| Plasma membrane NADPH oxidase(PM-NOX)plays an important role in regulating plant growth and responding to various biotic and abiotic stresses.In recent years,multiple NADPH oxidases have been reported in rice,but their function is still unknown.Previous studies have found that the expression levels of OsNOX3 and OsNOX5 in rice are affected by various abiotic stresses and hormone treatments.This study focused on the roles of OsNOX3 and OsNOX5 in rice growth and development and abiotic stress responses.Their promoter activity,protein subcellular localization and interaction with two malectin receptor-like proteins(OsMLPK63 and OsMLP15)were investigated.In addition,transgenic plants that OsNOX5 was overexpressed or knocked down by RNAi in rice were generated.The main results obtained are as follows:1.Cloning of OsNOX3/5 genes and their promoters: Firstly,the CDS sequences of OsNOX3 and OsNOX5 genes were cloned,which are 2532 bp and 2856 bp in length respectively,encoding proteins containing 843 and 951 amino acids,respectively.Secondly,a 2000 bp upstream of the transcription start site for each cloned genes was cloned as the OsNOX3/5 promoter fragments,respectively.The promoter sequences were then analyzed using the bioinformatics tools to find the plurality of biotic,abiotic stress and/or hormone responsive elements.2.Subcellular localization and expression pattern analysis of OsNOX3/5: The subcellular localization vector was constructed and transiently expressed in tobacco epidermal cell and Arabidopsis protoplasts,respectively.It is found that OsNOX3 and OsNOX5 are localized in the plasma membrane,consistent to the reported NOX family members.The tissue expression pattern of OsNOX3/5 was analyzed by qRT-PCR.The results show that the expression levels of OsNOX3 are higher in the leaves of rice at the seedling and tillering stages,while the expression levels of OsNOX5 are the highest in the leaves of the plants at the tillering stage even though both the OsNOX3/5 genes are all mainly expressed in the leaves at every development stage.In addition,the expression of OsNOX3 and OsNOX5 are strongly induced by NaCl(salt stress)and 20% PEG-6000(simulated drought)and some hormones(ABA,MeJA,GA,SA)treatments,while the expression levels kept no significantly changes under MV(oxidative stress)treatment.The results from the dual luciferase reporter assay also suggest that ABA and MeJA treatments did enhance the activity of the OsNOX3 and OsNOX5 promoters.These results indicate that OsNOX3 and OsNOX5 participate in multiple abiotic stress responses of the plant.3.Generation of OsNOX5 transgenic plants and phenotypic analysis: Construction of OsNOX5 overexpression and RNAi vectors,as well as OsNOX3 and OsNOX5 Crispr-Cas9 knockout vectors,T2 generation OsNOX5 overexpression and RNAi vector transgenic materials were obtained.We observed that OsNOX5 overexpression plants are little bit higher than the wild type.Their seeds are also wider and longer than those of the wild type plants.These results have laid a well foundation for further research on the function of OsNOX5 gene in rice growth and development.4.Preliminary study on the interaction between OsNOX3/5 and two malectin-like domain containing proteins: OsMLPK63 and OsMLP15 are two malectin proteins which involve in the ROS production and signaling in rice.In this study,we preliminary proved that both of the OsMLPK63 and OsMLP15 can interact with OsNOX3 and OsNOX5,providing a new idea to study the signal network associated with OsNOX3 and OsNOX5.The results will be verified by CO-IP,yeast two hybrid and other test methods in the future.As a whole,in this study,two rice NOX family members,OsNOX3/5,and their promoters were cloned and their expression characteristics were investigated.OsNOX3 and OsNOX5 are involved in the growth regulation and abiotic stress responses.It was initially found that OsNOX3/5 interacts with two proteins of the Malectin-like family,OsMLPK63 and OsMLP15.Further studies will need to clarify their functional molecular mechanisms in details. |
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