Construction And Functional Study Of Multiple Mutants Of Genes From Steroleosins (HSD) Family In Arabidopsis

Oil bodies are ubiquitous spherical oil storage organelles in almost all organisms.HSD(hydroxysteroid dehydrogenase)is a steroleosin localized at the surface of oil bodies.Six HSD homologs(HSD1-6)have been found in the genome of Arabidopsis thaliana.Although At HSD1 have been reported to be important in regulating growth and development,promoting or regulating hormone and salt stress response,but the genetic mechanism is not clear.Several HSD genes are closely linked on the chromosomes,so it is difficult to construct multiple mutants using traditional method.This is the possible reason of the poor research results of other HSD homologs.In this study,the CRISPR/Cas9 system and traditional crosses were used to edit HSD genes and constructed hsd multiple mutants in which several HSD genes were knocked out.Transgenic plants overexpressing HSD genes were also constructed.Further functional analysis of the HSD gene family were performed by analyzing the phenotypes.The results may ultimately be useful for the understanding the function of steroleosin in plant development.Preliminary research on the function of HSDs in wax synthesis has also been carried out.The study has important theoretical significance and practical application value.The work mainly includes the following aspects:1.Construction of hsd multiple mutantsUsing CRISPR/Cas9 gene editing technology and artificial hybridization technology,two hsd1/4/5/6 homozygous quadruple mutant lines,two hsd1/2/3/4/5homozygous five mutant lines and two possible hsd1/2/3/4/5/6 homozygous hexa mutant lines(under identification)were obtained.2.Phenotypic analysis of hsd multiple mutants2.1 Observation of growth and development phenotypes under normal conditions,ABA treatment and salt stressUnder normal culture conditions,the germination,plant growth and development of hsd2/3,hsd1/4/5,hsd1/4/5/6 and hsd1/2/3/4/5 multiple mutants were not significantly different from those of the wild type.Germination experiments of hsd2/3 and hsd1/2/3/4/5 mutant seeds on MS + ABA and MS + Na Cl medium showed that the hsd multiple mutants were more sensitive to ABA and Na Cl than WT.2.3 The morphology of oil bodies in hsd multiple mutant cells is significantly different from that of wild typeThe oil body Nile red staining results showed that the number of oil bodies in the seedling root cells of hsd1/2/3/4/5 mutant was significantly increased and the volume decreased compared with WT.The results of transmission electron microscopy showed that the oil bodies in the cotyledons of mutants were smaller and the edges were more irregular than that of WT.It suggests that HSDs protein play a certain role in oil body morphology and biogenesis.3.Construction of transgenic plants overexpressing HSD gene familyA total of 6 plasmids of 35SPro::HSD(1-6)-YFP were constructed,and transgenic Arabidopsis plants were obtained by inflorescence dip method.Homozygous HSD3-OE,HSD2-OE and HSD5-OE plants have been obtained;HSD1-OE,HSD4-OE,HSD6-OE have been infiltrated and T1 plants were obtained,and the screening work is in progress.4.Analysis of growth phenotype and oil body morphology of overexpressed transgenic plantsUnder normal culture conditions,the growth of HSD3-OE plants was significantly slower than that of wild-type plants.The results of oil body Nile red staining showed that the fluorescence signal in HSD3 OE was weaker and the number of oil bodies was less than that of WT.Compared with WT,oil bodies of HSD3 OE cells are larger and edges of oil bodies are smoother under transmission electron microscopy.It is worth noting that some oil bodies have entered the vacuole to degrade,indicating that the lipid mobilization in HSD3 OE plants was earlier than that of WT,which was consistent with the results of Nile Red staining.5.Functional analysis of HSDs in the process of wax synthesisOur previous work suggested that HSDs may participate in the wax synthesis process together with the key protein CER3 in the wax synthesis pathway in leaf epidermis.It is speculated that there may be an interaction between the two proteins.Construction of recombinant plasmids c LUC-CER1,CER3-n LUC,c LUC-HSD1-6.Through luciferase bimolecular complementation experiments,it was found that all six members of the HSD family interacted with CER3.Innovation points of this experiment:1.CRISPR/Cas9 gene editing and hybridization technology were used to construct multiple hsd mutants and transgenic lines overexpressing HSDs.Phenotypic analysis was perform and the morphology of oil bodies was observed by electron microscopy.It was found that HSDs protein may play a role in the morphology and biodegradable of oil bodies.Related results have not been reported.2.It was discovered for the first time that HSDs interacted with CER3,a key protein in the process of wax synthesis,indicating that HSDs may play a role in the synthesis of wax in the leaf epidermis of Arabidopsis.