Cloning And Functional Expression Of LACS Gene In Vernicia Fordii

Seed oil of Vernicia fordii has very high economic value, it is an important industrial raw material, tung oil has plenty of fatty acids, such as Eleostearic Acid. Synthesis of fatty acid is a complex process, this process consists of many enzymatic reactions. Long chain acyl coenzyme A synthetase (LACS) is involved in fatty acid synthesis and decomposition, the synthesis of jasmonic acid and phospholipid, three acyl glycerin, and is also involved in the beta oxidation of fatty acid. Based on the key role of the LACS in fatty acid synthesis, to understand the function of LACS in tung oil synthesis is of great significance to the breeding of tung oil trees. This paper mainly carry out the tung tree LACS gene cloning, gene structure and gene expression studies, laid the foundation for the study of gene function, the main research results are as follows:1. cDNA cloning and bioinformatics analysis of LACSl, LACS4, LACS8 gene. Based on tung tree transcriptome data base, Vernicia fordii was taken as experimental materials and specific PCR primers was designed to get full-length cDNA sequence of LACS1, LACS4, LACS8 gene with the method of RT-PCR. LACS1 full-length cDNA is 1983 bp, coding 660 amino acids, the predicted protein molecular weight and isoelectric point is 74.59 kDa and 6.07, GenBank number is KT921224; LACS4 full-length cDNA is 1989 bp, coding 662 amino acids, the predicted protein molecular weight and isoelectric point is 73.79 kDa and 6.60, GenBank number is KP996689; LACS8 full-length cDNA is 2199 bp, coding 732 amino acids, the predicted protein molecular weight and isoelectric point is 80.5 kDa and 7.46, GenBank number is KT362743.2. Genome Molecule Cloning of LACS1 and LACS8 from Vernicia fordii. DNA was extracted from the leaf tissues of Vernicia fordii as a template, and two specific primers were designed, full-length genome sequences of LACS1 and LACS8 were cloned by using RT-PCR technique. Full-length genome sequences of LACS1 and LACS8 is 4783bp and 5191bp. The transcriptome analysis of cDNA sequence and genome sequence shows that LACS1 genome has 18 introns, LACS8 genome has 10 introns.3. Analysis of correlation of temporal and spatial expression of LACS gene and oil accumulation. Using qPCR technology to analysis relative expression of LACS1,LACS4,LACS8 in different tissues and different developmental stages of seed from Vernicia fordii. The relative expression of LACS1 in stem and leaf is relatively high and get highest in the old leaves, the expression level reached the highest on August 15 in the developmental seed; Relative expression of LACS4 in flowers and old stem is relatively high, especially in the stamen was the highest and expression peak of LACS4 in seed was happened on October 10; Relative expression of LACS8 was the highest in pistil, the expression level in seed was gradually increased in September 20 to October 20 and reached the maximum on October 20.The oil synthesis law of Vernicia fordii is like a "s" curve, The peak of oil synthesis in seed of Vernicia fordii is from August to October. The correlation of expression of LACS8 and Oil synthesis law showed that LACS8 may be involved in the synthesis of fatty acid.4.Eukaryotic expression of LACS4 from Vernicia fordii. The Eukaryotic expression vector pCAMBIA1300-35S-LACS4 was constructed successfully and was transformed into agrobacterium GV3101. Pollen tube pathway was used to infect Arabidopsis thaliana, after screening positive plants through culture medium with hygromycin resistance, the T1 generation positive plants were successfully obtained by the amplification of the hygromycin gene in the Eukaryotic expression vector from the DNA templates which were extracted from positive plants.