Mutant Screen And Analysis In Rice (Oryza Sativa L.) Tagging Library

Rice (Oryza sativa L.) is one of the most important staple crops in the world, and a model plant for molecular biology studies of gramineous crops and monocotyledons. Now the sequencing of rice genome is nearly completed, and more than 40,000 putative genes have been identified from it. But only several dozen genes have been cloned up to date. T-DNA insertion mutants library will provide a main resource to study gene function.Our main work mainly circles around the enhancer trapping and activating tagging library being constructed. This paper deals with the mutant efficiency evaluation of two mutant library and screen of different mutants, while a high efficiency system of amplifying rice flanking sequence has been set up. The exact content followed:We have set up a high efficiency genetic transformation system. 100 thousand T₂ seeds including enhancer trapping and activating tag lines were collected. 4,081 rice mutants with obvious mutant morphology were observed in T₁ progeny. The mutant morphology dealed with plant height, leaf color, heading date,leaf shape,tiller, lesion mimic, grain shape and etc.Some very special rice mutants have been discovered,such as brittle culm mutant, defective spike, plastochron.The constrast between the enhancer trapping and activating tag library showed the mutant efficiency of the enhancer trapping library was 5.42% and the mutant efficiency of activating tag library was 6.42%, thus the activating efficiency in activating library as 1%, the result was confirmed by T₀ progeny mutant variation efficiency.Through modified PCR-Walking and Tail-PCR methods, over 1,000 rice flanking sequences were gained. The analysis of insertion site rhythm showed tag site has 53% in the intragenic region. By biological information analysis,4cloned genes have been tagged in our mutant library by T-DNA or Tos17 and their phenotype were similar as our former reports. For example, Hd₁ had been reported by Japanese Masahiro Yano and were inserted by one new Tos17 site .Hd₁ was reported as a quantitative trait. But heading date in this mutant was ahead of two month than wild type. Moreover, 5 candidate genes have been verified by co-separation experiment. 10 candidate genes with at least two loci insertion and the same phenotype have being verified.As a result, the above data and analysis showed that our mutant library has higher mutant efficiency and would be effective for gene function research.