Cloning And Mechanisms Of Enhanced Drought Resistance Of Upland Rice(Oryza Sativa L.)Mutant Idr1-1

Rice(Oryza sativa L.)production consumes a large quantity of water,and it is of great importance to find new germplasms with high drought tolerance for the rice drought-resistant breeding.In this study,we investigated the IAPAR-9(wild type)and its mutant idr1-1(increase drought resistance1-1)and found the mutant exhibited significantly increased drought tolerance compared to the wild type.We furthermore used molecular biology methods to explore the underlying molecular mechanisms of why idr1-1 mutation give IAPAR-9 increased drought tolerance.The main results are listed as follows:1.By comparing different drought-resistant evaluation methods usually used in paddy rice and upland rice,we developed a new method that can be used to evaluate the drought tolerance degree of upland rice cultivars,which is named DTD(drought tolerance degree)method.DTD is defined as the mean of the ratios of green leaf length to total leaf length of the top three leaves in every plant after severe drought treatment.DTD values thus vary from zero to one.Our results showed that the DTD method is a simple,direct and relatively accurate evaluation method for drought-resistance breeding of upland rice.2.To reveal the molecular mechanisms of increased drought tolerance of idr1-1 mutant,we generated a F2 mapping population developed from a cross between idr1-1 mutant and HD 119.The drought resistance individuals and sensitive individuals in the F2 population demonstrate 1:3 phenotype after drought treatment.The DTD method was used to identify drought tolerance of each individual in the F2 mapping population.In the end,the idr1-1 mutation was mapped to the long arm of chromosome 5,at an interval of?30.7 kb between the Indel markers IM63 and IM59.Based on the genes located in this region in Nipponbare genome,four candidate genes in idr1-1 mutant were selected for further analysis.After sequencing,only LOC_Os05g26890 was found to carry a 1-bp deletion in exon 5,which resulted in a premature stop codon within exon 6.Complementation and overexpression of wild-type IDR1 amplified from IAPAR-9 all rescued the developmental and decreased drought-resistant phenotypes,suggesting that IDR1 is indeed the gene that is responsible for the mutant phenotypes.Sequence analysis demonstrated that IDR1 encoded the G?,it was involving in ethylene signaling transduction in the plant cell.3.To understand transcriptional changes between IAPAR-9 and mutant,leaf tissues of IAPAR-9 and idr1-1 undergoing severe drought stress treatments were subjected to mRNA-Seq analysis.Functional annotations of these drought related genes are mainly involved in several biological aspects,such as drought tolerance,ethylene and Ca2+ signaling,lignin biosynthesis,osmotic adjustment,ROS scavenging,maintenance of cellular water and so on.In addition,qRT-PCR further confirmed the expression patterns of those genes involved in enhanced drought tolerance of idr1-1 as revealed by mRNA-Seq.4.Direct measurement of ethylene accumulation indicate that ethylene accumulation in idr1-1 mutant leaves are significantly higher than that in wild-type leaves after severe drought stress,suggesting that mutation of IDR1 leads to excessive accumulation of ethylene which in turn remarkably improves the drought tolerance of idr1-1 mutant.5.To better understanding the molecular mechanisms of enhanced drought tolerance in idr1-1 mutant,we used protein-protein interaction techniques to screen for candidate proteins that are able to interact with IDR1.By using firefly Luciferase Complementation Imaging(LCI)technique,we screened a collection of proteins which are involved in biosynthesis and signaling of ethylene,brassinolide(BR)and phospholipid D(PLD)for candidates interacting with IDR1.Our results revealed a direct interaction between IDR1 and OsERS2(a receptor of ethylene signaling).The result implying that IDR1 is presumably implicated in ethylene signal transduction.Taken together,our investigations preliminarily established a relationship between idr1-1 mutation and ethylene signaling,and will contribute to breed new rice cultivars that are of high drought resistance in the near future.