Separation And Cloning Of The Expressed Gene Fragments Related To Corn Resistance To Setosphaeria Turcia

Expressed genes in corns with major different resistant genes, B37,B37Htl and E37Ht2, had been separated and partially cloned after inoculated with race 0 and race 1 of Setosphaeria turcica by DDRT-PCR. The experimental results will help to elucidate the number of genes differentially expressed in compatible or incompatible interactions, and lay a solid foundation for establishing expressed sequence tags (ESTs) and study their function in the future by means of recovering, cloning and sequencing the differentially expressed genes related to corn disease-resistance.The main results in the study were as follow:1. An optimum protocol for corn leaf mRNA differential display was established.(1) The cDNA reverse-transcripted from total RNA isolated by UNIQ-10 column Trizol isolation kit ranged from 300bp to 1000bp. which was suitable for DDRT-PCR.(2) Compared with the one-step amplification method, the two-step amplification method was much better for DDRT-PCR, which can acquire clear amplification bands with the number of 40-60 and the length of 150-1000bp.2. Three-anchor primers and 12 random primers were used to DDRT-PCR amplification in corn cDNA, which was obtained from corn near isogenic lines induced by S. turcica. 148 differential expressed gene fragments were obtained by polyacrylamide gel electrophoresis (PAGE) and silver stain detection. 138 fragments out of the 148 differential bands were successfully recovered from PAGE gels and were effectively amplified again, and the recovery percent was up to 93.2%.3. Through reverse Northern blotting, 63 positive fragments were obtained with the positive percentage of 42.6%. Among them, 17 fragments from B37 were probably related to corn susceptibility to race 0, and 7 fragments from B37Htl related to corn susceptibility to race 1. 5 fragments specific to the resistance of B37Ht2 to race 1 and 16 fragments from both B37Htl and B37Ht2 probably related to corn resistance to race 0 were also found.4. Five fragments specific to the resistance of B37Ht2 to race 1 were cloned,regulation factors, and membrane transport protein to some extent. To make sure the definite function of the fragments, the full-length genes should be obtained and their functional complement experiments should be performed in the next step.