Glyphosate-Resistant Rice via EPSPS Site-Directed Mutation

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Solution Overview

Problem

Traditional rice breeding methods are time-consuming and lack sufficient germplasm resources, and current herbicides reduce rice yield by 5-30% due to weed competition, necessitating the development of herbicide-resistant rice varieties.

Innovation Solution

A method involving site-directed nucleotide substitution using CRISPR/Cas9 technology to modify the EPSPS protein in rice by substituting threonine at position 8 with isoleucine and proline at position 12 with serine, rendering the rice resistant to glyphosate herbicide.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional breeding methods are used to develop herbicide-resistant rice, then germplasm resources can be utilized, but the breeding process is time-consuming and efficient

Engineering Contradiction:

Improveherbicide resistanceVSAvoidbreeding time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The invention changes the amino acid sequence parameters of the EPSPS protein by substituting specific residues (Thr8Ile and Pro12Ser) to confer glyphosate resistance. This direct parameter modification through site-directed mutagenesis achieves herbicide resistance without time-consuming traditional breeding, resolving the contradiction between reliability and time loss.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention replaces the mechanical breeding process (crossing, selection, and generation advancement) with a molecular-level genetic modification approach. By directly editing the EPSPS gene sequence to introduce specific amino acid substitutions, the method eliminates the time-consuming mechanical breeding steps while achieving the desired herbicide resistance trait.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Object-affected harmful factors

If herbicides are applied to control paddy weeds, then weed control effectiveness is improved, but rice yield is reduced by 5-30% due to weed competition

Engineering Contradiction:

Improveweed control effectivenessVSAvoidrice yield

Core Design Contradiction:

Object-affected harmful factorsVSProductivity

Solution Approach 1:

The invention converts the harmful effect of glyphosate (which normally kills rice by inhibiting EPSPS) into a beneficial outcome by modifying the EPSPS protein so that it is no longer inhibited by glyphosate. The rice plant gains resistance to the herbicide, allowing weed control without yield loss, thus converting the potential harm into a benefit.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The invention applies local quality modification by making only specific amino acid positions (8 and 12) in the EPSPS protein different from the wild type. These localized changes at critical positions confer glyphosate resistance while maintaining the enzyme's normal function in the rice plant, enabling selective weed control without affecting rice productivity.

Inventive Principle:

Principle #3Local quality

3Reliability

If site-directed nucleotide substitution is used to modify EPSPS protein, then glyphosate resistance is achieved, but the complexity of the genetic modification process increases

Engineering Contradiction:

Improveglyphosate resistanceVSAvoidgenetic modification complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention segments the complex task of achieving glyphosate resistance into two specific, targeted amino acid substitutions (Thr8Ile and Pro12Ser) at defined positions in the EPSPS protein. This segmentation simplifies the genetic modification process by focusing on only two critical sites rather than requiring comprehensive gene replacement or multiple random mutations.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention uses precise parameter changes at two specific positions in the EPSPS protein sequence to achieve glyphosate resistance. By changing only the amino acid type at positions 8 and 12 through site-directed mutagenesis, the method achieves the desired trait with minimal genetic modification complexity, avoiding the need for entire gene replacement or complex transgenic approaches.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The method effectively confers glyphosate resistance on rice plants, reducing yield loss from weeds and simplifying weed control, thereby increasing rice yield and reducing herbicide usage.

Implementation Method 1

The genome editing technology is a rising new technology in recent years, and mainly includes three types of sequence specific nucleases: zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas9) system. These artificial nucleases can generate DNA double-strand breaks (DSBs) at DNA target sites

Methodology Applied

Scientific EffectCRISPR/Cas9 genome editing:

Implementation Method 2

the DSBs generated after damage of DNA activate intracellular inherent non-homologous ending-joining (NHEJ) or homologous recombination (HR) as two different repair mechanisms to repair the damaged DNA

Methodology Applied

Scientific EffectNon-homologous end joining (NHEJ):

Implementation Method 3

Methodology Applied

Scientific EffectHomologous recombination (HR):

Implementation Method 4

Its mechanism of action mainly lies in competitively inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway, resulting in the block of aromatic amino acid synthesis

Methodology Applied

Scientific EffectCompetitive inhibition:

Implementation Method 5

only substituting threonine (T) at position 8 of the amino acid sequence of a conserved region of endogenous EPSPS protein of a target plant with isoleucine (I), and substituting proline (P) at position 12 with serine (S) to obtain a plant, i.e., a glyphosate-resistant plant

Methodology Applied

Scientific EffectSite-directed nucleotide substitution:

Data Source

PatentUS11767536B2Method for obtaining glyphosate-resistant rice by site-directed nucleotide substitution

Publication Date: 2023.09.26 INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI

AI summary

The present invention discloses a method for obtaining glyphosate-resistant rice by a site-directed nucleotide substitution, and also relates to a method capable of generating a site-directed nucleotide substitution and a fragment substitution. The method for obtaining a glyphosate-resistant plant provided by the present invention comprises the following steps: only substituting threonine (T) at position 8 of the amino acid sequence of a conserved region of endogenous EPSPS protein of a target plant with isoleucine (I), and substituting proline (P) at position 12 with serine (S) to obtain a plant, i.e., a glyphosate-resistant plant. The method provided by the present invention is of great significance in breeding new herbicide-resistant plant varieties. The present invention also discloses a method for utilizing a CRISPR-mediated NHEJ pathway to substitute a region between two gRNA sites by designing the two gRNA sites, thereby realizing a site-directed mutation of a target nucleotide and site-directed substitution of a fragment.