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Method for Heterologous Synthesis of γ-polyglutamic Acid in Plants to Improve Plant Stress Resistance and Yield

A technology of polyglutamic acid and heterologous synthesis, applied in the fields of synthetic biology, genetic breeding, and genetic engineering, to achieve the effects of improving salt resistance, increasing biomass, and broad application prospects

Active Publication Date: 2022-05-17
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the heterologous synthesis of γ-polyglutamic acid in crops and the study of its effects on crop drought resistance, salt tolerance, yield, and quality.

Method used

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  • Method for Heterologous Synthesis of γ-polyglutamic Acid in Plants to Improve Plant Stress Resistance and Yield
  • Method for Heterologous Synthesis of γ-polyglutamic Acid in Plants to Improve Plant Stress Resistance and Yield
  • Method for Heterologous Synthesis of γ-polyglutamic Acid in Plants to Improve Plant Stress Resistance and Yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Obtaining the amino acid sequences of pgsA, pgsB, and pgsC

[0083] The three key enzyme genes PgsA, PgsB, and PgsC for the synthesis of γ-PGA were cloned by known methods from the γ-polyglutamic acid-producing strain Bacillus licheniformis; wherein, the GenBank ID of the gene PgsA: AIA08848.1 , its amino acid sequence is shown in SEQ ID NO.1; the Genebank ID of gene PgsB: AIA08846.1, its amino acid sequence is shown in SEQ ID NO.2; the Genebank ID of gene PgsC: AIA08847.1, its amino acid sequence is as SEQ ID NO.1 Shown in ID NO.3.

Embodiment 2

[0084] Example 2: Codon optimization

[0085] According to the obtained amino acid sequences of PgsA, PgsB, and PgsC genes, through codon bias analysis in maize or other plants to be transgenic and combined with CpG dinucleotides content, GC content, mRNAsecondary structure, Cryptic splicing sites, Premature PolyA sites, Internal Chisites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat), Restriction sites that may interfere with cloning, etc. are analyzed to finally determine the optimized sequence, and according to the sequence De novo synthesis of optimized PgsA, PgsB, and PgsC genes; wherein, the codon-optimized PgsA gene nucleotide sequence is shown in SEQ ID NO.4; the codon-optimized PgsB gene nucleotide sequence is shown in Shown in SEQ ID NO.5; the nucleotide sequence of the codon-optimized PgsC gene is shown in SEQ ID NO.6.

Embodiment 3

[0086] Embodiment 3: plant expression vector construction

[0087] The PgsA, PgsB, and PgsC genes after the codon optimization were connected into the plant expression vector pU130-bar containing the bar gene screening marker in a known method (the vector map is as follows: Figure 10 shown), obtain the plant expression vector containing the gene of interest, named as the plant expression vector PGA001, the vector map is as follows figure 1 shown; wherein the nucleotide sequence of the plant expression vector PGA001 is shown in SEQ ID NO.7; each functional element of the expression vector is described as:

[0088]

[0089]

[0090] The above-mentioned plant expression vector PGA001 is constructed using a multi-segment recombinase method, and the specific construction steps are:

[0091] 1 μg of vector pU130-bar was digested with restriction endonucleases Hindlll and Spel, reacted at 37°C for 1±0.2 hours, and the digested vector product was recovered using DC301 kit (com...

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Abstract

The invention discloses a method for heterologously synthesizing gamma-polyglutamic acid in plants to improve plant stress resistance and yield, which is to clone three key enzyme genes PgsA and PgsA for synthesizing gamma-PGA from a strain producing gamma-PGA PgsB, PgsC, and re-synthesize the codon-optimized gene sequences of PgsA, PgsB, and PgsC according to the codon preference in plants, and connect them into the vector pU130‑bar to obtain the plant expression vector PGA001, which was transferred into the In plants, a new line of transgenic plants producing γ-polyglutamic acid is obtained. Experiments have proved that the new line of transgenic maize obtained by the method of the present invention can not only improve drought resistance but also salt resistance, and can also significantly increase the biomass of maize under drought stress conditions and normal growth conditions. The above results indicate that the method of the present invention is a green, efficient and sustainable solution to the stable or reduced yield of crops under drought, water shortage, and saline-alkali stress. It can be widely used in drought-resistant, salt-tolerant molecular breeding and new Breeding.

Description

technical field [0001] The invention relates to a method for improving stress resistance and yield of plants, in particular to a method for heterologously synthesizing gamma-polyglutamic acid in plants to improve plant stress resistance and yield, which belongs to genetic engineering, genetic breeding, and synthetic biology field of technology. Background technique [0002] Abiotic stresses such as drought and soil salinization have become the main limiting factors of crop production in many countries and regions in the world. In China, about half of the country's land area is in arid and semi-arid regions. As the global temperature rises, my country's water resources are becoming more and more scarce, and droughts have an increasing impact on crop yields. In addition, soil salinization is also a major environmental factor affecting crop yield. At present, about 20% of the cultivated land and nearly 50% of the irrigated land in the world are seriously harmed by salinizati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/53A01H4/00A01H5/00A01H5/10A01H6/46
CPCC12N15/8261C12N15/8273C12N15/8205C12N15/52C12N9/0016C12N9/0014A01H4/001A01H4/008C12Y104/01013C12Y104/01014C12Y104/07001
Inventor 夏涛马海珍李灿
Owner QILU UNIV OF TECH
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