| Lindera communis has another name is Xiangguo and belonging to the family Rubiaceae, it is an evergreen broadleaf arbor or shrub, it is a fast-growing tree species which widely distributed in southern China, can be used as landscaping trees, the plant might serve as a species for conservation of soil and water as well as amelioration of soil. Its fruits and seeds contain rich oil, and they are a good raw material to extract lauric acid and produce biodiesel, it has huge prospects to development and application in China South and Southwest region. Cloning of fatty acid biosynthesis genes and functional identification of L.communis using molecular biology techniques, it help L. communis improve breeds. However, those studies have only addressed its fatty acid composition, biodiesel traits, seedling development, volatile oils, ecological functions, and afforestation, little is known about the regulatory mechanisms involved in the production of the saturated fatty acid rich oil in the developing seeds of L. communis. Molecular biology research on this species remains limited. FatB is a kind of important regulatory factors that biosynthesize fats in plant, and it is closely related to oils accumulation and ingredients. Therefore, we cloned L. communis FatB by RT-PCR and RACE, analyzed sequence structure, issue-specific expression, prokaryotic expression, and eukaryotic expression for functional identification, the main results are as follows:1) Because L. communis seed contain high oil, polysaccharides, polyphenols and other secondary metabolites, RNA extraction is difficult, no previous experiment can be learn to extract RNA from L. communis, we have used the modified CTAB method to successfully extract L. communis seed RNA, it can be used for follow-up biological experiments.2) Based on conserved amino acid sequences identified by the alignment of FatB proteins from other plant species, two degenerate oligonucleotide primers were designed, a full-length cDNA of L.communis FatB was cloned using RT-PCR and RACE for the first time, the nucleotide sequence of the cDNA is1788bp with an open reading frame (ORF) of1260bp, named LcFatB, NCBI’s registration number is:KF543781.3) Sequence analysis of LcFatB predicted a polypeptide of419amino acids, calculated molecular mass and predicted isoelectric point is46.09kDa and6.48respectively, LcFatB belongs to the family Acyl-ACP-TE, the deduced amino acid sequence showed61-73% identity to proteins in the FatB class of plant thioesterases, three residues (N315, H317, and C352) that are essential for TE catalytic activity are present in the mature protein, a phylogenetic tree based on the alignment of47TE sequences obtained from GenBank which showed a close relationship between LcFatB, UCFatB and CCFatB from L.communis, U. californica and C. camphora respectively.4) Expression of the LcFatB gene in developing seeds and vegetative tissues of L.communis was analyzed by qRT-PCR, transcripts were detected in all tissues examined, expression was highest in seeds and lowest in roots, LcFatB transcript levels were higher at75d (day after flowering) DAF than other stages of seed development after flowering30,45,60and90days.5) Predicted LcFatB protein has61amino acids in length chloroplast transit peptide using ChloroP1.1, after removing the transit peptide, the LcFatB gene was inserted into the T7-based expression vector pET-30a, the recombinant plasmid was transformed into BL21(DE3)△fadE that was deleted acyl-CoA dehydrogenase, SDS-PAGE analysis showed that a band of40.5kDa corresponding to the predicted size of mature protein. Gas chromatography analysis showed contents of the decanoic acid and lauric acid were increased significantly in transgenic bacteria, content of the lauric acid reached16.59%and is higher3.68-fold than control bacteria, content of the decanoic acid reached6.28%.6) To further understand the metabolic function of LcFatB in plant, the LcFatB gene was inserted into a plant binary expression vector pGreen-0029to construct the recombinant expression vector pGreen-LcFatB, it was transformed into Arabidopsis thaliana via Agrohacterium-mediated transfer methods and overexpressed under the control of the cauliflower mosaic virus (CaMV)35S promoter. The transgenic plants were selected in cultures with50ug/mL kanamycin and PCR tests were conducted to confirm that the LcFatB gene had been successfully integrated into the arabidopsis genome. Gas chromatography analysis of transgenic A. thaliana seed fatty acid composition found C12:0, C10:0increased significantly, reached2.32mg/g and0.99mg/g respectively. |
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