. Construction of vectors

[0066] Codon optimization of the Arabidopsis thaliana virus SMASh tag sequence (entrusted by Beijing Quanshijin Company) to obtain the sequence shown in SEQID NO:28, which was synthesized as a plant SMASh tag coding sequence (entrusted by Beijing Quanshijin Company) .

[0067]By fusion PCR method, the plant SMASh tag coding sequence was combined with the C-terminus of GFP (using primers 5'-AAGAGACAGGATCCACTAGTATGGTGAGCAAGGGCGAGGAG-3' (forward primer, SEQ ID NO: 1), and (5'-GCGGTGGCGGCCGCTCTAGACTAGTACAAAACCTCTATC-3'( Reverse primer, SEQ ID NO: 2)) or N-terminal sequence (use primer 5'-AAGAGACAGGATCCACTAGTATGGATTATAAAGATGATGATGATAAG-3' (forward primer, SEQ ID NO: 29), and (5'-GCGGTGGCGGCCGCTCTAGATTACTTGTACAGCTCGTCCATG-3' (reverse primer ,SEQ ID NO:30)) fusion, through homologous recombination method, using pJL12 binary vector (obtained from Institute...

Embodiment 2

[0095] Example 2. Utilizing plant-based SMASh tags to regulate endogenous gene protein levels in Arabidopsis 1. Construction of vectors and transformation mediated by Agrobacterium

[0096] Using the Arabidopsis genome as a template, using the high-fidelity DNA polymerase FastPfu, (using primers 5'-AAGAGACAGGATCCACTAGTATGGAGGGTTCGTCCAAAGGGCTG-3' (forward primer, SEQ ID NO: 31), and (5'-CCAGATCCACCTCCACTAGTATCAAATTTCACAGTCTCTCCATCGAAAAGACTC-3' (reverse Primer, SEQ ID NO:32)) amplifies an approximately 1.5 kb genomic DNA fragment containing the MYB75 (At1g56650) coding sequence, which does not contain a stop codon at the 3' end of the fragment and adds 3×HA by fusion PCR Tag. By the fusion PCR method, the SMASh sequence (primer 5'-AAGAGACAGGATCCACTAGTATGGAGGGTTCGTCCAAAGGGCG-3' (forward primer, SEQ ID NO: 5) and 5'-CCACTAGTATCAAATTTCACAGTCTCTCCATCGAAAAGACTC-3' (reverse primer, SEQ ID NO: 6)), and cloned into the pJL12 binary expression vector to construct a vector driven by the 3...

Embodiment 3

[0118] Example 3. Combination of plant-based SMASh tags and CRISPR / Cas9 systems

[0119] 1. Construction of vectors

[0120] The rice codon optimization was performed on the AcrIIA4 sequence to obtain the sequence encoding the target protein AcrIIA4 (see SEQ ID NO: 27 for the nucleotide sequence) and synthesize the sequence (Beijing Quanshijin Company), which was passed through the fusion PCR method using primers (5'-CAAAGCTTGTCGACGGATCCATGAACATCAATGACCTCATCAG-3' (forward primer, SEQ ID NO: 15) and 5'-GATTTCAGCGTACCGAATTCCTAGTACAAAACCTCTTCTATCAG-3' (reverse primer, SEQ ID NO: 16)) were fused to the carboxy terminus of the SMASh protein, using the same The method of originating from the same group was cloned into the pJIT163-Ubi-hGFP vector (obtained from the research group of Gao Caixia, Institute of Genetics and Development, Chinese Academy of Sciences, see the vector map Figure 5 ), a vector driven by the Ubi promoter was constructed, and the resulting vector was named Ubi...

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Abstract

The present invention provides methods for direct regulation of protein levels in plants. According to the method, the level of the target protein in the plant can be rapidly, accurately and reversibly reduced. The methods of the present invention can achieve dose-dependent reduction of target protein levels in plants and can be used in combination with other biological expression modulation methods, such as methods that utilize the CRISPR / Cas9 system, thereby achieving finer modulation.

Description

technical field [0001] The invention relates to a method for regulating protein levels in plants, which can directly reduce protein levels in plants. Specifically, the present invention relates to a method of using SMASh (Smallmolecule-associated shutdown), a small molecule-associated shutdown system suitable for plants. The present invention also provides the combined use of the method and the method using the CRISPR system, and the expression vector constructed by these methods. Background technique [0002] The regulation of gene expression and protein stability is an important means to study gene function in biology. It is generally carried out at the three levels of DNA, RNA and protein. For plants, there are mainly the following methods. [0003] At the DNA level, T-DNA insertion, gene editing technology, etc. can be used to further study the function of proteins after obtaining plants with corresponding phenotypes, but the disadvantage is that the cycle is long, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/65C12N15/82C12N9/22A01H5/12
CPCC07K14/00C07K14/415C07K14/43595C12N15/65C12N15/8218C12N9/22C07K2319/00
Inventor 邱金龙李盟鸥张倩伟尹康权
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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