Screening And Analysis Of Rolled-leaf And Small Grain Mutants In Rice

As one of the most important staple crops in the world, Rice (Oryza sativa L.) wasa model plant for molecular biology studies of gramineous crops and monocotyledons.With accomplishment of the rice genome sequence project, how to elucidate thebiological functions became another challenge in post genomic era. Only several dozenfunctional genes had been cloned up to now. As this, construction of T-DNA insertionmutants' library would be a main method to research gene functions. The mutants ofrolled-leaf and small grain were screened, and a PCR-Walking system for amplifyingT-DNA flanking sequence was established, with which partical flanking sequence wereobtained and some sequences were analyzed The results of the research weresummarized as follows:1 312 rolled-leaf mutants and 203 small grain mutants were selected from 35,000 ricematerials with T-DNA inserted, by investigating and screening the rice morphologyin the field.2 Rolled-leaf mutants were researched from styles of rolled-leaf, crimpy rate andanalysis of heredity. The result showed that the rolled-leaf was mainly positive andmiddle rolled, and was controlled by recessive genes. Small grain mutants accordedwith the phenotypic standard of satiety, small and ellipsoidal. The kilo-weight ofthe small grain mutants was lower than standard of nipponbare. The mutationfrequency of enhancer trapping and activating tagging was 0.519% and 0.671%,which showed that the mutation frequency of small grain mutants was low.3 T-DNA flanking sequences of the selected mutants were amplified by PCR-Walking.In the optimized system, the time was saved with combining the cleavage andligation steps, and the second PCR reaction system was adjusted from 20μL to40μLmake for amplifying of large flux flanking sequences. Then an effectivemethod of amplifying the T-DNA flanking sequences was set up.4 541 and 381 rice flanking sequences were obtained from rolled-leaf mutants andsmall grain mutants by modified method of PCR-Walking, respectively. It showedthat Tosl7 trend to insertion gene while Tosl7 had no preference by the study of the insert position in the genes. Only several sequences had known functions, via blastsequences with T-DNA and Tos17 inserted-genes. Two candidate genes wereverified in small grain mutants by co-separation experiment.