Plant genetics and molecular biology 1400-125GBMR

Lecture Topics

1. Introduction to Plant Molecular Biology:
Fundamental concepts and methodology of molecular biology; model plants in molecular research.
2. Plant Genomes – Structure, Evolution, and Diversity:
Organization of nuclear, chloroplast, and mitochondrial genomes; the role of whole genome duplications (WGDs) in plant evolution; significance of transposons and mobile elements in shaping plant genomes.
3. Omics in Plant Research:
Genomics, transcriptomics, proteomics, and metabolomics – an integrated systems approach; DNA microarrays, RNA-seq, and global gene expression analysis.
4. Functional and Regulatory Analysis of Genes:
Classical and reverse genetics – from mutant to gene and from gene to function; insertional mutagenesis, T-DNA, RNA interference (RNAi), virus-induced gene silencing (VIGS); modern genome editing techniques: CRISPR/Cas9, Cas12a.
5. Regulation of Nuclear and Organellar Gene Expression:
Coordination between nuclear and organellar genomes in regulating gene expression.
6. Chromatin and Epigenetic Inheritance:
Histone modifications, DNA methylation, and non-coding RNAs; mechanisms of epigenetic control of development and stress responses; epigenetic inheritance in the context of plant adaptation.
7–8. Regulation of Plant Development and Physiological Responses:
Genetic control of plant developmental processes.
9. Cell Signaling and Plant Hormones:
Major classes of phytohormones; hormonal signaling pathways illustrated by gibberellins (GA) and abscisic acid (ABA); integration of hormonal signals with gene expression regulation.
10. Types and Roles of Small RNAs (miRNA, siRNA, tasiRNA):
Mechanisms of small RNA biogenesis and function; their roles in regulating development and stress responses; applications of small RNAs in plant biotechnology.
11. Plants in a Changing Environment – Responses to Biotic and Abiotic Stresses:
Defense mechanisms against pathogens (PTI, ETI); responses to drought, salinity, low temperature, and nutrient deficiency; signaling networks and stress-responsive genes.
12. Plant Biotechnology and Ethical Aspects:
Genome engineering and gene editing; development of plants with enhanced resistance and nutritional value; directions of plant biotechnology development in the context of climate change.
13. Discussion and Case Studies:
Examples of translational research and biotechnological applications in plant science.

Laboratory Description
During laboratory sessions, students independently (individually or in pairs) conduct experiments addressing various aspects of plant molecular biology. The exercises are designed to familiarize students with, and develop their practical skills in, the handling and analysis of insertional mutants, CRISPR/Cas9-edited lines, and transgenic Arabidopsis thaliana plants.
Students will employ a range of molecular biology techniques, including nucleic acid isolation from plant tissues (genomic DNA, total RNA, and small RNA fractions), transcript level analysis, nuclear protein isolation, visualization and detection of GFP-tagged fusion proteins, and investigation of protein–protein interactions.

Description of Laboratory Experiments
1. Characterization of Arabidopsis Mutants in Genes Encoding Linker Histones (H1)
Students become familiar with the phenotypic effects caused by insertional mutations and genome deletions generated using CRISPR/Cas9 in Arabidopsis, as well as with methods for identifying these mutations.
Techniques used: genomic DNA isolation, PCR amplification, and agarose gel electrophoresis.
2. Analysis of H1 Gene Expression Levels
Students analyze the transcript levels of H1 genes using RT-qPCR in wild-type and mutant plant lines.
Techniques used: total RNA isolation, cDNA synthesis, and real-time PCR.
3. Analysis of Small RNA Fractions
Students investigate small RNA levels in wild-type plants, h1 mutants, and mutants defective in small RNA biosynthesis pathways.
Techniques used: isolation of small RNA fractions and separation of small RNAs by polyacrylamide gel electrophoresis.
4. Analysis of H1.1-GFP and H1.2-GFP Fusion Protein Levels
Students examine the levels of H1-GFP fusion proteins in transgenic Arabidopsis lines using Western blotting and visualize the GFP-tagged H1 proteins with fluorescence microscopy.
Techniques used: nuclear protein isolation, SDS-PAGE protein separation, immunodetection (Western blot), and fluorescence microscopy.
5. Analysis of Protein–Protein Interactions
Students analyze interactions between linker histone H1 and core histone H3 using the co-immunoprecipitation (Co-IP) method.
Techniques used: nuclear protein isolation, protein immunoprecipitation, and Western blotting.