Molecular Cloning Of CaFAD2 And Its' Function Analysis

As an important renewable energy, Biodiesel develops fast in recent years with high price, lacking of plant oil and low quality. Because of low combusting value, high viscosity and instability of plant-derived biodiesel, thus it's important to cultivate oil plants with suitable components of fatty acids.According the report(Kevin J. Harrington, 1986), the ideal material plant oil contianing high unsaturated fatty acids features a long, unbranched carbon chain with only one double bond at the end of the molecule, on the other side, more polyunsaturated fatty acids can protect the plant from cold.FAD2 is an important enzyme in the first step of polyunsaturated fatty acid synthey, located in endoplasmic reticulum. It catalyzes conversion of oleic acid to linoleic acid. Caragana intermedia is an important bio-energy plant distributing widly in the north China and its seeds can be made biodiesel, and it must be improved to decrease linoleic acid to increase oleic acid for polyunsaturated fatty acid of its oil is as high as 60.1%. And increasing the polyunsaturated fatty acid can protect it from low temperatures. Furthermore, the structure, function and regulation of FAD2 enzyme are far from being elucidated.In this work, According to published sequences, CaFAD2, an FAD2 homolog, was cloned by RT-PCR and RACE from total RNA of Caragana intermedia. The full-length of CaFAD2 is 1411bp (accession No. AY957393), and the longest open reading frame in the CaFAD2 encodes a polypeptide of 350 amino acids. The deduced amino acid sequence of CaFAD2 shows high identity to other plant delta-12 fatty acid desaturases, including the three histidine motifs, and two long stretches of hydrophobic residues which indicates of an integral membrane protein spanning membrane six times. To verify the function of CaFAD2, sense- and antisense of CaFAD2 were transformed into tobacco via Agrobacterium tumefaciens., and some transgenic plants with CaFAD2 were established in the population of the primary transformants. Although transgenic tobacco grew as wildetype tobacco, the analyses of fatty acid profile of the transgenic line RTCO(antisense-CaFAD2) and TC0(sense CaFAD2) showed that CaFAD2 has important effect on the polyunsaturated fatty acid synthesis. Adrastic increase of monounsaturated fatty acid especially oleic acid in total leaf lipid of RTC0 indicated that antisense-CaFAD2 decreased FAD2 expression in tabacco while CaFAD2 over expressing promoted the synthesis of polyunsaturated fatty acid, increase linolicl. It was possible CaFAD2 expressed constituitively in Caragana intermedia for that CaFAD2 expressed in both transgenic tobacco vegetative and seed tissues while the expressing level is higher in leaf than in stem in Caragana intermedia.To compare FAD2 from different plant species, SoyFAD2 was cloned from soybean. CaFAD2 and SoyFAD2 share 90% identity in deduced amino acid sequence. The longest ORF of SoyFAD2 encodes a polypeptide of 380 amino acids, including the three histidine motifs and two long stretches of hydrophobic residues. Sense of SoyFAD2 was transformed into tobacco. And in the transgenic line TS0, the amount of polyunsaturated fatty acid increased, which is the same of that in TC0.Oil biosynthesis is a complex biological process. There must be exist a network of genes playing different roles to control the components of fatty acids. In this study, the function of FAD2 from oil plant was investigated in detail, which may give more understanding in oil synthesis and provides genes to improve fatty acids components suitable for biodiesel.