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Method for indentifying non-toxic effect protein to identify signal peptide having secretion function by using anti-disease gene

A technology of effector proteins and disease-resistant genes, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of cumbersome operation process, low efficiency, long cycle, etc. Short time, low cost effect

Active Publication Date: 2018-08-03
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yeast system requires yeast transformation and verification on various media, the operation process is cumbersome and expensive
Overexpression of secreted proteins fused with tagged proteins in pathogenic bacteria requires the transformation of pathogenic bacteria, which is inefficient, long-term, and expensive

Method used

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  • Method for indentifying non-toxic effect protein to identify signal peptide having secretion function by using anti-disease gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) The signal peptide sequence (amino acid sequence such as SEQ ID NO.4) of the avirulent effector protein Avr1b of Phytophthora sojae and the signal peptide sequence (amino acid sequence such as SEQ ID NO.5) of the secreted protein SsSSVP1 of Sclerotinia sclerotiorum were connected respectively The C-terminal sequence of Avr3a (nucleotide sequence SEQ ID NO.2, amino acid sequence SEQ ID NO.3), constructing plant transient expression vectors, obtaining vectors that can express Avr1bSP-Avr3a-C and SsSSVP1SP-Avr3a-C on plants .

[0024] The specific process is as follows: design specific primers according to the fragment sequence, SP1bF (SEQ ID NO.8) and LapSP1b-3aR (SEQ ID NO.9) amplify the signal peptide region of Avr1b, obtain the amplification product A, LapSP1b-3aF (SEQ ID NO.9) ID NO.10) and Avr3aR (SEQ ID NO.11) amplify the C-terminal region of Avr3a to obtain amplified product B; SPSsSSVP1-F (SEQ ID NO.12) and LapSP-SsSSVP1-3aR (SEQ ID NO.13) The signal peptide ...

Embodiment 2

[0028]In order to verify the reliability of the method, the secretory function of the predicted secreted protein GME5899 in rice smut and the avirulent effector protein AvrPiz-t signal peptide in Magnaporthe grisea were selected for detection. By the method of (1) in Example 1, the GME5899 (amino acid sequence such as SEQ ID NO.6) and AvrPiz-t (amino acid sequence such as SEQ ID NO.7) signal peptide region and the C-terminal region of Avr3a are fused to construct a plant transient The expression vector is transformed into Agrobacterium by electric shock, and the Agrobacterium of the target gene expression vector is co-injected in tobacco, and it is observed whether the injection site is necrotic after 4-6 days. The occurrence of necrosis indicates that the protein cannot be secreted outside the cell, and the signal peptide has no secretion function; the absence of necrosis indicates that the protein can be secreted outside the cell, and the signal peptide has the secretion func...

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Abstract

The invention provides a method for indentifying non-toxic effect protein to identify signal peptide having a secretion function by using an anti-disease gene. The method comprises the following steps: 1) the to-be-identified signal peptide is connected with a C terminal construction expression carrier of phytophthora infestans nontoxic effect protein Avr3a, wherein the C terminal construction expression carrier of phytophthora infestans nontoxic effect protein Avr3a is shown as SEQ ID NO.3; 2) transforming the expression vector constructed in the step 1) to competent cell; and 3) injecting the competent cell in the step 2) and the competent cell containing the anti-disease gene in plant cells, and observing the necrotic condition of an injection site. If the injection site is necrotic, the to-be-detected signal peptide does not have the secretion function, and if the injection site has no necrotic condition, the to-be-detected signal peptide has the secretion function.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a method for identifying signal peptides with secretion function by using the characteristics of disease-resistant genes and non-toxic effector proteins in cells to identify and cause cell necrosis. Background technique [0002] Secreted proteins refer to proteins that are synthesized in cells and can be secreted to function outside cells. The signal peptide is located at the N-terminal of the secreted protein and generally consists of 15-30 amino acids. After the signal peptide sequence is synthesized, it is recognized by the signal recognition particle, and the protein synthesis is suspended or slowed down. The signal recognition particle carries the ribosome to the endoplasmic reticulum, and the protein synthesis resumes. Under the guidance of the signal peptide, the newly synthesized protein enters the cavity of the endoplasmic reticulum and is finally tr...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/82
CPCC12N15/8205C12N15/8279
Inventor 阴伟晓罗朝喜谢松霖林杨田振东
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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