Isolation Of The CEL I Nuclease And Creation Of Mutant Library For TILLING Resarch In Rice

Targeting Induced Local Lesions IN Genomes (TILLING) is a new reverse genetic technique that allows effective detection of induced or natural DNA mutation by the combination of PCR-based screening and a mismatch-specific endo-nuclease. The technique can be applied not only to model organisms but also to economically important crops, and may have wide and bright application future in rice. The CEL I nuclease is a key enzyme and the mutant library is an essential base to TILLING research. In order to carry out TILLING research for functional genomic study and variety improvement in rice, effects on the extractive amount for the CEL I of different factors were analyzed first time and the mutant library of rice variety 9311 (Oryza sativa L.ssp. Indica) was created in this dissertation.Effects of varieties, organs, developmental stages of celery and pH of extraction buffer were analyzed on the extraction amount of the CEL I. The isolation amount varied greatly in celery varieties. Among the 3 varieties, Thailand celery showed the highest extractive amount of CEL I, followed by Wentula and Xuebai. While among different organs from same variety, the isolated amount of CEL I from celery leaves was higher than that stalks and roots showed the lowest extraction amount. The differences of CEL I amounts extracted among varieties and organs were significant at 5% and 1% level, respectively. The effect of developmental stages on the CEL I amount was also comparied by using Shanhai Huangxin as extractive material. The CEL I amount extracted from celery plants at reproductive stage was higher than that at vegetative stage. The value of pH in extraction buffer also showed obvious effect on the CEL I amount extracted. When using the extraction buffer with pH 7.7, the higher amount was obtained than that with pH 5.0 and pH 7.0. Such results were helpful to establish the method for CEL I extraction with higher efficiency.Activity assay of CEL I crude extraction was studied. Incision at mismatches of single nucleotide suggested that CEL I could effectively detect mutation of eGFP gene at T→C base transversion and the digested DNA could be separated by agrose gel and observed intuitively. Therefore, the extracted enzyme can be used in TILLING This result is important to develop the easy and effective detection method of point mutation.In order to construct the mutants library, the rice seeds of 9311 (Oryza sativa L. ssp. Indica) was treated and induced by EMS (ethyl methane sulphonate) and mutations displayed in M₂ generation consisting of 887 lines were examined and recorded. Totally 1683 plants with mutated trait(s) were observed, distributing in 696 lines. The mutation type induced by EMS was abundant, being visible in the leaf, stem, ear and physiological characters. Totally 1472 mutants were obtained and some of them were new types, such as mutants with color pericarp and the mutant with very shot length of flag leaf, which have not been reported in research of mutations induced by EMS updately. Characters of some mutant were also analyzed, eg. the internode length of dwarf mutants, main agronomical and quality characters of mutants with color pericarp. Furthermore, DNA was extracted from each plant in M₂ population and about 1 000 DNA pools were formed, each containing equal amount of DNA from 8 individual plants. So the mutant library of rice variety 9311 was created, which would be important to TILLING research in rice.In conclusion, the result obtained in the dissertation is of great significance to function geneomic research and variety improvement in rice by TILLING...