| The oil bodies are the main organelle of storing oil in plants.they are sphere filling triacylglycerol formed by half unit membrane(TAG).The half unit membrane composes by monolayer phospholipid molecule and oil body binding protein.Oil bodies provide carbohydrate and energy for seed germination and seedling development through Fatty acid ?-oxidation process of glyoxylic acid cycle before the photosystem establishment.They also play an important role in abiotic stress response of plants.Oil bodies binding proteins include oleosin,caleosin and steroleosin.Oleosin is widely studied in three kinds of proteins,but caleosin and steroleosin are less studied.The caleosin family participates in many processes,such as stable oil body structure,affecting oil synthesis and degradation,signal transduction regulated by calcium ion,membrane fusion and fat body fusion,and abiotic stress etc in Arabidopsis thaliana.In Arabidopsis thaliana,there are eight members in the caleosin family,which are divided into H-Caleosin and L-Caleosin.H-Caleosin includes At CLO4-7,L-Caleosin including At CLO1-3 and At CLO8.In this study,Arabidopsis thaliana was used to construct caleosin family members mutants by using CRISPR-Cas9 gene editing technique and hybridization.The functions of the family were also discussed.The main tasks are divided into two parts:1.Study about multiple mutants of caleosin gene family.1.1 Construction of multiple mutants of caleosin gene family1.1.1 Construction of double mutantsThe CRISPR-Cas9 gene editing technique was used to edit the highly homologous At CLO5 and At CLO7 genes in Arabidopsis thaliana.Two sg RNA acting on two sites of the two genes were designed.and the large fragment deletion lines were found.A clo5/7-1 homozygous line was obtained.According to the same principle,clo4/6-1 and clo4/6-2 were obtained.Cas9 have been removed in all mutants.1.1.2 Construction of four genes mutantsclo5/7-1 and clo4/6-1 or clo4/6-2 were hybridized,then F2 generation was screened to identified clo4/5/6/7,clo4/5/6/7-1 and clo4/5/6/7-2 had been obtained in F2 generation.1.1.3 Construction of six genes mutantsOn the basis of clo4/5/6/7-1,two homozygous lines clo1/2/4/5/6/7-1 and clo1/2/4/5/6/7-2 were constructed through knockout of At CLO1 and At CLO2.The homology of At CLO3 and At CLO8 was low.By designing two sg RNA role respectively in the two genes,based on clo4/5/6/7-1 gene we knockout At CLO3 and At CLO8,.A pure and isolated clo3/4/5/6/7/8-1 lines had been obtained in which Cas9 have been removed.1.1.4 Construction of eight genes mutantsTwo mutants,clo1/2/4/5/6/7-1 and clo3/4/5/6/7/8-1 were hybridized,clo1/2/3/4/5/6/7/8-1 had been obtained in F2 generation.1.2 Functional study of caleosin gene family1.2.1 Phenotypic observation of L-CaleosinWe continuously observed the growth phenotype of L-Caleosin mutant and Col-0 during the whole development process under long sunshine condition.There was no significant difference between mutant and wild-type during the whole growth and development under normal conditions.1.2.2 Germination experiment of L-Caleosin mutants in stress environmentsIn this study,Col-0,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 were used to investigate the germination response of L-Caleosin mutants under stress.Under the ABA treatment,The results showed when treated with 1 ?M ABA,The germination of mutants was more sensitive to ABA than that of Col-0.There was no significant difference between the mutant and Col-0 under different concentrations of mannitol,Na Cl and different degrees of low temperature stress.1.2.3 Roots response of L-Caleosin mutant roots to stressAfter 4 days of normal growth in 1/2 MS,Col-0,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 seedlings were transferred to medium containing 150 m M mannitol,50 ?M ABA or 100 m M Na Cl,respectively.The root length of seedlings was counted after 6 days.Compared with Col-0,the root elongation of clo4/5/6/7-1 was shorter than Col-0 in mannitol medium.clo4/5/6/7-1 was more sensitive to mannitol.Compared with Col-0,the elongation of clo4/6-1 roots was longer than Col-0 in Na Cl medium.clo4/6-1,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 were more sensitive to to ABA compared with Col-0 in mannitol medium.1.2.4 Water loss test of L-Caleosin mutant leaves in vitroCol-0,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 was cultured for 22 days,and the water loss test on the leaves was carried out in vitro.Three groups of parallel tests were set up.The results showed that clo4/6 had certain tolerance to drought.Compared with Col-0,the clo4/6-1 water loss was less,1.2.5 Sucrose dependence test of L-Caleosin mutantsThe roots' length of Col-0,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 was counted after 6 days under 1/2 MS without sucrose medium.There was no significant difference in root elongation between mutants and Col-0.1.2.6 Extension length of hypocotyl of L-Caleosin mutants under dark treatment with or without sucroseThe hypocotyls' length of Col-0,clo5/7-1,clo4/6-1 and clo4/5/6/7-1 was counted after 8 days in dark condition under 1/2 MS without sucrose medium,The results showed there was no difference in the elongation of hypocotyls between mutants and Col-0.1.2.7 Observation of L-Caleosin mutant using Nile Red stainingThe seed coat of Arabidopsis thaliana seed was removed under stereoscopic microscope.The lipid mobilization of germinated seeds was observed by laser confocal microscope with 1 ?g/ml Nile red staining for 0 h,24 h,48 h,and 72 h.The results showed there was no significant difference between clo5/7-1,clo4/6-1,clo4/5/6/7-1 and Col-0.1.2.8 Experiment on sucrose dependence of Caleosin mutantsCol-0,clo4/5/6/7-1 and clo1/2/4/5/6/7-1 was cultured on sucrose-free medium of 1/2 MS for 5 days,and the difference was most significant at 5 days.The root length of mutants was counted.And compared with Col-0,the growth of clo1/2/4/5/6/7-1 was slower than that of clo1/2/4/5/6/7-1.1.2.9 Quantitative analysis of fatty acids in Caleosin mutantsCompared with Col-0,the total fatty acid content of clo5/7-1,clo4/6-1,clo4/5/6/7-1 and clo1/2/4/5/6/7-1 was decreased.Study on the missing 95 mutant2.1 Phenotype analysis of 95 mutant95 mutant was obtained in the process of screening clo4/6.The mutant grew slowly,and had a long life cycle,small leaf area,late bolting,irregular flower,and insufficient stamens,which result insufficient pollination and insufficient seed.2.2 Localization of the 95 geneThe mutant 95 was hybridized with the wild type of Col-0 and Ler of different ecotypes,and the isolate line of F2 generation was obtained.The phenotype segregation ratio of F2 generation was 3:1.It was found that there was a linkage between the missing site and At CLO4/6 by rough localization analysis,and fine-mapping was in progress.Innovation of this thesisIn the genome of Arabidopsis thaliana,there are eight members of the oil body caleosin genes.Among them,At CLO4,At CLO6,At CLO5 and At CLO7 gene are closely linked and the gene sequences are highly homologous.It is difficult to obtain mutants with polygenes knockout simultaneously by traditional T-DNA insertion and hybridization.In this study,CRISPR-Cas9 gene editing technique and traditional hybridization method were combined to obtain multiple gene mutants of caleosin family members.This paper lays a foundation for further study on the function of caleosin gene family in plant growth and development. |
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