Genome-Wide Identification And Functionalanalysis Of The Oleosin Family In Tigernut (Cyperus Esculentus L.)

Cyperus esculentus L.,originating from Africa,is the only new oil crop known to accumulate high levels of oil in tubers.Revealing the molecular mechanism of oil accumulation in Yousha bean tubers and exploring the involvement of tuber oil accumulation not only increases molecular understanding of oil metabolism and regulation outside of seeds,but also creates conditions for efficient production of oil using nutrient tissues such as tubers and roots.Triacylglycerol(TAG)is the main component of vegetable oil,which is encapsulated by phospholipid monolayer and oil body related proteins to form liposomes and stored in the cytoplasm.Oleosin(OLE)is a type of small molecule protein(14-30 k Da)embedded in oil bodies,which plays an important role in the formation,stability,and degradation of oil bodies.To reveal the biological function of OLE in oil accumulation in soybean tubers,this study aims to systematically identify the OLE gene using the genome sequence of soybean,analyze its expression characteristics,reveal the subcellular localization and protein interaction patterns of the protein,explore the regulatory effect of transcription factor Ce WRI1 on CeOLE,and explore its function in oil accumulation using two systems:tobacco transient overexpression and Arabidopsis stable overexpression.The specific results are as follows:1)A total of 6 OLE genes were identified from the genome of the oil bean,named CeOLE1–6.Although there are significant differences in overall sequence similarity between them,they both contain highly conserved oleosin domains.Based on sequence characteristics and evolutionary relationships,these 6 genes can be classified into three groups: U,SL,and SH,each containing 1,2,and 3 members.Collinearity analysis showed that CeOLE2 and CeOLE3 were located in the collinearity region of the genome,presumably originating from the whole genome duplication;In comparison,CeOLE4 and CeOLE5 have higher sequence similarity,originating later and originating from scattered duplication.In addition,except for CeOLE5 and CeOLE6,other CeOLE genes have collinearity homologous genes in rice,suggesting that their functions are conservative.Interestingly,although the OLE gene usually does not contain an intron,CeOLE5 has obtained an intron.Six gene coding regions were cloned using RT PCR technology.Sequence analysis showed that CeOLE1 encodes 152 amino acids,CeOLE2 encodes 146 amino acids,CeOLE3 encodes 149 amino acids,CeOLE4 encodes 156 amino acids,CeOLE5 encodes 143 amino acids,and CeOLE6 encodes 154 amino acids.The analysis of physicochemical properties showed that the theoretical molecular weights of the six proteins were15.57,15.89,15.14,15.92,16.25,and 14.33 k Da,respectively.The isoelectric points were 9.46,9.90,9.63,9.89,10.19,and 10.12,respectively.The total average hydrophobic index was 0.595,0.318,0.323,0.314,0.277,and 0.715,and the lipid solubility index was 112.95,92.05,107.13,99.61,101.94,and 112.93,indicating that they are all alkaline hydrophobic proteins.2)Expression analysis showed that six CeOLE genes were mainly expressed in young leaves,mature leaves,leaf sheaths,roots,stolons,buds,and tubers;During the development of tubers,the expression level of CeOLE genes gradually increases with development,with CeOLE2 and CeOLE5 having the highest expression abundance and being the main effector genes.3)The fusion expression vector of six genes and GFP was constructed,and the tobacco leaves were transiently transformed through Agrobacterium mediated transformation.The confocal microscope observation showed that the six proteins were located in the endoplasmic reticulum,in addition to the oil,and also located in the endoplasmic reticulum,highly coincident with the marker protein RFP-HDEL of the endoplasmic reticulum.The interaction between the two main proteins CeOLE2 and CeOLE5 was further analyzed using bimolecular fluorescence complementary technology.The results showed that the two proteins could not only form heteropolymers,but also homopolymers.4)The promoters of CeOLE2 and CeOLE5 were isolated,and the dual Luciferase report experiment showed that the transcription factor Ce WRI1 activated both,although only the promoter sequence of CeOLE2 contained AW boxes;Compared to CeOLE5,Ce WRI1 has a stronger activation effect on CeOLE2.5)Six overexpression vectors of genes were constructed,and their transient overexpression in tobacco significantly increased the TAG content of leaves,which preliminarily confirmed that these genes have oil regulation functions.The study also obtained overexpressed Arabidopsis plants with 6 genes.q RT PCR showed that the target gene was expressed in the leaves of T2 generation Arabidopsis,and the thousand seed weight of T2 generation Arabidopsis seeds was slightly increased compared to the wild-type;The identification of T3 generation of Arabidopsis is still ongoing.To sum up,the research system has completed the whole genome identification of the OLE gene family,and found that it is mainly expressed in oil producing tubers;Its coding protein locates in the oil body and endoplasmic reticulum of tobacco leaves,and acts in the form of homologous and heterogeneous polymers;Its promoter is regulated by the transcription factor Ce WRI1;Transient overexpression of genes can increase the TAG content in tobacco leaves.This result lays the foundation for further elucidating the mechanism of oil accumulation in the tubers of Camellia oleifera,and also creates conditions for future genetic improvement.