Construction Of Rice Insertion-mutant Library And Analysis Of The Sequence Flanking The Insertions

Rice is one of the most important food crops worldwide. By using transgenic approaches, it is possible to increase resistance against pests and pathogens, and to improve the tolerance of rice plants to drought and salinity. New genes, including regulatory genes in the signal-transduction pathways, need to be discovered towards these objectives. With the completed sequencing of rice genome, a great deal of sequence data has been accumulated in gene bank, and the functions of lots of genes need to be determined. The research of the functional genomics is more and more urgent. Translating A,T,C,G into individual genes, isolating gene with ourselves knowledge property right become the focus of competition in all the world. With the developing of sequencing technology and increasing of isolating efficiency of the flanking sequence, construction of rice insertion-mutant library is an efficient method to study gene function?This research, we have selected Oryza sativa L subsup Japonica nipponbare , the variety be sequenced by International Rice Genome Project, as acceptor material. The transposon AC/DS of maize had been transferred into rice genome respectively by Agrobacterium tumefaciens mediated transformation, and constructed the transposon insertion mutant library of rice by using transposable two elements system. The main results of molecular analysis of the transformants, cloning and analysis of the sequence flanking the transposon insertion and the genetic analysis and bioinformatics research of the mutants are summarized as below:1. The transposon AC/DS of maize had been transferred into genome of nipponbare respectively by Agrobacterium tumefaciens mediated transformation. 1800 DS transformants and 50 transformants were generated.2. The molecular analysis showed that the positive rate of DS transformants is 95% , The result of PCR and basta analysis are identical, but positive rate of transformants with AC is only 40%.3. Seven kind of mutant types, l).color of leave and photosynthesis: etiolation in seedling ;pea green, bottle green; stripe leave; the content of chlorophyll.2).height of plant: special low(10cm)-special high (125cm).3).tillering: no tillering- more tillering 4).plant type:tight-lax.5).the date of tasseling: less 30 days-more 30days.6) sterile-fertile.7).grain: grain height, quality of grain, have been found through investigation and statistic analysis.4. 700 DS transformants are amplified by the method of Tail-PCR in order to gain the sequences flanking the DS insertion. 250 of those have amplified out special the rice genome sequence flanking the insertion, 109 of which have been sequenced.5. Analysis and location of the 109 flanking sequence showed that 26 of the flanking sequence insert the coding region of genes, 83 insert non-coding region between genes, and some of those lose the T-DNA right border. And a T-DNA insertion-map was constructed by locating forty-one single copy DS transformants into different region of rice chromosomes.6. Cloned and sequenced the flanking sequence of single copy DS homozygous transformants and compared them with Genebank data, We have obtained forty-one disperse in rice chromosomes transposable anchor lines.7. Amplified the flanking sequence by using inverse PCR, We have obtained 4 flanking sequence of 4 mutants. The flanking sequence of mutant T835 is identical with the mRNA which is coding for OsPTR transporter protein of Oryza sativa (japonica cultivar-group), and have 96% homology with Histidine transporter protein of Arabidopsis. The flanking sequence of mutant T319 were located between two exons of a gene which is predicted by using software FGENESH kl. But the flanking sequence of the T331 and T415 were located at non-coding region between genes which are predicted by using software FGENESH 1.1.