(1,3;1,4)-β-D-Glucan Synthase Gene Identification via cDNA
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Solution Overview
Problem
Current methods fail to identify and isolate genes encoding (1,3;1,4)-β-D-glucan synthases due to the difficulty in purifying membrane-bound enzymes, which hampers the modulation of (1,3;1,4)-β-D-glucan levels in cereal grains and vegetative tissues, essential for altering grain quality.
Innovation Solution
Identification of (1,3;1,4)-β-D-glucan synthases as members of the CslF gene family, allowing for modulation of their expression and activity to influence (1,3;1,4)-β-D-glucan production in cells, particularly in cereal crops.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional purification methods are used for membrane-bound enzymes, then enzyme activity can be maintained, but gene identification and isolation become extremely difficult and time-consuming
Solution Approach 1:
The patent creates cDNA copies of the mRNA transcripts that encode the polysaccharide synthase enzymes. By copying the genetic information into a searchable database format, researchers can identify and isolate the genes without needing to physically purify and characterize the membrane-bound enzymes themselves. This copying approach maintains enzyme activity requirements while dramatically reducing the time for gene identification.
Solution Approach 2:
The patent uses mRNA and cDNA as intermediary molecules between the membrane-bound enzymes and the final gene isolation. Instead of directly working with the difficult-to-purify membrane enzymes, the methodology uses their mRNA transcripts as intermediaries that can be extracted, converted to cDNA, and used for gene identification, thus mediating the complex process of gene isolation.
2Measurement precision
If membrane-bound polysaccharide synthase enzymes are purified for characterization, then enzyme function can be studied, but the process is hampered by technical difficulties in purification
Solution Approach 1:
The patent replaces the mechanical/physical purification process with a molecular biology approach. Instead of using chromatography and other physical separation methods to purify membrane-bound enzymes, the methodology uses mRNA extraction, reverse transcription to cDNA, and molecular cloning techniques to characterize enzyme function and identify genes, thereby substituting a difficult mechanical purification system with a more feasible molecular approach.
3Adaptability or versatility
If genes encoding polysaccharide synthases are not identified, then grain quality cannot be modified, but traditional methods are insufficient for gene identification
Solution Approach 1:
The patent performs preliminary actions by constructing a cDNA library and database before attempting gene identification. By pre-processing the genetic material into a searchable format and establishing the molecular tools needed, the methodology enables subsequent gene isolation and grain quality modification without requiring complex purification and characterization steps during the actual gene identification process.
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
Enables the modulation of (1,3;1,4)-β-D-glucan levels in cells, thereby altering grain quality and tissue characteristics, facilitating applications in food processing, animal feed, and dietary fiber enhancement.
Implementation Method 1
genes which encode the biosynthetic enzyme for (1,3;1,4)-β-D-glucans, referred to herein as '(1,3;1,4)-β-D-glucan synthases'
Data Source
AI summary
The present invention relates generally to polysaccharide synthases. More particularly, the present invention relates to (1,3;1,4)-β-D-glucan synthases. The present invention provides, among other things, methods for influencing the level of (1,3;1,4)-β-D-glucan produced by a cell and nucleic acid and amino acid sequences which encode (1,3;1,4)-β-D-glucan synthases.