| Tung oil tree as peculiar to our country economic forest, is our country famous industrial oil tree species, known as one of the four major woody oil species of China. Tung oil is mainly consist of eleostearic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and so on, but eleostearic acid content accounts for more than 70%. It is a kind of high-quality industrial oil. During the fatty acid biosynthesis metabolic of tung oil tree seed, it including a series of regulation of physiological and biochemical metabolism enzymes and genes. FAS and FAE are one of the most important compound enzyme system. This research take different developmental stages of tung tree cultivar’PUTAOTONG’seeds as material, on the basis of the digitized transcriptome database, the full-length cDNA cloned and functional analyzed of tung KAR were carried out, which is one of FAS compound enzyme. It will provide a scientific basis for directional genetic improvement of tung fatty acid biosynthesis pathway and tung oil tree species, even the oil tree species. The main results are as follow:1. Molecule Cloning and bioinformatics analysis of KAR gene from tung oil tree. With RT-PCR technique, two members cDNA sequence of tung oil P-ketoacyl-CoA reductase gene families are cloned, respectively named as Vf_KAR1 and Vf_KAR2. The open reading frame of Vf_KAR1 and Vf_KAR2 are respectively 993bp and 903bp in length, which respectively codes 330 and 300 amino acids. The estimated molecular weight of the putative protein are 35kD and 32kD, and the isoelectric point were 9.28 and 9.11. The analysis of fat coefficient and total average hydrophilicity show that Vf_KAR1 and Vf_KAR2 are hydrophobic proteins. Vf_KAR1 has an obvious transmembrane helix topology structure and a suspected transmembrane helix topology, but Vf_KAR1 has only two suspected transmembrane helix topology. Vf_KAR1 and Vf_KAR2 contain chloroplast transit peptide,several active site and NAD(P) blinding site. Vf_KAR1 and Vf_KAR2. are all along to the member of SDR.From thier secondary structure and 3D structure, Vf_KAR1 and Vf_KAR2 mainly compose of alpha helix and beta fold. Thier amin acid sequences have high homology with other species, and Vf_KAR1 has 74% similarly with Vf_KAR2. In conclusion, Vf_KARl and Vf_KAR2 are the member of tung KAR.2. The relative expression of Vf_KAR gene from tung in different organs and difernt development period of seeds. The use of qRT-PCR standard curve to optimize system, the relative expression of Vf_KAR gene in different organs and difernt development period of seeds were detected. Vf_KAR1 and Vf_KAR2 express in all different organs, and Vf_KAR1 mainly express in seed, which are relative with its function to systhic fatty acid, but Vf_KAR2 mainly express in stem and leave. The expression pattern of Vf_KAR1 in differnt development period of seeds is similar to fatty acid biosynthesis in tung seed, but The expression pattern of Vf_KAR2 has not difference in all period of seeds. Thereforce, it can infere Vf_KAR1 maybe participate in fatty acid biosynthesis in tung,and it has the best function.3.The research of subcellular localization in tung KAR protein. Vf_KAR1 and Vf_KAR1 are all co-located in Chloroplast. The result is consisted with the prediction. The reason maybe relate to the chloroplast transit peptide in thier amin acids.4.The research of interactive relations between Vf_KAR1 and Vf_KAR2 proteins. From the self-activation tests, the concentration of 3AT in pDEST32-Vf_KAR1× pDEST22-Vf_KAR2 and pDEST32-Vf_KAR2×pDEST22-Vf_KAR1 double plasmid diploid yeast cell growth are 25mM. The concentration of 3AT in pDEST32-Vf_KAR1×pDEST22 and pDEST32-Vf_KAR2×pDEST22 double plasmid diploid yeast cell growth are 20mM. Through all double plasmid diploid yeast be cultivated in SC-Leu-Try-Ura and the Chromogenic reaction of X-gal, it finds Vf_KAR1 and Vf_KAR2 have not interactive relations. |
PDF Full Text Request