| As an important food crop,rice（Oryza sativa L）is the staple food of more than half of the world’s population.The growth and development of rice will not only be affected by diseases and pests,but also restricted by abiotic stress conditions.Drought stress will destroy the steady state of rice cells and damage the normal physiological function of rice,which results in the limitation of growth and development and the reduction of rice yield.To excavate rice drought tolerance genes and study rice drought tolerance mechanism is important to provide gene resources for water-saving and drought resistant rice breeding.In this study,the recombinant inbred lines（RILs）of Zhenshan 97B/IRAT109 was used for drought tolerant QTL mapping.Except for plant biomass,the green leaf rate was identified by image technology.Based on the genetic map of bin marker,the QTL of drought tolerant genes in rice seedlings was detected,and the candidated gene were cloned and the molecular function were preliminary analyzed.The results are as follows:1.Biomass and green leaf rate of 200 RILs of ZS97B/IRAT109 were investigated at seedling stage under normal conditions,20%PEG6000 stress and 10 m M H2O2stress.Sixteen QTLs were identified on chromosomes 1,2,4 and 9.The LOD value of q Redw2-3was 61.167 and the contribution rate was 15.447%.A Os COX17 was considered as a candidate gene for drought tolerance in rice.2.The candidate gene Os COX17 was cloned.Base on the promoter sequence of the parents,two indels at-1369 and-1416 bp of promoter were found.Luciferase transient expression analysis showed that the transcriptional activity of Os COX17 promoter from ZS97B was stronger than that from IRAT109.3.A molecular marker Os COX17-indel40 was developed based on the two indels.The marker was verified to be co-segragation with the drought tolerant phenotype in the mapping population.4.The expression pattern of candidate gene Os COX17 in rice was analyzed by q RT-PCR.It was found that the expression of Os COX17 was upregulated under 10 m M H2O2,20%PEG-6000（polyethylene glycol）and 1 m M ABA treatment.5.Os COX17 was transformed into E.coli and induced to express.It was found that the express of Os COX17 inhibited the growth of E.coli under hydrogen peroxide stress.6.The fusion vector of Os COX17 and GFP was constructed and transferred into tobacco and rice protoplasts.The subcellular localization of Os COX17 was observed by confocal microscope.It was found that Os COX17 protein was located in cell membrane and cytoplasm in tobacco cells and in cell membrane,cytoplasm and mitochondria in rice cells.7.The knockout vector and overexpression（OE）vector of Os COX17 were constructed and transformed into rice Nipponbare.The mutant and the OE plant were verified by sequence and RT-PCR.Compared with wild-type plants,the content of hydrogen peroxide was decreased in Os COX17 OE plants under normal conditions. |
