Gene Silencing Flashcards
Important root anatomy
Root hairs
Area of cell differentiation
Area of cell elongation
Area of cell division
Root cap
Casparian strip
Specially differentiated endodermal cells with special cell walls. Water proofs central cylinder and
RNAi/ post transcriptional gene silencing
Double stranded rna cleaved by dicer like proteins. Argonauts proteins and associated risc complex bind and with activation of atp target mRNA substrate leading to RNA degradation and translation repression.
Transgene specific pcr
Doesn’t give copy number of transgene.
High chance of contamination or pcr not working properly
Southern hybridisation
Detects transgene copy number
Northern analysis
Measures mRNA abundance.
Less specific that’s qRT-PCR and requires mor rna and is more complex
Quantitative real time reverse transcriptase pcr
Measures mRNA abundance
mRNA is converted to cDNA and measure by quantitative pcr.
Cycle threshold number indicates the number of cycles to detect cDNA
Is highly sensitive( so very little sample is required) easy to replicate for statistical analysis
Evolutionary roles of gene silencing
Genome defence mediated by siRNA
Control of endogenous genes by miRNAs
Forward genetics
Strat with wild type, mutate plant and then map based cloning of causative gene
Reverse genetics
Start with mutant line transform mutant line with wild type transgene so that you have a mutant plant with wt phenotype
Dicer like contributions to RNAi and epigenetics in Arabidopsis
Dcl1: miRNAs
Dcl4: abundant 21nt siRNAs
Dcl2: low abundance 22nt siRNAs crucial for systemic RNAi
Dcl3: 24nt siRNAs involved in epigenetic silencing
Post transcriptional gene silencing
DCL4 and DCL2, AGO1 and RDR6 involved
Silencing suppressor proteins
Virus proteins such as p19 bind to siRNA and miRNA duplexes to suppress gene silencing
Epigentic gene silencing pathway
DCL3 cleaves short day RNA. So RNAs associate with AGO4 leads to DNA methylation.
