Construction And Analysis Of Large-scale Rice (Oryza Sativa) Enhancer-trapping Population

Rice (Oryza saliva) is one of the most important food crops. It is also a good model for studies of monocot plants because of its relative small genome (430Mb), syntenic relationship with other agronomically important cereal species, the availability of genome resources such as well-defined genetic and physical maps, well-rounded transformation system and the gene draft sequence of Japonica cv. and Indica cv. Moreover, IRGSP has declared on December 18, 2002 at Tokyo that the plan of IRGSP has been finished with 366,000 kb DNA sequenced totally, the accuracy reached at 99.99% and 63,435 genes have been predicted. Large-scale and systematic functional genome studies of rice have been performed in several laboratories based on the above achievements. Construction of rice mutant bank is an efficient tool for functional genome research while efficient transformation system composed the basis for it. In this study, a high efficient Agrobacterium-mediated transformation system has been established and a large-scale rice enhancer trapping population has been generated. Further, molecular definition for this population has been done and PCR-walking adapted to this population for the amplification of T-DNA flanking sequence has been set up, simultaneously, we have analyzed some flanking sequence.This study is one part of "rice mutant bank construction" sponsored by The National High-Tech Program (863 program). Several progresses have been achieved with the efforts of all the members of our team within 2 years. Detailed information presented as follows:1. tn our studies, we have found that the timing of subculture on selection medium after coculture has prominent effects on the generation of resistant calli and their normal growth. Subculture after 10-15 days selection has been confirmed as the best time and over 90% calli could generate resistant calli under this status which were growing fine. We also found that the subculture interval under selection has momentous effects on the frequency of later differentiation. They have higher frequency of differentiation if 4-12 days subculture on selection medium were chosen while their frequency of differentiation decreased sharply if the subculture interval is over 16 days or less than 2 days. As an example, the frequency of differentiation reached at 85% if resistant calli were subcultured for 8 days on selection medium. We have set up and optimized one set of transformation system with characteristics of high efficiency, easily being performed and adapted O. sativa spp. Japonica. This integrated the achievements on the induction of embryogenic calli, the determination of the most optimal concentration of inoculation Agrobacterium solution, the interval of coculture with Agrobacterium and selection of different medium types at different stages. It takes only 2.5 months from induction of calli to generation of transformants.2 65,707 T-DNA tagged enhancer-trapping lines have been obtained through Agrobacterium- mediated transformation by applying enhancer-trapping vector pFx-z24.2-15R and employing rice (O. Sativa Spp Japonic) as material. It is indicated that more than 95% transformants harbored T-DNA insertion fragment confirmed by PCR and Southern blot analysis. In all TO transformants, there are about 43% events has one copy of T-DNA insertion and the average copy number of T-DNA insertion is about 1.2 confirmed by Southern Blotting. 3, The histochemical staining of GUS activity has been performed on the leaves and immature seeds of11,560 TO lines at heading stage. The general GUS expression frequency in seeds and leaves are 27.07% while 11.01% in the different parts of embryo, 8.28% in seed capsule and 3.42% in endosperm. 210 enhancer trapping lines with strong expression at this developmental stage have been selected form this population. The samples have been harvested and the DNA has been extracted for further studies.4. The T-DNA flanking sequences of 328 mutant lines have been amplified by PCR-walking. 82 lines of this group have been sequenced and analyzed pr...