Codon-Optimized Bt Cry2Ai Sequences for Plant Expression
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Solution Overview
Problem
The challenge is to achieve high-level expression of Bacillus thuringiensis (Bt) insecticidal crystal protein in plants to effectively combat insect pests, as native Bt genes have low expression due to high A/T content and codon usage mismatch with plant genes, leading to inefficient mRNA processing and translation.
Innovation Solution
Codon optimization of the Bt Cry2Ai gene to align with plant-preferred codon usage, specifically for monocot and dicot plants, to enhance protein expression, using synthetic nucleotide sequences that are operably linked with plant-specific promoters and terminators, resulting in higher levels of insecticidal protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native Bt cry gene is expressed in plants, then insecticidal activity is provided, but expression level is extremely low due to high A/T content and codon usage mismatch
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of the Bt cry gene, specifically adjusting codon usage frequency and A/T content to match plant gene characteristics. This involves substituting rare codons with preferred codons and reducing A/T content while maintaining the same amino acid sequence, thereby enabling high-level expression in plant systems without altering the insecticidal protein function
Solution Approach 2:
The patent creates a synthetic copy of the Bt cry gene with optimized nucleotide sequence. Instead of directly using the native bacterial gene, a redesigned copy is synthesized that mimics plant gene characteristics in terms of codon usage patterns and base composition, allowing it to be efficiently transcribed and translated by plant cellular machinery while still producing the functional insecticidal protein
2Reliability
If Bt cry gene with high A/T content is introduced into plants, then insecticidal function is achieved, but mRNA stability is reduced due to presence of termination sites and instability motifs
Solution Approach 1:
The patent extracts and removes harmful sequence elements from the Bt cry gene nucleotide sequence. Specifically, it eliminates polyadenylation signals (AATAAA), mRNA instability motifs (ATTTA), and cryptic splicing sites that are inherent in the high A/T content bacterial gene. By taking out these detrimental sequences while retaining the coding information through codon optimization, the resulting mRNA is stable and suitable for plant expression systems
Solution Approach 2:
The patent changes the nucleotide sequence parameters to reduce A/T content and eliminate specific motifs associated with mRNA instability. By adjusting the codon composition and removing problematic sequence patterns, the mRNA transcript becomes more stable and resistant to degradation, thereby extending its functional duration in plant cells without compromising the insecticidal protein function
3Object-affected harmful factors
If chemical pesticides are used to control insect pests, then crop protection is achieved, but environmental hazards and pollution increase
Solution Approach 1:
The patent enables plants to produce their own insecticidal proteins through genetic transformation. The transgenic plants self-generate Bt cry proteins that provide protection against insect pests, eliminating the need for external chemical pesticide applications. This self-service approach integrates the pest protection function directly into the plant's biological system, avoiding environmental contamination while maintaining effective crop protection
Solution Approach 2:
The patent introduces a biological intermediary (Bt cry protein) that mediates between the plant and insect pest. Instead of using chemical substances that pollute the environment, the plant produces a biologically derived protein that specifically targets insect pests. This intermediary substance provides effective pest control while being environmentally benign, replacing harmful chemical pesticides with a natural biological agent
Data Source
AI summary
The present disclosure provides codon optimized synthetic nucleotide sequences encoding Bacillus thuringiensis (Bt) insecticidal crystal Cry2Ai protein having insecticidal activity against insect pests including, but not limited to insect pests belonging to the order Lepidoptera. The present disclosure also relates to expression of these sequences in plants. The disclosure further provides a DNA construct, a vector, and a host cell comprising the codon optimized synthetic nucleotide sequences of the invention. Also it provides use of the codon optimized synthetic nucleotide sequences for production of insect resistant transgenic plants, and a composition comprising Bacillus thuringiensis comprising the codon optimized synthetic nucleotide sequence of the present invention.