Functional Characterization Of Two Stressrelated Genes OsEBP89 And OsRMT1 In Rice

With the development of the world economy and the emission of greenhouse gases,the global temperature rises and extreme weather occurs frequently.Drought,high temperature,flood and chilling injury are common adversity factors that bring serious threat to crop production.Rice is the staple food in China,and it is of great significance to ensure the high and stable yield of rice for China's food security.However,during the rice production from sowing to harvesting,it is vulnerable to various abiotic factors such as drought and high temperature.Many important genes involved in drought and high temperature resistance of rice have been identified.However,it is rarely reported thatabiotic these genes were applied in rice breeding because of its small effect.Therefore,the stress tolerance genes should be further explored in order to deeply understand the resistance mechanism of abiotic stress.In this study,the function of two genes was further analyzed.One was OsEBP89 encoding AP2/ERF transcription factor,the other was OsRMT1 encoding E3 ubiquitin ligase with zinc finger domain.OsEBP89 was derived from the interaction network of OsSn RK1,and OsRMT1 was a candidate gene derived from the drought-resistant QTL interval of chromosome 4 mapped using a recombinant inbred lines population derived from the cross between IRAT109 and Zhenshan 97 B in our laboratory.In this study,the function of the two stress-related genes OsEBP89 and OsRMT1 were evaluated,and the mechanism of plant resistance to abiotic stress was further understood,providing new genetic resources for rice stress tolerance breeding.The specific research results are as follows:1.RT-q PCR analysis showed that at the seedling stage,OsEBP89 was mainly expressed in roots,while the expression level of OsEBP89 in leaves and stems was very low.In flowering stage,the expression level of OsEBP89 in roots and stems was significantly increased,indicating the expression of OsEBP89 gene has spatio-temporal expression specificity.The expression of OsEBP89 gene was inhibited under 20% PEG and ABA treatments.On the contrary,the expression of OsEBP89 was induced by ET and submergence.Subcellular localization assay displayed that OsEBP89 located in the nucleus.We constructed the OsEBP89 overexpression vector and CRISPR/Cas9 gene-edited vector,and then transformed the two constructs into japonica varieties Nipponbare,respectively.Finally,the 26 individuals of overexpression plants and 3 homozygous mutants were acquired at T0 generation.2.Transgenic rice was treated with 200 m M mannitol,20% PEG osmotic stress at the bud stage and seedling stage respectively,and treated with drought stress at the booting stage.The results indicated that OsEBP89 knockout mutant exhibited enhanced drought resistance compared to WT at these three stages,while the overexpression plants showed the opposite phenotype.3.For the three-leaf OsEBP89 transgenic rice plants after 20% PEG treatment,it was found that the content of MDA and H2O2 of OsEBP89 knockout lines were lower than those of WT,but the Pro and SOD activity of OsEBP89 knockout lines were significantly higher than those of WT.It indicated that the oxidation level and damage degree of membrane lipid of the OsEBP89 knockout lines were lower than those of WT and osmotic protective substance and ROS scavenging ability of OsEBP89 knockout were significantly higher than those of WT.The water loss rate of detached leaves at booting stage of OsEBP89 overexpressed lines was higher than that of WT and knockout plants.4.Transcriptomic analysis for the WT and knockout plants was conducted at the4-week-old seedling stage under normal conditions and 20% PEG treatment.Transcriptome sequencing analysis showed that four GO items(i.e.response to stress,oxidation reduction,response to water,and response to stimulus)were associated with stress response.8 candidates were selected from the 4 GO items,which were Os03g0285700(OsAPX1),Os03g0358100(Glutathione Peroxidase domain containing protein),Os02g0527300(OsHsf A3),Os04g0423400(OsASR2,Anabscisic Acid-stress-ripening-inducible 2 Protein),Os05g0211100,(OsCYP51G3),Os05g0455500(OsP5CS),Os01g0256500(SENESCENCE-ASSOCIATED GENE 21)and Os08g0120600(Fructose-Bisphospate Aldolase isozyme).It has been reported that OsEBP89 is capable of combining the promoter region GCC box and GCC-like box,so the promoter of these 8 genes was analyzed.The results showed that there were GCC box cis-elements in the promoter region of OsAPX1 and Senescenceassociated gene ARG2,GCC-Box and GCC-like box in the promoter region of OsP5 CS and OsHsf A3,suggesting that these 4 genes may act as downstream target genes of OsEBP89 transcription factor.5.The seeds of OsEBP89 transgenic plants were flooded during seed germination and the results showed that germination rates of OsEBP89 knockout lines were higher than that of WT and overexpression lines under submergence for 4 days.Further,the bud length of OsEBP89 knockout lines was significantly higher than that of WT and overexpressed plants under submergence for 7 days.It suggested that OsEBP89 negatively regulated rice submergence tolerance.6.The interaction between different OsEBP89 truncated segments and OsSnRK1 were analyzed by yeast two hybrid assay.The results showed that only the full length of OsEBP89 could interact with OsSn RK1,suggesting that OsEBP89 requires a complete structure to maintain interaction with OsSn RK1.Pull down experiment indicated that OsEBP89 could interact with OsSn RK1 in vitro.Luciferase complementary experiment in tobacco leaf further demonstrated that OsEBP89 interact with OsSn RK1 in vivo.The phosphorylation experiment in vitro showed that OsSn RK1 can phosphorylate OsEBP89 transcription factors.7.In order to comprehensively study the function of OsRMT1,the CRISPR/Cas9 gene editing system was used to construct OsRMT1 knockout vector.Then the OsRMT1 knockout vector was transformed into the Nipponbare.27 plants with gene editing were obtained and 3 homozygous mutation types were included.8.The expression of OsRMT1 was induced by high temperature and salt stress.OsRMT1 overexpressed plants exhibited enhanced high temperature and salt tolerance,while the OsRMT1 RNAi lines were in the opposite way.The survival rate of the OsRMT1 knockout mutants was lower than that of the knockdown lines.It indicated that OsRMT1 postively regulated the tolerance to high temperature.Under high temperature stress of 45? for 1 d at seedling stage,there was no significant difference in ABA content,while JA content in the mutants was significantly higher than that of WT plants.9.We fused OsRMT1 gene into PGBKT7-BD vector and screened the c DNA library from rice leaf.We obtained 6 interaction proteins.The interaction relationship between these 6 proteins and OsRMT1 was further confirmed by the bimolecular luciferase complementary experiment in Nicotiana tabacum L.The six genes are Os03g0390900(expressed protein),Os04g0376700(ER lumen protein retaining receptor),Os04g0650000(oryzain alpha chain precursor),Os08g0104600(2Fe-2S iron-sulfur cluster binding domain containing protein),Os10g0419500(1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase protein),Os11g0128800(homoserine dehydrogenase).The expression profiles of these 6 genes were analyzed at specific time points under high temperature stress of 45?.Among them,4 genes were induced by high temperature,but 2 genes were inhibited.Subcellular localization showed that these interacting proteins were expressed in both the nucleus and cytoplasm,which were similar with OsRMT1.