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A Comprehensive Guide to Yeast-Based Screening for Stress Resistance Genes
Release time:
2025-01-06
Biotic and Abiotic Stresses
Unlike animals, which can move freely to find suitable environments, plants are stationary organisms, rooted in the soil, and inevitably exposed to harsh environmental conditions. Environmental stresses can be divided into two main categories based on their nature: biotic and abiotic stresses.
Biotic Stress refers to all biological factors that negatively affect plant survival and development.
Abiotic Stress is caused by non-living environmental factors, including water-related stresses (drought, flooding), temperature extremes (high or low temperatures), salinity, and environmental pollution (such as heavy metals and pesticides).
Some plant genes respond to these stresses, thereby reducing the negative impact on the plant's survival and growth. These genes are known as stress resistance genes. When plants encounter adverse environmental conditions, gene expression is regulated through cellular signal transduction pathways, resulting in significant changes in protein expression. Studying these molecular mechanisms at the protein level is crucial for understanding how plants adapt to stressful conditions. So, what can be achieved through yeast-based stress resistance gene screening?
Why Yeast?
Yeast is a single-celled eukaryote that grows rapidly in culture media. Its expression system is similar to that of plants, and it also performs important processes such as glycosylation, disulfide bond formation, protein folding, and post-translational modifications. This similarity ensures that the proteins encoded by plant genes in yeast function properly, significantly reducing the likelihood of false positives in validating stress resistance genes.
By using yeast, we can screen for resistance-related genes across the entire genome through resistance gradient experiments. Candidate proteins can be identified using proteomics, followed by phenotypic validation at the protein level.
Yeast Stress Library Construction Process
1.Samples are taken from different growth stages and tissue types, and RNA is extracted to generate a high-coverage cDNA library.
2.The cDNA library is ligated with adaptors to create a three-frame library.
3.The library undergoes equalization treatment to reduce redundancy and enrich for low-abundance genes.
4.Using SMART technology, the cDNA library is inserted into the pYES 2-NTB vector, which is then electroporated into E. coli competent cells for cloning and amplification.
5.The library is checked for quality, plasmids are extracted, and NGS sequencing is performed to determine the sequences present in the library.
Yeast Stress Library Screening Process
1.Prepare yeast working cultures and transform the cDNA library into stress-sensitive yeast strains.
2.Verify the quality of the working cultures through colony PCR, setting up positive and negative controls.
3.Create five different concentrations of culture media to test various stress conditions and identify the appropriate screening conditions.
4.Spread 20 plates (15 mL each), extract 96 positive clones, and perform colony PCR and first-generation sequencing.
5.The sequencing results are analyzed by BLAST alignment with the GenBank database, followed by validation, NGS sequencing, and GO+KEGG analysis.
Phenotypic Validation of Stress Resistance Genes
1.Synthesize the target gene and insert it into the pYES 2-NTB vector.
2.Transform the successful constructs into stress-sensitive yeast strains, setting up a negative control group.
3.Perform gradient dilution on plates and subject them to stress conditions to evaluate whether the target gene confers stress resistance.
Case Study: Verification of Gene A’s Role in Improving Cadmium Resistance in Yeast Strain Δycf1
Experimental Results:The empty vector control group can grow under SG-U conditions with 0 µM, 40 µM, 50 µM, and 60 µM CdCl₂, but cannot grow under 80 µM or 100 µM CdCl₂. In contrast, the experimental group can grow normally under 0 µM, 40 µM, 50 µM, 60 µM, 80 µM, and 100 µM CdCl₂.
Conclusion:The pYES 2-NTB-A vector enhances the cadmium resistance of yeast strain Δycf1.
Q&A
1.How reliable is yeast for gene resistance screening or validation?
Yeast is a eukaryotic organism, and its expression system is quite similar to that of plants. It is capable of processes such as glycosylation, disulfide bond formation, protein folding, and post-translational modifications, which ensure that the proteins encoded by plant genes in yeast function properly. Yeast has been widely used in research for screening and validating stress-resistance genes. However, as a heterologous system, results from yeast can provide supportive evidence but should not be the sole validation system. It is recommended to combine yeast results with other systems for a more comprehensive validation.
2.How is a yeast stress library constructed?
Similar to yeast single-hybrid and two-hybrid systems, yeast stress libraries can be constructed using methods like SMART or Gateway, with differences in the vectors used. If a Gateway primary library exists, a stress library can be subcloned from it, reducing construction costs.
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