The Study On Transcription Factor Promoters Of Oryza Sativa L. By ECOTILLING

We detected transcription factor promoters between rice cultivars and Nipponbare by simplified ECOTILLING technology to find alleles correlated to agronomic traits. Main results were summarized as follows:The method for extracting crude CEL I enzyme was further simplified and the activity assay of different samples from every process were also investigated. Effective incision at mismatches of single nucleotide suggested that celery juice also had activity and the activity could be enhanced by purification. The digested DNA could be separated by agrose gel and observed intuitively. Therefore, the simplified ECOTILLING technology can be used in plant.469 primer pairs were designed according to the sequence information of rice transcription promoters and 202 primer pairs were found nucleotide polymorphisms between Nipponbare and materials by ECOTILLING. Association analysis between transcription factor markers and traits showed that in drought treatment, marker AP2-7-6 and AP2-7-9 related to ear length; maker HAP5-3 related to effective panicle and effective panicles per plant; maker AP2-9-13 related to the number of grain per ear and setting rate. In water treatment, maker G2-15 and HAP5-19 associated to ear length; maker AP2-1-5 and AP2-1-7 associated to the number of effective panicles per plant. Maker HAP3-9 associated to imperfect grain per panicle and setting rate. Maker Alfin-11,GRF-5 and AP2-7-4 related to drought coefficient and drought index; maker GRF-8,HAP3-17,AP2-2-11,AP2-8-10 and ZIM-14 related to drought resistance level. Maker GRF-5 and AP2-1-5 related to 100-seed weight in both treatments.The promoter sequences of AP2-7-6 and AP2-7-9 were analyzed to show that the promoter homology were 94%-99%, including TATA box, GC box/CAAT box and some positive regulatory elements which were necessary to mediate genes expression. The signal sites of promoter matched the function of its transcription factor. The same signal sites were found in promoter sequences of different transcription factors which have similar function.Expressions of four genes at different stages under the treatment of 20% concentration PEG 6000 were detected. The four genes were Alfin-11,AP2-7-4,GRF-5 and HAP3-17 which were related to drought resistance. The expression level was identical to the results of association analysis. In the same gene, the expression level was regular according to the different drought resistance index in each gene.