植物营养的分子生物学：Chapter3 general method in plant nutrition.pptVIP

Chapter 3 GENERAL METHODS USED IN MOLECULAR BIOLOGY of gene function 3.1 Cell Culture and Fractionation 3.1.1 cell culture Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called cell senescence because of a progressive shortening of the cell’s telomeres. Cell lines can often be most easily generated from cancer cells. One of common cell lines for plant research is BY2 originally from undifferentiated meristematic cells of tobacco PLANT TISSUE CULTUREhttp://www.liv.ac.uk/~sd21/tisscult/introduction.htm Cells can be fused together to form hybrid cells Fuse one cell with another to form a heterocaryon, a combined cell with two separate nuclei. Techniques: cell suspension is treated with certain inactivated viruses or with PEG (polyethylene glycol) or electronic shock to alter the plasma membranes. 3.2. Isolation, Cloning, Sequencing of DNA Restriction endonuclease recognition sites are short, usually palin-dromic, DNA sequences Actions of some restriction endonucleases Restriction mapping is the first step in characterizing an unknown sequence Two methods for looking at DNA sequence variation Plasmid (vector) Procedure for cloning a DNA fragment in a plasmid vector Difference between cDNA genomic DNA clones Colony hybridization assay for the identificationof bacterial colonies carrying a particular DNA clone Constructing cDNA libraries for screening a root phosphate transporter Optimise expression of phosphate transporters in plant roots Extract and purify full length mRNAs Use mRNAs as templates to synthesize double stranded cDNAs Ligate the cDNAs into shuttle vector Transform into E. coli and select for transformants on ampicillin media Extract plasmids from transformants and purify Screening genomic cDNA libraries Infect E. coli with phage(抗菌素） library and plate out Transfer phage to a nitrocellulose（硝化纤维） filter Denature phage and wash the filter Hybridise 32P-labelled probe to the DNA overnight Wash exces