| Elaeagnus mollis is a unique woody oil tree species in China.Its seed oil is rich in unsaturated fatty acids and vitamin E,which has high nutritional and health value.However,due to the lack of basic research in the field of molecular biology and the limited research on the biosynthesis pathway of vitamin E,the utilization of E.mollis with high vitamin E content in seed oil is limited to further improve vitamin E content,especially α-tocopherol content,by genetic engineering.In this study,the full-length transcriptome information of E.mollis was obtained through the third-generation sequencing.Through gene annotation,genes may relate to vitamin E synthesis were mined.Furthermore,a key gene named γ-TMT which regulating α-tocopherol synthesis was cloned and transformed into Arabidopsis thaliana for functional verification.The main results are as follows:The full-length transcriptome from the mixed samples(leaves,flowers,seeds and pericarps)of E.mollis was sequenced by pacbio sequel platform.The results showed that 135,627 high-quality transcripts were obtained,with an average length of 2.128 bp and N50 of 3,208 bp.By functional annotations in public databases,such as NR,NT,GO,KOG,KEGG,Swiss-Prot and Pfam,a total of 122,325 unigenes were annotated in at least one database,and 28,789 unigenes were annotated in all databases,accounting for 91.19% and21.23% of the total,respectively.In addition,13,346 CDS sequences with complete open reading frame were predicted.20,662 SSR(2～6 bp)loci were detected by MISA,with a mean distribution distance of 13.97 Kb between two loci in the full-length transcriptome.Furthermore,8,047 transcription factors which belonging to 78 transcription factor families were identified by iTAK software and 18,118 lncRNAs were all predicted by CNCI software,CPC software and Pfam database.Combined with the annotation in KEGG pathway,NR and Swiss-Prot databases,73 unigenes encoding 8 enzymes which may related to the synthesis of vitamin E in E.mollis were identified.Based on the unigene which was annotated as γ-TMT in the full-length transcriptome analysis,the specific primers were designed which were used to clone the Emγ-TMT gene in E.mollis.As a result,the Emγ-TMT gene is 1,047 bp in length,which encode 348 amino acids and with a CG content is 46.42%.The bioinformatics analysis showed that the molecular weight of the protein was 38,746.09 D,which was composed of 20 amino acids.Emγ-TMT protein belongs to hydrophilic protein,and there are some phosphorylation sites and disulfide bonds,but there is no transmembrane region.In addition,the analysis shows that Emγ-TMT is a non-secretory protein located on chloroplast,which belonging to the methyltransferase family.The prediction results of the secondary and tertiary structure of the protein showed that Emγ-TMT are composed of α-spiral,irregular curl and extension chain.By evolutionary analysis,the Emγ-TMT protein was similar to the γ-TMT from rubber tree,which indicating that they have relatively close relationship in possible.To further verify the function of Emγ-TMT,Emγ-TMT was overexpressing in Arabidopsis.The results showed that seven positive clones were identified.Of them,γ-TMT-2#,γ-TMT-5#,γ-TMT-7# were selected to determine the content of vitamin E and the components of tocopherol in seeds.The results showed that the content of α-tocopherol was about 4.71～5.40mg/100 g,the average content was 5.4 times of wild type.However,the total content of vitamin E was not increased much,which was about 1.09 times higher than that of wild type.It was indicated that Emγ-TMT promoted the content of α-tocopherol significantly,but wasn’t the promotion of total content of vitamin E.In conclusion,the full-length transcriptional sequence of E.mollis is carried out,which provides data support for further molecular biology research,the cloning and transformation analysis of Emγ-TMT provides important basis for improving tocopherol content by usingγ-TMT through gene engineering technology. |
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