Construt Cotton Stress Induced Normarlized CDNA Library And Isolate Some Genes Involved In Stress Response

Detecting and responding to environmental perturbations are important for plant, plant are sessile and thus have to endure environmental challenges, such as soil salinity, drought, extremely temperatures, nutrition deficit, pest and disease attack etc, which influence plant growth even their lives greatly. As a result, to unravel the molecular mechanism underlying plant response to harsh environment should be the eventual goal of plant biologist and crop breeder, for it will facilitate to elucidate the signaling pathways used by plants, and it will benefit to identify key genes for modifying the responses of crops to environmental stresses. In this study we constructed a normalized stress-related cDNA library of G. barbadense cv.7124 by saturation hybridization with genomic DNA and analyzed the genes specifically expressed in Verticillium Wilt-reponse.The main results are as follows.1. We constructed six individual libraries of G. barbadense cv.7124 by high/low temperature, high salt, drought, potassium& phosphorus deficit and cotton Verticillium Wilt infectinon. The normalized fiber cDNA library by saturation hybridization with genomic DNA. Most inserts of the normalized library were 0.8 to 1.6kb, and the average length was 1.15 kb.2. randomly select clones to sequence, read sequence file by Phred software set the threshold at Q>20,aftere remove the vector sequence, we get 6707 high-quality sequence, which are assembled into 3647 unique sequence (3009 singletons and 638 contigs) ultimately. The average ESTS contained in each contig is 4.2,the most abundant contig contain 437 ESTS, the redundancy of library is 58.8%3. BLAST analysis is carried out against to Arabidopsis EST database, NCBI Non-redundant protein database and cotton EST database separately.11%、15%,7%, get no hits.4.1573 unique sequences were assigned to functional categories using gene ontology. Almost 51% unique sequences of belonged to metabolism functional categories and map to 173 biochemical and biophysical pathway.5. BLASTx searches revealed 453 (12.4% of unique sequences) with matches in PlnTFDB at E-value≤10-5 The 453 TFs fell into 68 families. The most abundant TF family was the MYB-related (35), followed by the C2H2 (25), FAR1 (22), bZIP(18), bHLH(17), MADS (17) AP2-EREBP(15), C3H group (9.9%) families, most of which are involved in stress-response.6. We make a array contain 1000 unique sequence derive from library based on bioinformatics analysis. Based on array analysis of the 1000 selected ESTs by cotton Verticillium wilt infection cDNA, we selected a batch genes which deduced to responsive to cotton Verticillium as candidate7. Cloning and characterization to the WRKY transcript factor family, and analysis the profiling under cotton Verticillium wilt infection. Construct express vector and do transformation to testify their molecular function.8. Cloning and genetic transformation of genes GbHAk5、GbPht1...