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Method for increasing transgene plant disease resistance by using antibacterial peptide fusion expression

A technology of transgenic plants and fusion expression, which is applied in the field of high-abundance expression of broad-spectrum and high-efficiency antimicrobial peptides, can solve the problems of degradation, limiting the efficacy of antimicrobial peptides, and low animal-derived antimicrobial peptides, and achieve enhanced expression efficiency, enhanced stability, and reduced The effect of the probability of degradation

Inactive Publication Date: 2014-04-09
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main problem encountered after the transfer of animal-derived antimicrobial peptide genes into plants is the low abundance of antimicrobial peptides, which limits the efficacy of antimicrobial peptides
Although the post-transcriptional expression of genes is very complicated and related to many factors, it is generally believed that the low expression of animal-derived antimicrobial peptides in plant cells is mainly due to the insufficient stability of exogenous antimicrobial peptides in plant cells, which are easily detected by intracellular proteases. Cause of degradation

Method used

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  • Method for increasing transgene plant disease resistance by using antibacterial peptide fusion expression
  • Method for increasing transgene plant disease resistance by using antibacterial peptide fusion expression
  • Method for increasing transgene plant disease resistance by using antibacterial peptide fusion expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Taking the antimicrobial peptide AMP1 as an example, the fusion expression of 6 copies of the homopolymeric antimicrobial peptide

[0026]Taking the cleavage site of the antibacterial peptide of Impatiens as the spacer sequence, its amino acid sequence and cleavage method are S↓NAADEVATPE↓DVEPG, and the sequence unit is named LP; the sequence of the antibacterial peptide AMP1 is gskkpvpiiycnrrtgkcqrm, and the signal peptide SP is used at the same time, and the sequence is maserqalmillillttffftikpsqa, secrete antimicrobial peptides into the intercellular space after expression.

[0027] After optimizing according to the codon usage frequency table of dicotyledonous plants and adding gene manipulation restriction sites, the chemically synthesized gene 1 sequence is:

[0028] ccatggcttctgagagacaggctcttatgcttatccttcttactactttcttcttcactatcaagccttctcagg ctggatctaagaagcctgttcctatcatctactgtaacagaagaactggaaagtgtcagagaatgagatct tagtctaga,其中碱基1-6为Nco I酶切位点,6-77编码SP,78-1...

Embodiment 2

[0030] Example 2: Fusion expression of 6 copies of heteromeric antimicrobial peptides taking antimicrobial peptides AMP1-AMP6 as an example

[0031] The sequence of antibacterial peptide AMP1 is the same as above, the sequence of AMP2 is FLPILASLAAKFGPKLFCLVTKKC, the sequence of AMP3 is GILDAIKAIAKAAG, the sequence of AMP4 is KWKLFKKIIGIGAVLKVLTTGLPALKLTK, the sequence of AMP5 is LEHMKWKLFKKIIGIGAVLKVLTTGLPALKLTK, the sequence of AMP6 is KKLFKKILKY, and the sequence of LKD is the same as above. 按照SP-AMP1-LP-AMP2-LP-AMP3-LP-AMP4-LP-AMP5-LP-AMP6融合表达的次序,密码子优化后合成基因3,序列为ccatggcttctgagagacaggctcttatgcttatccttcttactactttcttcttcactatcaagccttctcagg ctggatctaagaagcctgttcctatcatctactgcaacagaagaactggaaagtgtcagagaatgtctaac gctgctgatgaggttgctactcctgaggatgttgagcctggattccttcctatccttgcttctcttgctgctaa gttcggacctaagcttttctgtcttgttactaagaagtgctctaacgctgctgatgaggttgctactcctgag gatgttgagcctggaatccttgatgctatcaaggctatcgctaaggctgctggatctaacgctgctgatga ggttgctactcctgaggatgttgagcctggaaagtggaagcttttcaagaaga...

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Abstract

The present invention discloses a method for increasing antibacterial peptide expression abundance and enhancing transgene crop disease resistance by using fusion expression of a variety of antibacterial peptides or multi-copy fusion expression of the same type of the antibacterial peptide. The method comprises: 1) a AMP1-LP-AMP2-LP-AMP3......LP-AMPn fusion gene expression structure is adopted to construct a gene expression vector, wherein AMP1-AMPn can be different antibacterial peptides and can further be the same antibacterial peptide, n can be any natural number and is generally 3-10, and LP is a plant endopeptidase recognition and cleavage sequence; and 2) the constructed cleavable antibacterial peptide fusion expression vector is transformed into a crop to make the antibacterial peptide be efficiently expressed in the transgene crop, and the endopeptidase in the plant acts on the endopeptidase recognition site LP so as to make the fused antibacterial peptide be cleavaged into the single active antibacterial peptide unit. According to the present invention, the problems of not high expression abundance in the transgene plant and difficultly increased resistance of the antibacterial peptide can be solved.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method of using multiple or multiple copies of the same antimicrobial peptides to be fused and expressed in plant leaves or other organs and then cut into a single active antimicrobial peptide, which is highly abundant in crops. The technology of expressing broad-spectrum and high-efficiency antimicrobial peptides is used to enhance the disease resistance of crops. Background technique [0002] Antimicrobial peptides are a class of peptides produced in the defense system of organisms that have high-efficiency killing activity against exogenous bacteria, especially some animal-derived antimicrobial peptides, such as cecropins, melittins, and bombesin (Magainins), etc., have certain killing activity on various pathogenic bacteria such as bacteria, fungi, viruses, spirochetes, and even cancer cells, and long-term use will not cause drug resistance of pathogenic bacteria, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82A01H5/00
Inventor 刘仁虎王伏林郑滔郎春秀吴关庭石江华胡张华金卫陈锦清
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES