第五章第一节图位克隆基本原理解析.pptVIP

第五章、植物基因克隆 5.1 植物基因克隆的基本原理 5.2 苹果抗黒猩病基因的克隆 5.3 水稻主效基因的克隆 5.4 水稻米香基因的克隆 5.5 数量性状基因克隆 5.6 玉米主效QTL克隆和利用 5.1 植物基因克隆的基本原理 Strategies for gene cloning 1) Map-based gene cloning 2) Transposon tagging 3) Candidate gene approach 5.1.1 Map-based gene cloning Gene isolation without prior knowledge of the gene product Requirements: Population of individuals with genetic differences in the trait of interest Gene responsible can be mapped to a chromosomal position adjacent to molecular markers (e.g. RFLP, microsat markers) First step is to identify molecular markers that are close to the gene of interest Initially small population used (60-150) Then saturate the region with other markers Much larger population (300-1000) Use markers based on comparative mapping info e.g. Arabidopsis- Brassica colinearity to help find canola genes Looking for ones that rarely show recombination 5.1.1 Map-based gene cloning-continue 目标基因定位 R R Target gene M1 M2 P1 F1 P2 BC1 × BC1-1 BC1-100 BC1-99 BC1-98 BC1-2 M1 M2 目标基因精细定位 R R R R R R R R R R Target gene M1 M2 Map-based gene cloning -Chromosome walking Next, screen a large insert genomic library (e.g. BAC) with the markers to isolate clones that contain the marker Involves creating new markers Based on sequences at the ends of the selected clones Screen the library with the new markers Goal is to find a set of clones that cover the region harboring the locus of interest. Create a contig of BACs which are anchored between the closest markers flanking the locus of interest If you are lucky somebody else has already walked by and created a contig which can be used Map-based gene cloning -Chromosome walking Map-based gene cloning -Chromosome walking Identifying the target gene: YACs or BACs can contain several different genes Make a cDNA library from tissue that should be expressing the gene of interest identify YAC or BACs to cDNAs identify those that match the region represented by the YAC or BACs If more than 1 cDNA matches use high-