Rice T-DNA Insertional Mutant Screening And SDP1 Gene Function Analysis

Recently, it is widely applied to clone genes by mutant. We take a forward geneticstrategy to clone genes from a rice Enhancer trap population. By screening the T₁ lines,we found some morphological mutants which carried T-DNA insertions. Then thegenomic sequence flanking the T-DNA inserts were isolated to test whether the mutantphenotype co-segregate with the T-DNA insert loci, and provided a base for geneisolation and cloning. A semi-dwarf, delayed heading, pale green mutant, sdp1, wasanalysed in detail for its corresponding functional gene (SDP1) and agronomic traits. Themain results are as follows:1. By screening about 2,000 T-DNA insertion mutant lines in 2005, the results showedthe mutant frequency in seeding stage and in field were 4.40% and 13.42% respectivelly,the main mutant phenotypes were: albino leaf, yellow leaf, lesion minic, dwarf, thintiller,open tillers, earlier death, delayed heading, et al. We finally got 126 lines whosephenotype segregated with T-DNA insertions. Employing the TAIl-PCR, inverse PCRand plasmid rescue strategies, 142 flanking sequences were isolated and investigated byblastn and co-segregation test.2. The bioinformatics analysis of the flanking sequence of sdpl showed that T-DNAwas inserted into the terminal 8 bp of gene OS03g63050 which was located on Chr3 andincluded 3 exons, but no function prediction was found for this 809 bp length gene. Sdp1was identified to have a single copy of T-DNA insertion and exhibited a stableco-segration pattern in even T₃ generation. Complementary test(vector constructed byZhihuiChen) results showed: 24.36% of the transformants displayed a rescued phenotype,indicating the disruption of gene SDP1 caused the phenotype change. GUS which locatedon Enhancer trap vector (pFX-E24.2-15R) staining assay showed GUS was constitutivelyexpressed in root, internode, leaf, sheath and glume in all different stages, while RT-PCRanalysis revealed that SDP1 was not detected in root, internode, leaf, sheath and spiket inall different stages. A fused expression vector: pSDP1::GFP was constructed andtransformed into ZH11. By measuring the pigment content, we found that the decrease ofCh1 and the increase of Car caused the leaf change from green to pale green. Bymeasuring the plant height of ZH11, sdp1 wild type, heterozygote and mutant, weconcluded the fours changing trend following the time and confirmed SDP1 had influenceon plant height in 23 days after germination. Finally, the agronomic traits were cheked, many differences such as plant height, seed set, 1000-grain weight between the wild andmutant were found, and the first, the second internode made the main differences on plantheight.