园艺植物生物技术第七章.ppt

Genetic Engineering also called genetic modification, is the direct manipulation of an organisms genome using biotechnology.  New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or knocked out, using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations. An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria in 1973. Genetically modified crops (GMCs, GM crops).The first genetically modified plant was produced in 1982, using an antibiotic-resistant tobacco plant. The first genetically modified crop approved for sale in the U.S., in 1994, was the FlavrSavr tomato, which had a longer shelf life. 7.1 Instrumental Enzyme of Gene Engineering Restriction endonucleases (限制性内切酶) Ligase（连接酶） Polymerase（聚合酶） Nuclease（核酸酶） Terminal enzyme（末端转移酶） Methylase（甲基化酶） 7.1.1 Restriction Endonuclease 一类能识别双链DNA分子中某一特定核苷酸序列，并能对核酸内部的磷酸二酯键进行切割的一种内切核酸酶。 TypeⅠendonuclease：识别专一位点，切割不专一； TypeⅡendonuclease：识别专一位点，切割专一； TypeⅢ endonuclease：识别专一位点，切割不专一。 TypeⅡendonuclease Recognize：4~6个核苷酸，回文序列 Cuting result： a sticky ends(粘性末端)： Pst1 EcoR1 5’-CTGCA G -3’ 5’-G AATTC-3’ 3’-G ACGTC -5’ 3’-CTTAA G-5’ a blunt ends(平末端)：NruⅠ 5’ TCG CGA 3’ 5’ AGC GCT 3’ Isoschizomers(同裂酶)：指来源不同，但识别相同靶序列的核酸内切酶。 如：HpaⅡ和MspⅠ是一对同裂酶 (CCGG) Isocaudarner(同尾酶)：指来源不同、识别靶序列不同但产生相同的粘性末端的核酸内切酶。 BamHⅠ BclⅠ G GATC C T GATC A C CTAG G A CTAG T 7.1.2 DNA ligase（DNA连接酶） T4 DNA ligase： join blunt ends or sticky ends Escherichia coli DNA ligase：