Functional Study Of DIP2-an Interacting Protein Of The Rice Dwarf And Yellow Leaf Gene DRT1

Rice(Oryza sativa L.)is one of the most important crops,accounting for more than 40% of global food production.According to previous study,more than 90% of crop yield comes from photosynthesis.Therefore,improvement of the photosynthesis ability of crops has been suggested as an effective way to increase yields.In the previous study in the laboratory,the drt1 mutant was obtained by ethyl methanesulfonate(EMS)mutation technology.Through map cloning and other methods,it was revealed that DRT1 is a cytoskeletal protein,which is involved in the regulation of plant height and leaf color.In addition,it was found that DRT1 is involved in the regulation of chloroplast movement,but its specific molecular mechanism is not clear.In order to further explore the function of DRT1,the DRT1 interaction protein DIP2 was obtained by yeast two-hybrid screen library.It was found that DIP2 is a translation elongation factor.Based on the interaction between DRT1 and DIP2,this thesis conducts an in-depth study on the function of DIP2.The results are as follows:1.DIP2-N interacts with DRT1-FH2.The interaction between the DIP2-N and the DRT1-FH2 was confirmed by yeast two-hybrid experiment and firefly luciferase fragment complementation imaging experiment,laying a foundation for the subsequent functional studies of DIP2 and DRT1;2.DIP2 was located in chloroplast envelope.Subcellular localization and chloroplast subfraction separation showed that DIP2 was expressed in the outer membrane of chloroplast.The tissue-specific expression pattern of DIP2 indicated that DIP2 was specifically expressed in chloroplast-containing tissues;3.The phenotypes of wild type and dip2-ami mutant were significantly different.In order to further study the function of DIP2,the dip2-ami mutant was obtained by micro RNAs silencing technology,and two strains with great phenotypic differences were screened,named dip2-ami-w,dip2-ami-s.The plant height of dip2-ami was lower than that of the wild type in the whole growth cycle,and the difference between dip2-ami-s and wild type was particularly obvious.In the early stage of growth and tillering,the leaves of dip2-ami-s were obviously yellow,and the leaf color gradually tended to light green from tillering stage to mature stage.Compared with the wild type,the expression of chlorophyll synthesis genes and chloroplast development genes was significantly suppressed in dip2-ami mutant,indicating that DIP2 may be involved in chloroplast-related activities;4.DIP2 and its Arabidopsis homologous gene At DIP2 are involved in the accumulation of chloroplast movement.Chloroplast movement experiments showed that dip2-ami mutants and Atdip2 mutant were damaged in varying degrees in the accumulation reaction,which showed that DIP2 was involved in the regulation of chloroplast movement,and its homologues were evolutionarily conservative.The DIP2 overexpression vector fused with His-tag was transferred into the Atdip2 mutant,it was found that the transgene restored the defect of Atdip2 mutant in accumulation reaction,which further confirmed that DIP2 was involved in the regulation of chloroplast movement;5.Heat hypersensitivity of dip2.In the study of DIP2 involved in the response to heat stress,it was found that dip2-ami was more sensitive than the wild type,whether in vitro leaf heat treatment or in vivo heat treatment.Under heat stress,the ability of dip2-ami to scavenge reactive oxygen species was weakened,and the transcriptional activation of Os Hsf A2 and its target genes in the mutant was significantly inhibited,thereby reducing the heat tolerance of dip2-ami mutant.