Map-based Cloning And Functional Analysis Of A Drought Sensitive Mutant Dsm1 In Rice

In this study,our materials were taken from T-DNA Insertion Mutant Library found by our labotory(Nipponbare was used as genetic background).We screened them and obtained a drought sensitive mutant dsm1 in rice seedlings by using PEG.We simulated drought abiotic stresses,determined physical indexes,obtained the mutant site of the gene DSM1 by using map-based cloning technique for fine mapping,and did qRT-PCR experiments.The result of our study is as follows:1.Screening and Identifying drought sensitive mutants: By using PEG to simulate drought abiotic stresses and testing content of malonaldehyde(MDA)and water,resectively,we verified resistance to drought.The results showed that the mutant lines were extremely sensitive to adversity,especially drought,and indicated wilt even death after adding culture solutions again compared with wild type(WT)as controls.The relevant physiological signs trended to change apparently in mutants compared with WT as controls.2.Agronomic characters comparision between mutant dsm1 and WT: It is during trefoil stage that the mutant exhibited the sensitivity to drought abiotic stresses.In aging time,the phynotypes of the mutant dsm1,besides narrow leaf,were the same to the ones of the WT.Therefore,we used the narrow leaf phynotype as secondary agronomic character for screening mutants.3.Map-based cloning: Given the difficulty of cloning gene for the sake of drought phynotype belonging to QTLs characters,our study made ues of the narrow leaf phynotype in placing of drought phynotype for genetic research.We also mapped a main QTL gene within 80 kb region in No.4 chromosome by using recombinant inbred lines through initial location and fine location,respectively.We found that there is a SNP mutation in CDS arrays by sequencing candidate genes called corresponding lines,which induced cytimidine was transfered to guanine,and the gene number is LOC_Os04g52479,leading to the mutation of amino acid: an Arginine is transfered to a Glycine.4.The complementary experiments: Building vectors with CDS of LOC_Os04g52479.We transferred them to the mutant dsm1 and obtained 212 positive lines.In T1 stage,we screened the tolerance by using PEG.Parts of them were restored the resistance to drought.5.Overexpressive experiments: Building vectors with the CDS of LOC_Os04g52479.We transferred them to the wild type and obtained 128 positive lines.In T1 stage,we screened the tolerance by using PEG.Parts of them were raised the resistance to drought.In conclusion,we cloned the gene DSM1 and primary identified the resistance to drought owe to the expression of the gene,which pays foundation for further studying the mechanism of drought tolerance.