The Study On MiRNAs And Promoters Of Oryza Sativa L. By ECOTILLING

TILLING was widely used in genome functional research. We detected miRNAs and promoters between rice cultivars and Nipponbare by ECOTILLING to find alleles correlated to agronomic traits. Main results were summarized as follows:1. 95 materials were selected from 148 rice cultivars (Oryza sativa L.) judged by the drought resistance, including 3 deepwater rices. The genetic diversity of 30 InDels, 28 SSRs, 70 miRNAs among 95 materials were found from 392 markers; InDels and SSRs diversity implies that the marker polymorphisms between Nipponbare and 95 materials represent a large proportion of inter-subspecific diversity. All materials were divided into indica and japonica groups when clustered by InDels and SSRs. There were 5 materials different from indica and japonica groups when clustered by miRNAs;2. The method and activity assay of CEL I crude extraction were studied. Incision at mismatches of single nucleotide suggested that CEL I could effectively detect mutation of P221 GFP plasmid which AT base inserts BamH I and the digested DNA could be separated by agrose gel and observed intuitively. Therefore, the extracted crude enzyme can be used in TILLING; a method for simple detection of TILLING product was established, through the ellipsis of enzyme termination and purification. This result is important to develop the easy and effective detection method of point mutation;3. 70 miRNA markers polymorphic between Nipponbare and materials were selected from 210 miRNA markers that based on the sequence information. Correlation analysis between miRNA markers and traits shown that in water and drought treatments, miR439e associated to ear length; miR159c associated to number of effective spikes per plant; miR413 associated to 100-seed weight; miR156fl, miR159c, miR435 associated to biological yield, contribution rate were 9.58%-32.51%; miR156d, miR159f, miR167c, miR319b, miR393b, miR415 and miR819k associated to drought resistance stage, contribution rate were 13.80%-26.88%; miR156a and miR415 associated to relative setting rate, contribution rate were 10.47% and 9.07%.4. The promoter sequences of part of correlated miRNAs were analyzed to show that the average promoter homology were 95%, including TATA box and GC box.