Cloning And Function Expression Of Long-chain Acyl-CoA Synthetase Gene In Vernicia Fordii

Vernicia fordii belongs to Euphorbiaceae,which is one of China's four major woody oil species.The oil content of tung oil seeds is as high as 60%,individual more than 70%,tung oil fatty acid content mainly contains stearic acid,palmitic acid,eleostearic acid,linoleic acid,linolenic acid and oleic acid.Eleostearic acid is unique fatty acid to tung oil,accounting for about 80%of the total fatty acid content,on the physical and chemical properties of tung oil play a decisive role.Tung oil has good heat resistance,cold resistant,acid and alkali resistance,insulation and anti-corrosion properties,is a kind of environmental friendly chemical raw materials,can be used to make coating,paint,medicine,etc.Tung oil is also the important raw material for production of biodiesel.In this study,different tissues,organs and different seed development stages of tung cultivar'PUTAOTONG' seed were used as materials,on the basis of digitized transcriptome database,The full-length cDNA was cloned and analyzed by VLACS4,VfLACS6 and VfLACS7 genes in the long chain lipoxygenase A synthetase family gene.For further research of LACS gene families and provide theoretical basis for tung fatty acid metabolism.The main results are as follow:1.The cDNA cloning and bioinformatics analysis of VfLACS6 and VfACS7 gene in tung LACS gene families.The 'PUTAOTONG' variety nearly mature seeds were used as the material,in the experiment have been build tung transcriptome database,based on the conventional PCR amplification cloning technique,The cDNA sequence of the two member genes of the long-chain fatty acyl-CoA synthase gene family was obtained.Respectively named VJLACS6(GenBank number:KY012243)and VfLACS7(GenBank number:KY012244).The CDS of VfLACS6 is 2085bp which codes a predicted protein of 694 amino acids.Sequence analysis showed that VfLACS6 had four transmembrane domains,the protein molecular weight is 76.11kDa and isoelectric point is 6.55.The CDS of VfLACS7 is 2088bp which codes a predicted protein of 695 amino acids.Sequence analysis.showed that VfLACS7 had eight transmembrane domains,the protein molecular weight is 77.30kDa and isoelectric point is 6.64.Identified by amino acid comparison found that VfLACS6 and VfLACS7 genes have AMP binding link domain conservative sequence(ICYTSGTGDPK),shows that the two genes belonging to the family of LACS.2.The prokaryotic expression of VfLACS4 VfLACS6 and VfLACS7 genes in LACS gene family.On the basis of the VfLACS4,VJLACS6 and VfLACS7 gene cDNA sequence,and the conventional carrier to build technology,the recombinant expression vector,including pET30a-VfLACS4,pET30a-VfLACS6 and pET30a-VfLACS7,were conducted successfully by the pET30a and BL21(DE3)prokaryotic expression system,and obtained the target protein,whose apparent volume of about 74 kDa,76 kDa and 77 kDa.3.The yeast expression of VfLACS4 VfLACS6 and VfJLACS7 genes in LACS gene family.On the basis of the VfLACS4,VfLACS6 and VJLACS7 gene cDNA sequence,and the conventional carrier to build technology,the recombinant expression vector,including pYES2-VfLACS4,pYES2-VfLACS6 and pYES2-VfLACS7,were conducted successfully by the pYES2 yeast expression system,and drawed the corresponding growth curve,obtained the corresponding target protein of VfLACS6 and VfLACS7 genes.4.The research of interactive relations between VfLACS6 and VfLACS7 proteins.On the basis of the VfLACS6 and VJLACS7 gene cDNA sequence,the corresponding bait carrier pGBKT7-VfLACS7 and library carrier pGADT7-VfLACS6 had successfully built,Transferred into the yeast strains AH 109.Cultured by culture medium and X-ray gal chromogenic reaction experiment,it finds VfLACS6 and VfLACS7 have no interactive relations,it showed it was suggested that VJLACS6 and VJLACS7 proteins were independently involved in the corresponding metabolic and catabolic metabolism of tung oil.5.Analysis of correlation of temporal and spatial expression of VJLACS6 and VfLACS7.By real-time qPCR technology,this paper analyzes the tung different tissues and organs as well as seeds VJLACS6 and VJLACS7 gene expression in different development periods.The expression of VfLACS6 gene was highest in roots and stems,expressed in petals volume is relatively low.VJLACS6 genes are expressed in different tissues and organs of tung volume from large to small in order:Stamens>root>stem>leaf>calyx>stigma>ovary>petals,it showed that VfLACS6 genes play an important role in the cutin grease anabolism.The relative expression quantity highest on September 20 of VfLACS6 in seed,and the relative expression quantity minimum on October 24;The expression of VfLACS7 gene was the highest in leaves,followed by the stem,express the minimum in the stigma,VfLACS7 genes are expressed in different tissues and organs of tung volume from large to small in order:Stamens>leaf>stem>root>calyx>petal>ovary>stigma,Thus speculate that VfLACS7 gene may play an important role in the leaf fat synthesis.The VfLACS7 gene was the highest relative to the August 15 development of seed development,the rest of the time expression level is roughly the same,show VfLACS7 play an important role only at the beginning of the seed germination of fat synthesis.