Continued Transcriptome Sequencing Analysis And Cloning And Expression Of The Oil Body Protein Oleosin Gene

Euphorbia lathylris L.(Euphorbiaceae)is an energy plant with great exploitation potential for the high oil content in seeds and the compositon of fatty acids with the desired diesel substitutes.Oil body is the major organelle for lipids storage in the seeds.The size and oil content of oil body are closely related to the oleosins located on the surface.In this paper,the transcriptomics of E.lathylris,cloning and expression of oleosin genes,extraction and fatty acids composition of oil body were studied This study will provide theoretical references for molecular biology research and high oil breeding of E.lathylris.The main contents and results are as follows:1.E.lathylris organs,including roots,stems,leaves,flowers,seeds in early,mid-term and late development,were sampled after the repectively treatments of 10 ?M ABA,200 mM NaCl and 10 ?M GA on the plant for 12 h.The mixing sample was prepared by mixing all treated and untreated samples,and applied for transcriptome sequencing using Solexa sequencing technology.A total of 34,732 Unigenes were generated with a data size of 28.49 Mb.Funtional assignments were obtained by comparing with public genetic databases,such as NCBI,NR,SWISS-PORT,TREMBL,CDD,PFAM,KOG,KEGG,GO.In KOG,a total of 10,508 Unigene were assigned to 25 groups,the top 3 functional categories were general funtion prediction only,signal transduction mechanisms and posttranslational modification,protein turnover,chaperones.In KEGG,3079 Unigene gene were assigned to 329 pathways.In GO,a total of 19328 Unigene were assigned to biological process,cellular component,and molecular funtion.2.Oleosin genes of E.lathylris(EIOLE1-EIOLE4)were obtained based on the transcriptome sequencing.The encoded proteins(ElOLE1-ElOLE4)were used for bioinformatic analysis.The four predicted oleosins were low molecular proteins with an average molecular weight of 14.98 kDa.The proteins showed high isoelectric point and hydrophobicity.Using online analysis,their subcellular localization were on the plasma membrane.On the basis of homology analysis of amino acids by comparing with other oleosin famlies,El OLE1-ElOLE4 contained the highly conserved "Proline Knot" motif-"PX5SPX3P",and were highly homological with Euphorbiaceae plants.By alignment with OLE family of model plant Arabidopsis,ElOLEl,ElOLE2,ElOLE3 regarded as S-type OLE protein,and ElOLE4 regarded as SM-type OLE protein.3.Using RT-PCR and fluorescence quantitative PCR,ElOLE genes expression were detected in different organs with treated or untreated.The results showed that:EIOLE1,EIOLE2 and EIOLE4 were expressed in all organs,while EIOLE3 were mainly expressed in the seeds.During seed germination,the expression levels of the four genes in seed were higher than that in roots,stems and leaves.EIOLE1 and E1OLE2 had higher expression levels.NaCl promoted the expression of EIOLE1 and ElOLE4 in stem,while ABA and NAA treatment had no effect on the expression of the four ElOLE genes.The recombinant expression vector pET30-(ElOLEl-ElOLE4)were constructed in vitro,and expressed in Escherichia coli BL21.By SDS-PAGE,the recombinant proteins were intracellularly expressed with the molecular weights of about 18 kDa,which were greater than the deduced due to the fused expression with sequence tags.4.Using gas chromatography-mass spectrometry(GC-MS)technology,the composition and content of fatty acids from seed and seed oil body were measured in E.lathylris from two origins(Jiangsu Haian and Henan Zhuji).The results were as follows:two origin E.lathylris seed oil body fatty acids were mainly oleic acid(83.32%and 73.99%)and palmitic fatty acids(10.56%and 15.97%),linoleic acid and stearic acid were also detected with higher content.Two kinds of seed fatty acids were mainly oleic acid(82.04%and 75.63%)and palmitic acid(6.00%and 11.75%).Significant difference were found in content levels.The content of oleic acid and palmitic acid were negatively correlated,namely that oleic acid content was high while palmitic acid content was low in seed.The essential fatty acids of the oil body isolated from Haian and Zhuji seeds which polar lipids and neutral lipids were octadecenoic acid(oleic acid,an unsaturated double bond),followed by a palmitic acid.The neutral lipids were similar,while polar lipids were quite different,especially octadecenoic acid and palmitic acid,and both showed a negative relationship.