Molecular genetics of gene expression：基因表达的分子遗传学.pptVIP

Lecture 17 Chapter 9 Marker genes Neal Stewart Discussion questions 1. Why use marker genes? 2. What are some differences between selectable markers and scorable markers? 3. Discuss the relative merits of GUS and GFP as reporters. Does the profile of experimentation using these reporter genes overlap directly or partially? 4. What are the advantages, if any, for the use of the manA gene over the nptII gene as a selectable marker for food and feed crops, and would the use of the manA gene overcome public concern over the use of the nptII gene? Conversely, what are the disadvantages? Using marker genes helps answers Are my plants transgenic? Is the gene expressed? How is my promoter working? Selectable markers Scorable markers (reporter genes) Typically used to recover transgenic plant cells from a sea of non-transgenic cells Antibiotic resistance markers and herbicide resistance markers are most common Can help visualize transient expression Can help visualize if tissue is stably transgenic Useful for cellular and ecological studies Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed Barnase kills tapetum cells (and pollen)—negative non-conditional selection useful to engineer male-sterility Common reporter genes Beta glururonidase (GUS) uidA protein from Escherichia coli– needs the substrate X-gluc for blue color Luciferase proteins from bacteria and firefly yields light when substrate luciferin is present. Green fluorescent protein (GFP) from jellyfish is an example of an autofluorescent protein that changes color when excited by certain wavelengths of light. Firefly luciferase produced in tobacco 35S:GFP canola Green (and other color) fluorescent proteins FP properties Detection and measurement Anthozoan FPs Why red is better than green Why orange is best of all! What is fluorescence? LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence A brief FP history Anthozoan FPs in